Composition comprising combination of lactoferrin and sulfated polysaccharide

Abstract

The present invention relates to a composition for use in the treatment and / or prevention of microbiota (in particular vaginal microbiota) dysbiosis in a subject wherein the composition comprises lactoferrin and a sulfated polysaccharide.

Description

Technical Field

[0001] The present invention relates to a composition for treating and / or preventing dysbacteriosis of a subject, wherein the composition comprises lactoferrin and a sulfated polysaccharide. In particular, the present invention relates to a composition for treating and / or preventing dysbacteriosis of the vaginal microbiota of a subject. Background Art

[0002] The microbiota commonly associated with the human body has important influences on human development, physiology, immunity, and nutrition. The vast majority of indigenous microbiota exist in symbiotic relationships with their human hosts, while a minority are opportunistic pathogens capable of causing chronic infections and life-threatening diseases. These microbial communities are thought to constitute the first line of defense against infection by competitively excluding invasive non-indigenous organisms that cause disease.

[0003] The indigenous microorganisms of the vagina play a protective role in preventing colonization of the host by potentially pathogenic organisms, including those that cause symptomatic bacterial vaginosis, yeast infections, sexually transmitted infections (STIs), and urinary tract infections.

[0004] Lactobacilli species are considered keystones of the vaginal microbiota in women of childbearing age of certain ethnicities. The most abundant Lactobacilli species are Lactobacillus iners, L. crispatus, and L. gasseri. Other species, such as L. jensenii, L. gallinarum, and L. vaginalis, have been found in some women.

[0005] The protective effect of lactobacilli results from multiple mechanisms: competition for nutrients and tissue adhesion, lowering of vaginal pH to ~3.5-~4.5, modulation of immunity, and production of bioactive compounds. Among the bioactive factors of the cervicovaginal mucosa, lactoferrin (an iron-binding cationic glycoprotein) is a multifunctional glycoprotein with antibacterial, antifungal, antiviral, and antiparasitic activities.

[0006] When the vaginal environment is depleted of predominant Lactobacillus flora, non-indigenous bacteria can begin to colonize.

[0007] Chronic or acute disturbances of the vaginal microbiota, so-called dysbiosis, can result from antibiotic use, hormonal fluctuations associated with menstruation, pregnancy, breastfeeding and birth control pills, a weakened immune system, sexual activity, age, lifestyle, vaginal lubricants and douching.

[0008] A decrease in Lactobacillus can lead to adverse conditions such as premature birth, sexually transmitted infections, and pelvic inflammatory disease. In addition, a decrease in Lactobacillus can lead to bacterial vaginosis (BV), a disease characterized by a more diverse vaginal microbiota characterized by Gardnerella vaginalis, Atopobium vaginae, Megasphaera, and many other anaerobic or facultative anaerobic species.

[0009] A study by Otsuki et al. (2014) showed that the administration of vaginal lactoferrin was able to increase the presence of Lactobacillus at the vaginal flora level, suggesting a role for lactoferrin as a prebiotic.

[0010] While attempts have been made to develop products that utilize lactoferrin to prevent and / or treat dysbiosis of the vaginal microbiota, there remains a need for improved compositions that promote the growth of existing vaginal commensal flora (e.g., vaginal Lactobacillus strains) while not promoting the growth of undesirable organisms (e.g., Gardnerella, Candida, and other opportunistic pathogens).

[0011] The plasma membrane of almost all mammalian cells contains negatively charged high molecular weight carbohydrate receptors, such as heparan sulfate glycosaminoglycans. The initial attachment of many different pathogens to host cells involves docking with these carbohydrate receptors. After binding, the pathogen is able to infect the cell by invading through the cell membrane.

[0012] Heparan sulfate proteoglycans (HSPGs) are glycoproteins consisting of a core protein linked to one or more linear heparin sulfate (HS) chains. These chains consist of alternating D-glucosamine and uronic acid (D-glucuronic acid and L-iduronic acid), which are capable of variable N- and O-sulfation. The presence of sulfate groups at specific positions in HS imparts a high overall negative charge, and their arrangement in short segments on the chain creates binding sites for HS binding proteins.

[0013] HSPGs present a negative charge, which can electrostatically interact with HS binding proteins expressed by pathogens (e.g., with the basic amino acid portion of viral surface proteins). This interaction promotes pathogen attachment to host cells, mediates cell entry and increases infectivity. Therefore, heparan sulfate glycosaminoglycans can be viewed as acting as initial low-affinity co-receptors that concentrate pathogens on the cell surface and promote binding to secondary receptors that stimulate infection.

[0014] Therefore, disrupting the interaction between HS on the surface of mammalian cells and HS-binding proteins on pathogens is a viable therapeutic option to prevent infection.

[0015] Some suitable treatment options are based on disrupting the binding of pathogen-expressed HS-binding proteins to HS through competitive binding of positively charged polymers to HS. Suitable positively charged polymers include carrageenan and chitosan. These polymers rely on electrostatic interactions between their positively charged functional groups and the highly anionic sulfate and carboxylate moieties of HS.

[0016] Other promising compounds for use as therapeutics are based on the use of exogenous HSPG mimetic compounds that act as competitive inhibitors. Examples include, but are not limited to, sulfated polysaccharides that can block viral infection by physicochemically mimicking cell-surface-bound heparan sulfate, thereby competing with the initial attachment of viral particles to the cell surface.

[0017] There is a need for compositions suitable for vaginal use that both inhibit colonization by pathogenic microorganisms and take into account the natural commensal vaginal microbiota. Summary of the invention

[0018] The present inventors have surprisingly discovered that the combined use of lactoferrin and sulfated polysaccharides has a beneficial effect on influencing and maintaining a healthy vaginal microbiota.

[0019] According to a first aspect, a composition comprising lactoferrin and sulfated polysaccharide for preventing and / or treating dysbacteriosis in a subject is provided.

[0020] In some embodiments, the sulfated polysaccharide consists of or comprises uniform or different monomeric units selected from the group of the following compounds: xylose, arabinose, rhamnose, fucose, glucose, mannose, galactose, lactose, fructose, glucosamine, galactosamine, mannosamine, glucuronic acid, galacturonic acid, mannuronic acid, carrageenan, agaran, ulvan, fucoids, laminaran, alginate, lentinan, pluran, xylan, dextran, heparin, keratan sulfate, chondroitin 4-sulfate, chondroitin 6-sulfate, dermatan sulfate, heparan sulfate, hyaluronic acid, teichuronic acid, and partial hydrolysates of starch, cellulose, glycogen, chitin or pectin.

[0021] In some embodiments, the sulfated polysaccharide is derived from algae.

[0022] The carrageenan may be selected from kappa-(κ) carrageenan, iota-(ι) carrageenan and lambda-(λ) carrageenan. Preferably, the carrageenan is kappa-(κ) carrageenan.

[0023] In some embodiments, the composition contains about 5 μg / ml to about 50 μg / ml lactoferrin.

[0024] In some embodiments, the composition contains about 5 μg / ml to about 50 μg / ml carrageenan.

[0025] In some embodiments, the composition comprises about 10 μg / ml to about 30 μg / ml lactoferrin and about 10 μg / ml to about 30 μg / ml carrageenan.

[0026] In some embodiments, the composition is suitable for vaginal administration, preferably, the composition is a vaginal lubricant and / or a vaginal moisturizer.

[0027] In some embodiments, the composition is used to prevent qualitative-quantitative changes in the physiological vaginal microbiota, wherein the changes can cause infections and inflammatory processes.

[0028] In some embodiments, the composition is a pharmaceutical composition for treating vaginal and cervical-vaginal infections. The infection can be caused by bacteria, fungi or viruses. For example, the infection can be selected from the group consisting of vaginitis, vaginosis, herpes simplex, papillomavirus (HPV), thrush.

[0029] According to a second aspect of the present invention, there is provided a device for preventing and / or treating vaginal and cervicovaginal infections caused by dysbiosis of vaginal microbiota, wherein the device comprises the composition of the first aspect.

[0030] In some embodiments, the device is coated or impregnated with the composition of the first aspect. In some embodiments, the device is a vaginal pessary, a vaginal suppository, a tampon, a contraceptive, a spatula, or an intimate toy.

[0031] For example, the device may be a condom pre-coated with a lubricant comprising the composition of the first aspect.

[0032] According to a third aspect, a method for preventing and / or treating vaginal dysbacteriosis is provided, the method comprising the step of administering to a subject a composition comprising lactoferrin and sulfated polysaccharides. More specifically, the method can be used to prevent and / or treat vaginal and cervicovaginal inflammation and infection caused by dysbacteriosis of the vaginal microbiota.

[0033] Non-limiting examples of vaginal and cervicovaginal inflammation and infections that can be treated using the compositions of the present invention include bacterial vaginitis caused by Gardnerella, chlamydiosis caused by Chlamydia trachomatis, genital herpes caused by herpes simplex virus (HSV), genital warts caused by human papillomavirus (HPV), gonorrhea, thrush (candidiasis infection) caused by Candida. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Embodiments of the present invention will now be described, by way of example only, with reference to the following drawings, in which:

[0035] Figure 1 A checkerboard assay for evaluating the combined effects of lactoferrin and carrageenan on specific microorganisms is shown;

[0036] Figure 2 shows the prebiotic effect of (a) an exemplary composition of the first aspect containing 12.5 μg / ml of κ-carrageenan and 25 μg / ml of lactoferrin; and (b) an exemplary composition of the first aspect containing 25 μg / ml of κ-carrageenan and 25 μg / ml of lactoferrin on Lactobacillus crispatus 33197;

[0037] 3 shows the prebiotic effect of (a) an exemplary composition of the first aspect containing 12.5 μg / ml iota-carrageenan and 25 μg / ml lactoferrin; and (b) an exemplary composition of the first aspect containing 25 μg / ml iota-carrageenan and 25 μg / ml lactoferrin on Lactobacillus crispatus 33197;

[0038] Figure 4 shows the prebiotic effect of (a) an exemplary composition of the first aspect containing 12.5 μg / ml of κ-carrageenan and 25 μg / ml of lactoferrin; and (b) an exemplary composition of the first aspect containing 25 μg / ml of κ-carrageenan and 25 μg / ml of lactoferrin on Lactobacillus jensenii 25258;

[0039] 5 shows (a) an exemplary composition of the first aspect containing 12.5 μg / ml iota-carrageenan and 25 μg / ml lactoferrin; and (b) an exemplary composition of the first aspect containing 25 μg / ml iota-carrageenan and 25 μg / ml lactoferrin, the prebiotic effect on Lactobacillus jensenii 25258;

[0040] 6 shows the antimicrobial effects of (a) an exemplary composition of the first aspect containing 12.5 μg / ml κ-carrageenan and 25 μg / ml lactoferrin and (b) an exemplary composition of the first aspect containing 6.25 μg / ml κ-carrageenan and 12.5 μg / ml lactoferrin against Gardnerella vaginalis 14018.

[0041] 7 shows the antimicrobial effects of (a) an exemplary composition of the first aspect containing 12.5 μg / ml iota-carrageenan and 25 μg / ml lactoferrin; and (b) an exemplary composition of the first aspect containing 6.25 μg / ml iota-carrageenan and 12.5 μg / ml lactoferrin against Gardnerella vaginalis 14018.

[0042] definition

[0043] "Dysbiosis" or "dysbiotic state" refers to an imbalance in the types of organisms present in the body's natural microbiome, which is thought to contribute to a range of adverse health conditions.

[0044] "Ecological balance" or "ecological equilibrium" refers to the optimal balance of the types of organisms present in the natural microbiome of the human body and is believed to lead to a range of adverse health conditions.

[0045] "Non-human lactoferrin" refers to lactoferrin produced or obtained from a source other than human breast milk, such as bovine lactoferrin.

[0046] "Personal lubricants" are specialized lubricants used during intimate acts (such as sexual intercourse and masturbation) to reduce friction between the penis and the vagina, anus, or other body parts, or used in sex toys to reduce friction or facilitate insertion. These are different from surgical or medical lubricants or gels, similar to personal lubricants that can be used for medical purposes (such as speculum insertion).

[0047] "Reconstituted solution" refers to a solution prepared by adding a diluent (e.g., water, saline, etc.) to an ingredient (e.g., powder, solution, gel, suspension, paste, solid, liquid, liquid concentrate, etc.). When the term "reconstituted solution" is used in reference to lactoferrin and carrageenan, this refers to a solution prepared by adding a diluent (e.g., water, saline, etc.) to a certain form of lactoferrin / lactoferrin / carrageenan, carrageenan-containing composition (i.e., in the form of a powder, solution, gel, suspension, paste, solid, liquid, liquid concentrate, etc.).

[0048] Unless otherwise indicated, all percentages, parts and ratios used herein are recited by weight of the total formulation.All amounts specified for "daily" administration may be administered in a single unit dose, a single dosage, two or more doses or dosages over a 24 hour period.

[0049] Whenever a singular feature or limitation is mentioned in the present disclosure, it shall include the corresponding plural feature or limitation, and vice versa, unless otherwise stated or the context of the reference clearly implies otherwise.

[0050] All combinations of method or process steps disclosed herein can be performed in any order unless otherwise indicated or clearly implied to the contrary by the context of the referenced combination.

[0051] The formulations of the present disclosure may comprise, consist of, or consist essentially of any of the components described herein, as well as any additional useful ingredients.

[0052] All features disclosed in this specification (including any accompanying claims, abstract and drawings), and / or all steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of the features and / or steps are mutually exclusive.

[0053] Unless expressly stated otherwise, each feature disclosed in this specification (including any accompanying claims, abstract and drawings) may be replaced by an alternative feature having the same, equivalent or similar purpose. Therefore, unless expressly stated otherwise, each feature disclosed is only an example of a series of equivalent or similar features. DETAILED DESCRIPTION

[0054] Lactoferrin is a polypeptide belonging to the transferrin family. In nature, lactoferrin is present in many mucus secretions, including vaginal secretions. The concentration of lactoferrin in human vaginal fluid is equivalent to 1μg / ml to 3μg / ml (Valenti P, 2018), and the content in cervical mucus plugs is extremely high (100μg / ml) (Heim, M, 2002)

[0055] The antimicrobial activity of lactoferrin has been demonstrated to be effective against bacteria, viruses, fungi and parasites (Farnaud S, 2003).

[0056] Antimicrobial activity of lactoferrin and its effect on iron ions (Fe 3+ The combination of lactoferrin and iron ions in mucus secretions regulates the proliferation and aggregation of bacteria and viruses to the cell membrane.

[0057] Lactoferrin of any aspect detailed herein can include human lactoferrin produced by a genetically modified organism, humanized lactoferrin, non-human lactoferrin, or any combination thereof. Non-human lactoferrin can include bovine lactoferrin, porcine lactoferrin, horse lactoferrin, buffalo lactoferrin, goat lactoferrin, murine lactoferrin, or camel lactoferrin.

[0058] In some embodiments, the sulfated polysaccharide is a sulfated polysaccharide of marine origin. Examples include, but are not limited to, carrageenan from red macroalgae, fucoidan from brown macroalgae, ulvan from green macroalgae, and agar from red macroalgae (composed of two polysaccharides, agarose and agaropectin).

[0059] Carrageenan is a general term for linear galactosyl polysaccharides extracted from seaweed (Rhodophyceae). Carrageenan consists of repeating units of sulfated D-galactose and 3,6-anhydro-D-galactose. There are more than 10 structurally different carrageenans, and their properties depend on the seaweed species from which they are extracted. There are three main types of carrageenan, which differ in the degree of sulfation, with kappa (κ) carrageenan having one sulfate group per disaccharide, iota (ι) carrageenan having two sulfate groups, and lambda (λ) carrageenan having three.

[0060] The food, cosmetic and pharmaceutical industries use carrageenans extensively as thickeners, gelling agents and stabilizers. They are classified as “generally recognized as safe” (GRAS) by the Food and Drug Administration (FDA). Carrageenans have favorable physical properties and also show antiviral activity against a range of viruses, including human papillomavirus (HPV).

[0061] In addition to interacting with many different viral particles and bacteria, carrageenan forms a mucoadhesive layer on the surface of the vaginal epithelium, thereby creating a shield on its surface that prevents interaction with the epithelium and infection of cells.

[0062] Carrageenan’s advantageous combination of low coefficient of friction and antimicrobial properties has led to its inclusion in some brands of vaginal lubricants and spermicides.

[0063] The carrageenan used in the present invention is commercially available, but can also be prepared by extracting from seaweed plants according to extraction methods known in the art.

[0064] The carrageenan of the present invention is a homopolymer or a heteropolymer. The homopolymer of carrageenan is composed of only one type of subunit among kappa, iota and lambda carrageenan, while the heteropolymer contains two or more types of carrageenan subunits.

[0065] The lactoferrin content of the composition may be about 5 μg / ml to 50 μg / ml. In some embodiments, the lactoferrin content of the composition may be 10 μg / ml to about 40 μg / ml, 15 μg / ml to about 35 μg / ml, or about 20 μg / ml to about 25 μg / ml.

[0066] In some specific embodiments, the composition comprises a combination of lactoferrin and carrageenan.

[0067] In some specific embodiments, the composition comprises a combination of lactoferrin and kappa-carrageenan.

[0068] Lactoferrin may be present in an amount of about 5 μg / ml to about 50 μg / ml. In some embodiments, the composition may contain lactoferrin in an amount of 10 μg / ml to about 40 μg / ml, 15 μg / ml to about 35 μg / ml, or about 20 μg / ml to about 25 μg / ml. κ-carrageenan may be present in an amount of about 5 μg / ml to about 50 μg / ml. In some embodiments, the composition may contain κ-carrageenan in an amount of 10 μg / ml to about 40 μg / ml, 15 μg / ml to about 35 μg / ml, or about 20 μg / ml to about 25 μg / ml.

[0069] In some specific embodiments, the composition comprises a combination of lactoferrin and iota-carrageenan.

[0070] Lactoferrin may be present in an amount of about 5 μg / ml to about 50 μg / ml. In some embodiments, the composition may contain lactoferrin in an amount of 10 μg / ml to about 40 μg / ml, 15 μg / ml to about 35 μg / ml, or about 20 μg / ml to about 25 μg / ml. ι-carrageenan may be present in an amount of about 5 μg / ml to about 50 μg / ml. In some embodiments, the composition may contain ι-carrageenan in an amount of 10 μg / ml to about 40 μg / ml, 15 μg / ml to about 35 μg / ml, or about 20 μg / ml to about 25 μg / ml.

[0071] In some specific embodiments, the composition comprises lactoferrin, kappa-carrageenan and iota-carrageenan. Advantageously, such a composition is used as a broad-spectrum prebiotic. For example, the combination of lactoferrin and kappa-carrageenan has been shown to have a prebiotic effect on Lactobacillus crispatus, while iota-carrageenan has been shown to have a prebiotic effect on Lactobacillus jensenii.

[0072] Lactoferrin may be present in an amount of about 5 μg / ml to about 50 μg / ml. In some embodiments, the content of lactoferrin in the composition may be 10 μg / ml to about 40 μg / ml, 15 μg / ml to about 35 μg / ml, or about 20 μg / ml to about 25 μg / ml. κ-carrageenan may be present in an amount of about 5 μg / ml to about 50 μg / ml. In some embodiments, the content of κ-carrageenan in the composition may be 10 μg / ml to about 40 μg / ml, 15 μg / ml to about 35 μg / ml, or about 20 μg / ml to about 25 μg / ml. ι-carrageenan may be present in an amount of about 5 μg / ml to about 50 μg / ml. In some embodiments, the content of ι-carrageenan in the composition may be 10 μg / ml to about 40 μg / ml, 15 μg / ml to about 35 μg / ml, or about 20 μg / ml to about 25 μg / ml.

[0073] In some specific embodiments, the composition contains about 10 μg / ml to 30 μg / ml of lactoferrin and about 10 μg / ml to 30 μg / ml of κ-carrageenan.

[0074] In some specific embodiments, the composition contains about 25 μg / ml lactoferrin and about 12.5 μg / ml kappa-carrageenan.

[0075] In some specific embodiments, the composition contains about 25 μg / ml lactoferrin and 25 μg / ml kappa-carrageenan.

[0076] In some specific embodiments, the composition contains about 10 μg / ml to 30 μg / ml lactoferrin and about 10 μg / ml to 30 μg / ml iota-carrageenan.

[0077] In some specific embodiments, the composition contains about 25 μg / ml lactoferrin and about 12.5 μg / ml iota-carrageenan.

[0078] In some specific embodiments, the composition contains about 25 μg / ml lactoferrin and 25 μg / ml iota-carrageenan.

[0079] In some specific embodiments, the composition contains about 10 μg / ml to 30 μg / ml of lactoferrin, about 10 μg / ml to 30 μg / ml of kappa-carrageenan, and about 10 μg / ml to 30 μg / ml of iota-carrageenan.

[0080] In some specific embodiments, the composition contains about 25 μg / ml lactoferrin, about 12.5 μg / ml kappa-carrageenan, and about 12.5 μg / ml iota-carrageenan.

[0081] In some specific embodiments, the composition contains about 25 μg / ml lactoferrin, about 25 μg / ml kappa-carrageenan, and about 25 μg / ml iota-carrageenan.

[0082] The composition of the first aspect may be administered to a subject by topical intravaginal administration into the vagina.

[0083] The composition of the first aspect may be in the form of a powder, solution, gel, suspension, paste, solid, liquid, liquid concentrate or reconstituted solution. In some specific embodiments, the composition is administered to the subject in the form of a personal lubricant.

[0084] According to a second aspect of the invention, a device comprising the composition of the first aspect is provided. In some embodiments, the composition is coated or impregnated on a device inserted into the vagina. The device can be inserted into the vagina permanently or temporarily. For example, the device can be a vaginal pessary, a vaginal suppository, a tampon, a contraceptive device, a spatula or a private toy. The contraceptive device can be a condom. The condom can be a male condom and / or a female condom.

[0085] In some embodiments, the composition is attached to the surface of the device, for example, by physical and / or chemical attachment. In some other embodiments, the composition is used to lubricate the device.

[0086] The compositions described herein may be used prophylactically by a subject to maintain an ecologically balanced vaginal microbiota.Thus, the compositions of the first aspect may be used non-therapeutically.

[0087] The composition can be used therapeutically by a subject to treat dysbiosis of the vaginal microbiota in an attempt to push the microbiota toward a state of ecological balance.

[0088] Therefore, according to the third aspect, a method for preventing and / or treating vaginal flora imbalance in a subject is provided, the method comprising the step of administering the composition of the first aspect to the subject. More specifically, the method can be used to prevent and / or treat vaginal and / or cervical-vaginal infections.

[0089] In some embodiments, the method can include administering the composition to a subject with sufficient frequency and for sufficient time to promote the growth of at least some species of vaginal lactobacilli while reducing or preventing the growth of pathogenic species. Thus, the composition enhances the natural lactobacilli population, thereby promoting an environment that is harmful to vaginal pathogens.

[0090] More specifically, the method can include administering the composition to the subject with sufficient frequency and for sufficient time to promote the growth of Lactobacillus crispatus and / or Lactobacillus jensenii while reducing or preventing the growth of Gardnerella vaginalis and / or Candida albicans.

[0091] The frequency of administering the composition to a subject can vary according to the needs of the subject. For example, the composition can be administered daily.

[0092] The composition may also contain probiotics. In particular, the probiotics may be selected from lactobacilli, bifidobacteria, pediococci and mixtures thereof. Preferably, at least one of the following is used as the lactobacillus: Lactobacillus plantarum, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus bulgaricus, Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus acidophilus, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus crispatus, Lactobacillus iners, Lactobacillus jensenii, Lactobacillus gallinarum and Lactobacillus vaginalis, and as the bifidobacteria, at least one of the following is used: Bifidobacterium lactis, Bifidobacterium bifidum and mixtures thereof.

[0093] The invention is not limited to the details of the above embodiments. The invention extends to any novel feature or any novel combination of features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel step or any novel combination of steps of any disclosed method or process.

[0094] The scope of the present invention is defined by the appended claims. It should be appreciated that a skilled person may modify the scope of the claims without departing from the scope of the present disclosure.

[0095] Example

[0096] method

[0097] A stock solution of bovine lactoferrin was prepared at 8 mg / mL by adding the appropriate amount of sterile MilliQ water to the weighed compound, followed by vortexing and filter sterilization.

[0098] 2 mg / mL stock solutions of carrageenan (κ-, ι-, λ-) were prepared by adding appropriate amount of sterile MilliQ water to the weighed compound, incubating at 37°C for 30 min, then vortexing until clear and filter sterilizing.

[0099] like Figure 1 As shown, a 96-well microtiter plate checkerboard assay was used to evaluate the synergistic combination effect of different doses of lactoferrin and / or carrageenan (κ-, ι- or λ-) on specific microorganisms. The use of a checkerboard assay enables the evaluation of whether a specific combination of ingredients has a prebiotic effect or an antimicrobial effect.

[0100] The lactoferrin stock solution is serially diluted along the rows of a microtiter plate in a broth-based medium appropriate for the growth of the specific microorganism to be tested.

[0101] A stock solution of carrageenan (κ-, Ι-, or λ-) is serially diluted along the columns of a microtiter plate in a broth-based medium appropriate for the growth of the specific microorganism to be tested.

[0102] The final volume of culture medium in each well was 0.1 mL.

[0103] In each well containing lactoferrin and / or carrageenan (κ-, ι-, or λ-), 5x10 5 Logarithmic phase bacteria / yeast were inoculated at a concentration of colony forming units (CFU) / well.

[0104] Positive (growth) controls consisted of the relevant microorganism inoculated in broth-based medium.

[0105] Negative (no growth) controls consisted of broth-based medium in the absence of any microorganisms.

[0106] A summary of the examples is given in Table 1 below.

[0107] Table 1

[0108]

[0109] After adding the relevant microorganisms, the plates were sealed with optical quality films and incubated in a plate reader at a temperature suitable for the growth of the microorganisms to be tested. The optical density (OD) was recorded every 15 minutes for 48 hours to assess microbial growth and biomass formation.

[0110] result

[0111] Prebiotic effect of a composition containing lactoferrin and carrageenan

[0112] Examples 1 to 4 demonstrate that the combination of lactoferrin and different types of carrageenan has a prebiotic effect on Lactobacillus crispatus 33197 and Lactobacillus jensenii 25258.

[0113] Example 1: Effect of the combination of κ-carrageenan and lactoferrin on Lactobacillus crispatus 33197

[0114] The prebiotic effects of different concentrations of lactoferrin and kappa-carrageenan, alone and in combination, on Lactobacillus crispatus 33197 were evaluated. Lactobacillus crispatus is a commensal bacterium in the vaginal microbiota, therefore compositions having a prebiotic effect on this bacterium are particularly advantageous.

[0115] Figure 2a The effect of 12.5 μg / ml kappa-carrageenan and / or 25 μg / ml lactoferrin is shown. Figure 2b The effect of 25 μg / ml kappa-carrageenan and 25 μg / ml lactoferrin is shown.

[0116] 25μg / ml lactoferrin and 12.5μg / ml ( Figure 2a ) or 25 μg / ml ( Figure 2b The combination of κ-carrageenan and β-carrageenan showed a synergistic effect on the growth of Lactobacillus crispatus 33197. The combination has a prebiotic effect, not only promoting growth, but also shortening the initial lag phase. The lag phase is one of the earliest stages of the bacterial growth cycle and is the stage when bacteria tend to adapt themselves to growth conditions; maturing but not dividing.

[0117] Embodiment 2: ι-Carrageenan Effects of the combination of 1,2-dihydrotestosterone and lactoferrin on Lactobacillus crispatus 33197

[0118] Different concentrations of lactoferrin and iota-carrageenan alone and in combination were evaluated for their prebiotic effects on Lactobacillus crispatus 33197. Lactobacillus crispatus is a commensal bacterium in the vaginal microbiota, therefore compositions having a prebiotic effect on this bacterium are particularly advantageous.

[0119] Figure 3a The effect of 12.5 μg / ml iota-carrageenan and / or 25 μg / ml lactoferrin is shown. Figure 3b The effect of 25 μg / ml iota-carrageenan and 25 μg / ml lactoferrin is shown.

[0120] The combination of 25 μg / ml lactoferrin and 25 μg / ml iota-carrageenan ( Figure 3b ) has a synergistic effect on the growth of Lactobacillus crispatus 33197, and this effect becomes obvious in about 10 hours. The composition has a prebiotic effect, not only promoting growth, but also shortening the lag phase.

[0121] Example 3: Effect of the combination of κ-carrageenan and lactoferrin on Lactobacillus jensenii 25258

[0122] The prebiotic effects of different concentrations of lactoferrin and kappa-carrageenan, alone and in combination, were evaluated on Lactobacillus jensenii 25258. Lactobacillus jensenii is a commensal bacterium in the vaginal microbiota, therefore compositions having a prebiotic effect on this bacterium are particularly advantageous.

[0123] Figure 4a The effect of 12.5 μg / ml kappa-carrageenan and / or 25 μg / ml lactoferrin is shown. Figure 4b The effect of 25 μg / ml kappa-carrageenan and 25 μg / ml lactoferrin is shown.

[0124] like Figure 4a As shown, the combination of 25 μg / ml lactoferrin and 12.5 μg / ml κ-carrageenan had less effect on the growth of Lactobacillus jensenii 25258 than testing 12.5 μg / ml κ-carrageenan alone. Figure 4b As shown, it was observed that the combination of 25 μg / ml lactoferrin and 25 μg / ml κ-carrageenan improved the prebiotic effect.

[0125] Embodiment 4: ι-Carrageenan Effects of the combination of 1,2-dihydrotestosterone and lactoferrin on Lactobacillus jensenii 25258

[0126] The prebiotic effects of different concentrations of lactoferrin and iota-carrageenan, alone and in combination, were evaluated on Lactobacillus jensenii 25258. Lactobacillus jensenii is a commensal bacterium in the vaginal microbiota, therefore compositions having a prebiotic effect on this bacterium are particularly advantageous.

[0127] Figure 5a The effect of 25 μg / ml lactoferrin and 12.5 μg / ml ι-carrageenan is shown. Figure 5b The effect of 25 μg / ml lactoferrin and 25 μg / ml iota-carrageenan is shown.

[0128] As shown in FIG5 , a combination of 25 μg / ml lactoferrin and 12.5 μg / ml ι-carrageenan ( Figure 5a ) or a combination of 25 μg / ml lactoferrin and 25 μg / ml iota-carrageenan ( Figure 5b ) has a synergistic effect on the growth of Lactobacillus jensenii 25258. The combination keeps Lactobacillus jensenii 25258 alive in the stationary phase of the bacterial growth cycle, whereas, in contrast, when the bacteria are exposed to lactoferrin or iota-carrageenan alone, the bacteria enter a death phase.

[0129] Antimicrobial effects of compositions comprising lactoferrin and carrageenan

[0130] Examples 5 and 6 demonstrate the antimicrobial effect of lactoferrin in combination with different types of carrageenan against Gardnerella vaginalis 14018. Gardnerella vaginalis is the bacterium that causes bacterial vaginosis (BV), and therefore compositions having an antimicrobial effect against this bacterium are particularly advantageous.

[0131] Example 5: Effect of the combination of κ-carrageenan and lactoferrin on Gardnerella vaginalis 14018

[0132] The antimicrobial effects of different concentrations of lactoferrin and kappa-carrageenan alone and in combination against Gardnerella vaginalis 14018 were evaluated.

[0133] Figure 6a The effect of 25 μg / ml lactoferrin and 12.5 μg / ml κ-carrageenan is shown. Figure 6b The effect of 12.5 μg / ml lactoferrin and 6.25 μg / ml kappa-carrageenan is shown.

[0134] As shown in FIG6 , a combination of 25 μg / ml lactoferrin and 12.5 μg / ml κ-carrageenan ( Figure 6a ) or a combination of 12.5 μg / ml lactoferrin and 6.25 μg / ml κ-carrageenan ( Figure 6b ) has a synergistic effect in inhibiting the growth of vaginal Gardnerella 14018.

[0135] Embodiment 6: ι-Carrageenan Effects of the combination of 1,2-dihydrotestosterone and lactoferrin on vaginal Gardnerella 14018

[0136] The antimicrobial effects of different concentrations of lactoferrin and iota-carrageenan alone and in combination against Gardnerella vaginalis 14018 were evaluated.

[0137] Figure 7a The effect of 25 μg / ml lactoferrin and 12.5 μg / ml iota-carrageenan is shown. Figure 7b The effect of 12.5 μg / ml lactoferrin and 6.25 μg / ml iota-carrageenan is shown.

[0138] As shown in FIG. 7 , a combination of 25 μg / ml lactoferrin and 12.5 μg / ml ι-carrageenan ( Figure 7a ) or 12.5μg / ml lactoferrin and 6.25μg / ml ( Figure 7b ) inhibited the growth of Gardnerella vaginalis 14018 compared to the positive control, but only to levels comparable to lactoferrin or iota-carrageenan when used alone.

[0139] in conclusion

[0140] It would be desirable for a composition for vaginal administration (eg, a personal lubricant) to prevent and / or treat dysbiosis of the vaginal microbiota by promoting the growth of commensal bacteria while inhibiting the growth of pathogenic bacteria.

[0141] The above examples show that in particular, a composition comprising a combination of lactoferrin and kappa-carrageenan is particularly advantageous, not only showing a synergistic prebiotic effect against Lactobacillus crispatus 33197 (see Example 1), but also showing a synergistic antimicrobial effect against Gardnerella vaginalis 14018 (see Example 5).

[0142] Compositions comprising a combination of lactoferrin and iota-carrageenan are advantageous, showing a synergistic prebiotic effect against Lactobacillus crispatus 33197 (see Example 2), but also showing an antimicrobial effect against Gardnerella vaginalis 14018 (see Example 6) (comparable to lactoferrin or iota-carrageenan alone).

[0143] References

[0144] Farnaud, S. and Evans, RW Lactoferrin: a multifunctional protein with antimicrobial properties. Molecular Immunology. 2003; 40(7): 395-405.

[0145] Hein M.et al.Antimicrobial factors in the cervical mucus plug.Am.J.Obstet.Gynecol.2002Jul;187(1):137-44.

[0146] Valenti P.et al.Role of lactobacilli and lactoferrin in the mucosalcervicovaginal defense.Front Immunol.2018;9:376.

[0147] Otsuki K.et al.Administration of oral and vaginal prebioticlactoferrin for a woman with refractory vaginitis recurring preterm delivery:Appearance of lactobacillus in vaginal flora followed by term delivery.TheJournal of Obstet.Gynaecol.Res.2014Feb;40(2):585-585.

Claims

1. A composition comprising lactoferrin and sulfated polysaccharide for use in treating and / or preventing dysbacteriosis in a subject.

2. The composition according to claim 1, wherein The sulfated polysaccharide is composed of or includes uniform or different monomer units selected from the group of the following compounds: xylose, arabinose, rhamnose, fucose, glucose, mannose, galactose, lactose, fructose, glucosamine, galactosamine, mannosamine, glucuronic acid, galacturonic acid, mannuronic acid, carrageenan, agar, ulvan, fucoidan, laminarin, alginate, lentinan, pullulan, xylan, dextran, heparin, keratan sulfate, 4-chondroitin sulfate, 6-chondroitin sulfate, dermatan sulfate, heparan sulfate, hyaluronic acid, uronic acid teichoic acid, and partial hydrolysates of starch, cellulose, glycogen, chitin or pectin.

3. The composition according to claim 1, wherein The sulfated polysaccharide is derived from seaweed.

4. The composition according to claim 2 or 3, wherein The carrageenan is selected from the group consisting of kappa-(κ) carrageenan, iota-(ι) carrageenan and lambda-(λ) carrageenan.

5. A composition as claimed in any one of the preceding claims, wherein The composition comprises about 5 μg / ml to about 50 μg / ml lactoferrin.

6. The composition according to any one of claims 2 to 5, wherein The composition comprises about 5 μg / ml to about 50 μg / ml of carrageenan.

7. The composition according to claim 6, wherein The composition comprises about 10 μg / ml to about 30 μg / ml of lactoferrin and about 10 μg / ml to about 30 μg / ml of carrageenan.

8. Composition according to any of the preceding claims, for use in preventing qualitative-quantitative changes in the physiological vaginal microflora, wherein said changes are capable of causing infectious and inflammatory processes.

9. The composition of claim 8, wherein The composition is suitable for vaginal administration, preferably wherein the composition is a personal lubricant.

10. A composition as claimed in any one of the preceding claims, wherein The composition is a pharmaceutical composition.

11. The composition according to claim 10, wherein The pharmaceutical composition is used to treat vaginal and cervicovaginal infections.

12. The composition according to claim 11, wherein The infection is caused by bacteria, fungi or viruses.

13. The composition of claim 12, wherein The infection is selected from the group consisting of vaginitis, vaginosis, herpes simplex, human papillomavirus (HPV), and thrush.

14. A device for preventing and / or treating vaginal and cervicovaginal infections, wherein the device comprises the composition of any one of claims 1 to 13.

15. The device of claim 14, wherein: The device is coated or impregnated with the composition, preferably wherein the device is a vaginal pessary, vaginal suppository, tampon, contraceptive device, spatula or intimate toy.

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