COMPOSITION FOR USE IN THE TREATMENT OF GROUP-B STREPTOCOCCUS (GBS) INFECTIONS

Abstract

The present invention concerns the field of bacterial infections, and in particular Group-B Streptococcus (GBS) infections during and after pregnancy. The present invention relates to the use of a composition comprising a strain of Lactobacillus acidophilus, a strain of Lactobacillus rhamnosus, and lactoferrin in the preventive and/or curative treatment of Group-B Streptococcus (GBS) infections.

Claims

1-13. (canceled)

14. A method for preventing or treating Group-B Streptococcus (GBS) infections in a subject in need thereof, said method comprising: administering to said subject a composition comprising a strain of Lactobacillus acidophilus, a strain of Lactobacillus rhamnosus, and lactoferrin, wherein said Group-B Streptococcus (GBS) infections occur during or after pregnancy, and wherein said strain of Lactobacillus acidophilus has a 16s region having at least 95% identity with the sequence of SEQ ID NO:1.

15. The method according to claim 14, wherein said strain of Lactobacillus acidophilus and said strain of Lactobacillus rhamnosus are each independently of each other in a total concentration in a range from 10.sup.7 to 10.sup.12 CFU/dose.

16. The method according to claim 14, wherein said strain of Lactobacillus acidophilus is selected from the group consisting of L. acidophilus deposited with deposit number LMG S-29159, L. acidophilus GLA-14 and L. acidophilus La 14.

17. The method according to claim 14, wherein said strain of Lactobacillus rhamnosus is selected from the group consisting of L. rhamnosus deposited with deposit number SD5675, L. rhamnosus Lr-32, L. rhamnosus GG, L. rhamnosus GR-1 and L. rhamnosus SP-1.

18. The method according to claim 14, wherein said strain of Lactobacillus acidophilus has a 16s region having 100% identity with the sequence of SEQ ID NO:1.

19. The method according to claim 14, wherein said strain of Lactobacillus acidophilus is deposited with deposit number LMG S-29159 and said strain of Lactobacillus rhamnosus is deposited with deposit number SD5675.

20. The method according to claim 14, wherein said lactoferrin is in a total concentration in a range from 5 mg/dose to 300 mg/dose.

21. The method according to claim 14, further comprising at least one additional component selected from the group consisting of: minerals, vitamins, probiotics, prebiotics, proteins and mixtures thereof.

22. The method according to claim 21, wherein said minerals are selected from the group consisting of calcium, phosphorus, copper, magnesium, potassium, iron, selenium, sodium, zinc, manganese, chloride and iodine, said vitamins are selected from the group consisting of vitamin A, vitamin B1 (thiamin), vitamin B2 (riboflavin), vitamin B3 (niacin) vitamin B6, vitamin B8 (biotin), vitamin B12, vitamin C, vitamin D, vitamin E and folic acid, said probiotics are selected from the group consisting of Bifidobacterium or lactic acid bacteria selected from the group consisting of Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus bifidus Lactobacillus reuteri, Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus plantarum, Lactobacillus jensenii, Lactobacillus delbrueckii, Lactobacillus fermentum, Lactobacillus helveticus, Lactobacillus iners and said proteins are selected from whey and casein.

23. The method according to claim 14, comprising Lactobacillus acidophilus in a range from 10.sup.7 to 10.sup.12 CFU/dose, Lactobacillus rhamnosus in a range from 10.sup.7 to 10.sup.12 CFU/dose, lactoferrin in a range from 5 mg/dose to 300 mg/dose and pharmaceutically acceptable excipients.

24. The method according to claim 14 wherein said composition is used in a daily dosage in the range from 10.sup.7 to 10.sup.12 CFU/dose in combination with lactoferrin in the range from 5 mg/dose to 300 mg/dose.

25. The method according to claim 14, wherein said composition is for oral administration.

26. The method according to claim 14, wherein said composition is in solid, liquid or semi-liquid form, selected from the group consisting of a tablet, a capsule, a powder, a granulate, a soluble stick and a liquid suspension.

27. The method according to claim 14, wherein said strain of Lactobacillus acidophilus and said strain of Lactobacillus rhamnosus are each independently of each other in a total concentration of 10.sup.9 CFU/dose.

28. The method according to claim 20, wherein said lactoferrin is bovine lactoferrin.

29. The method according to claim 20, wherein said lactoferrin, is in a total concentration of 50 mg/dose.

30. The method according to claim 24 wherein said composition is used in a daily dosage of 10.sup.9 CFU/dose.

31. The method according to claim 24 wherein said composition is in combination with lactoferrin in an amount of 50 mg/dose.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The characteristics and advantages of the present invention will be apparent from the detailed description reported below, from the Examples given for illustrative and non-limiting purposes, and from the annexed FIGS. 1 and 2, wherein:

[0029] FIGS. 1A, 1B and 1C: show graphs reporting the growth inhibition of GBS (10.sup.6 CFU) after different treatment with L. acidophilus, L. rhamnosus, the mixture of both lactobacilli, and the mixture in combination with lactoferrin (in the range of 0.1 to 10 mg/ml). Both lactobacilli strains were used at 10.sup.7 CFU as described in Example 7.

[0030] FIGS. 1A, 1B and 1C show the growth inhibition of GBS after 6, 12 and 24 hours, respectively.

[0031] FIG. 2: shows a graph reporting the growth of GBS in presence of lactoferrin (Lf).

DETAILED DESCRIPTION OF THE INVENTION

[0032] The present invention concerns a composition comprising a strain of Lactobacillus acidophilus, a strain of Lactobacillus rhamnosus, and lactoferrin for use in the preventive and/or curative treatment of Group-B Streptococcus (GBS) infections. The composition comprising a strain of Lactobacillus acidophilus, a strain of Lactobacillus rhamnosus, and lactoferrin is suitable for use in the preparation of a medicament for the preventive and/or curative treatment of Group-B Streptococcus (GBS) infections. Lactobacilli, such as Lactobacillus acidophilus and Lactobacillus rhamnosus, are microorganisms exerting several beneficial effects for human health when administered in adequate amount. For this reason, they are considered probiotics. Lactobacillus spp. represents the microorganism category which is predominant in the healthy vaginal ecosystem. Lactobacilli contribute to the vaginal homeostasis by balancing the different bacterial populations through the production of antimicrobial substances able to prevent the growth of pathogenic microorganisms. In addition, they also produce lactic acid, which maintains physiological low vaginal pH, thus contributing to a healthy vaginal environment. Alterations in the microbial composition of vaginal ecosystem are linked to several adverse health outcomes such as bacterial vaginosis (BV) and aerobic vaginitis (AV).

[0033] Lactoferrin (Lf) is a safe, natural component of most exocrine biological fluids, including tears, milk, saliva and vaginal secretions, and deserves attention as a possible therapeutic agent. Lf is a ˜80 kDa iron binding multifunctional glycoprotein constituting one of the major immunomodulatory components of the innate immune system. Other biological activities of Lf described in the scientific literature include antimicrobial activity against a wide range of pathogenic bacteria, fungi, protozoa and viruses, as well as anti inflammatory, and iron carrier. As other milk glycoconjugates, Lf functions as soluble receptor mimetic that inhibits pathogen binding to the mucosal cell surface. Despite Lf's broad spectrum of antimicrobial and immunomodulatory activities mentioned above, relatively little is known about Lf's capacity to modulate the growth of vaginal bacteria.

[0034] Without being bound to any theory, the mechanism of action responsible for the prevention of GBS infection in women is the direct effect due to the antimicrobial effect of the composition of the invention in the gut. In fact, the intestine represents the reservoir of many pathogen microbes causing diseases in humans, such as lower genito-urinary infections. Our composition is able to drastically reduce the GBS's proliferation and consequently its migration and colonization of the lower urogenital tract.

[0035] In our previous experimental studies we found that a combination of lactobacilli (i.e. L. acidophilus and L. rhamnosus) exerted antimicrobial effects by inhibiting the proliferation and growth of some specific pathogen and opportunistic bacteria. In particular, we showed that the probiotic combination induced a significant effect Staphylococcus aureus and Escherichia coli which are considering the most common cause of lower urinary tract infections. On the contrary, nothing is known about the effects of this complex against GBS.

[0036] Based on the above mentioned evidence, we investigated the ability of selected Lactobacilli, mainly Lactobacillus acidophilus and Lactobacillus rhamnosus, alone or in combination, and in the presence or absence of Lf, preferably bovine lactoferrin, on the growth and proliferation of GBS.

[0037] Surprisingly the inventors found that when combining together a strain of Lactobacillus acidophilus, a strain of Lactobacillus rhamnosus, and lactoferrin in a composition, the composition had a synergic effect on GBS.

[0038] The results shown in FIGS. 1 and 2 and described in Example 7 show that neither Lactobacillus acidophilus nor Lactobacillus rhamnosus, inhibited the GBS growth neither alone nor in combination after 6 hours of incubation. A slight inhibition can be seen for L. acidophilus after 12 hours, whereas no significative inhibitory effects are exerted by lactoferrin alone at any of the incubation times.

[0039] Nevertheless, when combined together, the inhibitory effect of the lactobacillus strains resulted stronger and appeared earlier and were significantly enhanced when in combination with Lf, highlighting a synergic effect.

[0040] In a preferred aspect, the invention provides the use of a composition comprising a strain of Lactobacillus acidophilus, a strain of Lactobacillus rhamnosus, and lactoferrin, wherein said strain of Lactobacillus acidophilus and said strain of Lactobacillus rhamnosus are each independently of each other in a total concentration in a range from 10.sup.7 to 10.sup.12 CFU/dose, preferably 10.sup.9 CFU/dose.

[0041] In a further aspect, in the composition of the invention the strain of Lactobacillus acidophilus is chosen from the group consisting of L. acidophilus deposited with deposit number SD5212 (American Type Culture Collection or ATCC) and deposit number LMGS-29159 (Belgian Coordinated Collections of Microorganisms or BCCM/LMG of the Ghent University in Belgium), and other lactic acid bacteria belonging to the same species such as, but not limited to, L. acidophilus NCFM, L. acidophilus La-5, L. acidophilus Lafti L-10, L. acidophilus W22 and Lactobacillus acidophilus BIFOLAC 5 strains and the strain of Lactobacillus rhamnosus is chosen from the group consisting of L. rhamnosus deposited with deposit number SD5675 (ATCC), and other lactic acid bacteria belonging to the same species such as, but not limited to, L. rhamnosus Lr-32, L. rhamnosus GG, L. rhamnosus GR-1 and L. rhamnosus SP-1 strains.

[0042] In a still further aspect, in the composition of the invention said lactoferrin, preferably bovine lactoferrin, is in a total concentration in a range from 5 mg/dose to 300 mg/dose, preferably 50 mg/dose.

[0043] According to another aspect, the described invention provides the use of a composition comprising a strain of Lactobacillus acidophilus, a strain of Lactobacillus rhamnosus, and lactoferrin, said composition further comprising at least one additional component selected from the group consisting of: minerals, vitamins, probiotics, prebiotics, proteins or any mixtures thereof.

[0044] Preferably said minerals are selected from the group consisting of calcium, phosphorus, copper, magnesium, potassium, iron, selenium, sodium, zinc, manganese, chloride and iodine, said vitamins are selected from the group consisting of vitamin A, vitamin B1 (thiamin), vitamin B2 (riboflavin), vitamin B3 (niacin) vitamin B6, vitamin B8 (biotin), vitamin B112, vitamin C, vitamin D, vitamin E and folic acid, said probiotics are Bifidobacterium or lactic acid bacteria selected from the group consisting of Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus bifidus, Lactobacillus reuteri, Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus plantarum, Lactobacillus jensenii, Lactobacillus delbrueckii, Lactobacillus fermentum, Lactobacillus iners, Lactobacillus helveticus, and said proteins are selected from whey and casein.

[0045] In a more preferred aspect, the invention relates to the use of a composition comprising Lactobacillus acidophilus in a range from 10.sup.7 to 10.sup.12 CFU/dose, Lactobacillus rhamnosus in a range from 10.sup.7 to 10.sup.12 CFU/dose, lactoferrin in a range from 5 mg/dose to 300 mg/dose and pharmaceutically acceptable excipients.

[0046] Such pharmaceutically acceptable excipients can be but are not limited to, hydroxypropylmethylcellulose, starch, magnesium stearate and silicon dioxide. Other excipients are known to the person of ordinary skill in the art that can prepare the compositions according to the Examples.

[0047] Under another aspect the composition comprising a strain of Lactobacillus acidophilus and a strain of Lactobacillus rhamnosus, can be used in a daily dosage in the range from 10.sup.7 to 10.sup.12 CFU/dose, preferably 10.sup.9 CFU/dose where the ratio between L. acidophilus and L. rhamnosus ranges between 20:1 and 2:1, preferably 4:1 and L. acidophilus is always higher than L. rhamnosus.

[0048] Lactoferrin, preferably bovine lactoferrin, can be used in a daily dosage in the range of 5 mg/dose to 300 mg/dose preferably 50 mg/dose.

[0049] Such dosage is used in our ongoing multicentric clinical trial with 410 pregnant women randomized into 2 groups according to the treatment (verum or placebo). They will take one capsule of formulation per day from 32° to 37° week of pregnancy. Each capsule contains 5×10.sup.9 CFU of Lactobacilli mixture (i.e. L. acidophilus and L. rhamnosus) and 50 mg of lactoferrin. In a preferred aspect, the composition for use according to the present invention is for oral administration, wherein said composition is in solid, liquid form, chosen from the group consisting of a tablet, a capsule, a powder, a granulate, a soluble stick, a liquid suspension.

[0050] The composition herein described is advantageously used in the prevention and/or the treatment of Group-B Streptococcus (GBS) infections occur during or after pregnancy. Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

EXAMPLES

[0051] Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention.

Example 1

Preparation of the Composition According to the Invention (Capsules)

[0052] The composition according to the invention was prepared by weighing each raw material which are L. acidophilus, L. rhamnosus, lactoferrin, magnesium salts of fatty acids and silicon dioxide in a workshop with controlled humidity in the range of 20-30%. After that, each ingredient is sieved and introduced into the mixing machine and mixed in presence of nitrogen in order to remove oxygen as much as possible. The mixture (powder) is loaded into a hopper of a capsule machine for the production of HPMC capsules under controlled environmental conditions (temperature and humidity). After filling, capsules are transported to the blistering machine for the primary packaging in alu/alu blister or suitable jars.

Composition

[0053] Lactoferrin 54.3 mg

[0054] L. acidophilus+L. rhamnosus: 5×10.sup.9 CFU

[0055] Hydroxypropyl methylcellulose 80 mg

[0056] Starch 53.6 mg

[0057] Magnesium salts of fatty acids 5 mg

[0058] Silicum dioxide 3.7 mg

Example 2

Preparation of the Composition According to the Invention (Coated Tablets 1q)

[0059] Lactoferrin 60 mg

[0060] L. acidophilus 20×10.sup.9

[0061] L. rhamnosus 5×10.sup.9

[0062] Calcium phosphates 300 mg

[0063] Microcrystalline cellulose 150 mg

[0064] Magnesium salts of fatty acids 10 mg

[0065] Silicum dioxide 5 mg

[0066] Coating agent 50 mg

[0067] A blend of Hydroxypropyl methylcellulose with different viscosity (200 mg) and Methylcellulose 125 mg

Example 3

Preparation of the Composition According to the Invention (Sachet 2 g)

[0068] Lactoferrin 100 mg

[0069] L. acidophilus 10×10.sup.9

[0070] L. rhamnosus 2×10.sup.9

[0071] Maltodextrin 1780 mg

[0072] Silicon dioxide 20 mg

Example 4

Preparation of the Composition According to the Invention (Oral Stick Pack—Ready to Use 1.5 q)

[0073] Lactoferrin 50 mg

[0074] L. acidophilus 15×10.sup.9

[0075] L. rhamnosus 5×10.sup.9

[0076] Fructo-oligosaccarides 1000 mg

[0077] Sorbitol 335 mg

[0078] Biossido di silicio 15 mg

Example 5

Preparation of the Composition According to the Invention (Suspension in Oil 10 ml)

[0079] Lactoferrin 30 mg

[0080] L. acidophilus 6×10.sup.9

[0081] L. rhamnosus 3×10.sup.9

[0082] Sunflower oil up to 10 ml

[0083] Mono-diglycerides of fatty acids 5 mg

[0084] Vitamin E 15 mg

Example 6

Preparation of the Composition According to the Invention (Liquid Suspension)

[0085] Vial 10 ml

[0086] Plug: Lactoferrin 25 mg; Lactobacillus mixture 15×10.sup.9 (L acidophilus; L rhamnosus) 75 mg, silicon dioxide 5 mg

[0087] Vial: water, fructose, potassium sorbate, sodium benzoate, flavor, citric acid.

Example 7

In Vitro Analysis of Activity of the Composition of the Invention: Liquid Co-Culture Assay.

[0088] The capability of L. acidophilus and L. rhamnosus, alone and in combination with lactoferrin, to interfere with the growth of the GBS was evaluated by a liquid co-culture assay.

[0089] Lactobacillus strains (for example L. acidophilus and L. rhamnosus) were stored in milk yeast extract (MYE) at −80° C.

[0090] Before the experiments, each strain was transferred from the frozen stock culture to MRS (De Man Rugosa Sharpe) broth incubated at 37° C. under non-agitated conditions. Streptococcus agalactiae was cultured in Brain Heart Infusion (BHI) broth.

[0091] The co-culture test was performed by incubating in Defined Medium Simulating Genital Tract Secretions (DMSGTS) (capable of sustaining the growth of both probiotics and pathogens) different concentrations of the probiotic strains (10.sup.7 and 10.sup.8 cfu/mL), alone or in combination, with different concentrations (10.sup.6 and 10.sup.7 cfu/mL) of the target pathogen. Probiotic strains were tested alone and in combination with lactoferrin at the following final concentrations: 0.1-1-5-10 mg/ml.

[0092] Controls were carried out by inoculating DMSGTS with the bacteria alone.

[0093] To check whether the pathogen was inhibited or killed, 0.05 mL of coculture suspensions were diluted and seeded on specific agar medium. After an incubation period at 37° C. for 6-24 h, bacterial growth was evaluated. No growth was interpreted as microbicidal activity (100% inhibition).

[0094] Statistical analysis was performed by Student's t-test for unpaired data. Data were expressed as the mean and SD and P values of <0.05 were considered significant.

Results

[0095] Results are shown in FIGS. 1 and 2.

[0096] Lactobacilli showed different effects on GBS growth when tested alone. In particular, L. acidophilus caused a slight growth inhibition after 12 hours of incubation with pathogen bacterium. On the contrary, L. rhamnosus did not reduced the bacterial growth after neither 6 nor 12 hours, and a slight inhibition was shown after 24 hours. Lactoferrin did not affect GBS growth significantly (FIG. 2). Surprisingly, when Lf was added to the lactobacilli mixture, the complex exerted more marked inhibition, which was significant already after 6 hours of incubation.

[0097] Such an effect was independent from the initial probiotic count (similar results were obtained with either 10.sup.7 and 10.sup.8 cfu/ml).

Conclusions

[0098] The results shown in FIGS. 1 and 2 clearly demonstrate the efficacy of the composition according to the invention (formulation of 2 lactobacilli, preferably L. acidophilus and L. rhamnosus, in combination with lactoferrin) for preventing and reducing the growth of GBS pathogen bacterium.

[0099] Neither Lactobacillus acidophilus nor Lactobacillus rhamnosus, inhibited the GBS growth alone nor in combination after 6 hours of incubation. A slight inhibition can be seen for L. acidophilus after 12 hours, whereas no significative inhibitory effects are exerted by lactoferrin alone at any of the incubation times.

[0100] Surprisingly, the effect of the lactobacilli combination was enhanced by lactoferrin, highlighting a synergic effect.

[0101] Considering that GBS is one of the most common and pathogenic bacteria causing severe conditions in both pregnant women and mainly newborns, the composition of the present invention may represent a potential alternative approach for the prevention and treatment of GBS infection during pregnancy.

Example 8

Ongoing Clinical Study

[0102] A multicentric, double-blind, randomized, placebo-controlled clinical trial is ongoing, in order to assess the efficacy of the composition according to the invention in the prevention of GBS infection in pregnant women.

[0103] The study will start only after a written approval obtained from the Independent Ethics Committee at respective sites and communication to the Italian Ministry of Health. The trial will be conducted in accordance with the Declaration of Helsinki, (Fortaleza, Brazil, and October, 2013) and Good clinical practice.

[0104] 410 pregnant women (from 32.sup.nd to 37.sup.th week of pregnancy) will be enrolled and randomly divided into 2 groups (verum or placebo) according to the treatment. The recruitment will be performed at the University of Modena (coordinator), University of Milan, and University of Trieste.

[0105] All women will be recruited if they are adult (>18 years) and pregnant at low obstetric risk with rectal-vaginal swab positive to GBS; pregnancy within the 33.sup.rd week and prevision of vaginal delivery.

[0106] Exclusion criteria include pregnant women with urine positive to GBS; women with previous infants with early sepsis; use of antibiotics one month before enrolment; incapacity to understand the study and sign the informed consent.

[0107] The primary endpoint is the rate of pregnant women with GBS colonization (carriers) at the pre-partum screening (35-37.sup.th week).

[0108] The secondary endpoints include the rate of women treated with antibiotics during the delivery; the rate of women with early rupture of membranes; Apgar score >8 at 5{circumflex over ( )} minute; the rate of newborns receiving antibiotics during the first 48 hours of life; the rate of newborns with early GBS sepsis; and safety and tolerability of investigational product/placebo.

[0109] The treatment consists in oral administration of 1 capsule per day of either investigation product (both lactobacilli in combination with lactoferrin) or placebo from 32.sup.nd to 37.sup.th week of pregnancy. Each capsule of verum contains 5×10.sup.9 CFU of lactobacilli (i.e. L. acidophilus and L. rhamnosus) and 50 mg of lactoferrin.

[0110] During the study, rectal and vaginal swabs will be collected from each woman for the GBS analysis according to the current clinical practice. A follow-up visit will be performed after the delivery in order to assess the GBS colonization in newborns.

[0111] In addition, on a sub-group of 20 women per each center, an additional vaginal swab will be collected during the assessment of 35-37.sup.th week. It will be used for demonstrating the vaginal colonization of 2 lactobacilli strains included in investigational product and for assessing the vaginal microbiota and cytokines profiles by molecular approach (extraction and isolation of bacterial DNA from the swabs; 16S rDNA sequence; RT-PCR analysis).

[0112] Safety will be assessed by recording adverse events.

[0113] All data will be showed as absolute or relative frequencies. The primary and secondary endpoints will be compared between the 2 study arms by using a 2 tailed Z test.

[0114] From the above description and the above-noted examples, the advantage attained by the composition described and obtained according to the present invention are apparent.