STRAIN OF SACCHAROMYCES CEREVISIAE VAR. BOULARDII FOR TREATING INFECTIOUS DISEASES OF THE ORAL CAVITY
20220062359 · 2022-03-03
Assignee
Inventors
Cpc classification
A61K45/06
HUMAN NECESSITIES
A61K9/006
HUMAN NECESSITIES
International classification
A61K45/06
HUMAN NECESSITIES
A61K9/00
HUMAN NECESSITIES
Abstract
A yeast strain of Saccharomyces cerevisiae var. boulardii for the treatment and/or the prevention of infectious diseases of the oral cavity, such as dental caries and periodontal diseases. This strain can be used either alone or in combination with the inactive dry form of a yeast strain of Saccharomyces cerevisiae.
Claims
1-17. (canceled)
18. A method for preventing or treating an infectious disease of the oral cavity in a subject, the method comprising a step of administering an effective amount of a Saccharomyces cerevisiae var. boulardii yeast strain, or its combination with the inactivated dry form of a Saccharomyces cerevisiae yeast strain.
19. The method according to claim 18, wherein the Saccharomyces cerevisiae var. boulardii yeast is in live dry form.
20. The method according to claim 18, wherein the Saccharomyces cerevisiae var. boulardii yeast strain is the strain deposited, on Aug. 21, 2007, at the CNCM under number I-3799.
21. The method according to claim 18, wherein the Saccharomyces cerevisiae yeast strain is the strain deposited, on Oct. 17, 2007, at the CNCM under number I-3856.
22. The method according to claim 20, wherein the Saccharomyces cerevisiae yeast strain is the strain deposited, on Oct. 17, 2007, at the CNCM under number I-3856.
23. The method according to claim 18, wherein the Saccharomyces cerevisiae var. boulardii yeast strain, or its combination with the inactivated dry form of a Saccharomyces cerevisiae yeast strain, is comprised in a dietary supplement.
24. The method according to claim 23, wherein the dietary supplement is in the form of a lozenge to be sucked, a candy, a chewing gum, an orodispersible powder or a powder to be diluted in water in the form of a stick or sachet, a tablet to be sucked or chewed, a gum, a capsule, a tablet, drops, a vial with a measuring cap.
25. The method according to claim 18, wherein the Saccharomyces cerevisiae var. boulardii yeast strain, or its combination with the inactivated dry form of a Saccharomyces cerevisiae yeast strain, is comprised in a parapharmaceutical or cosmetic composition.
26. The method according to claim 25, wherein the parapharmaceutical or cosmetic composition is in the form of a toothpaste, a mouthwash, an oral spray, an oral cream or gel, an orodispersible sheet, a powder to be sprinkled directly into the oral cavity, an orodispersible powder or a powder to be diluted in water in the form of a stick or sachet, a vial with a measuring cap.
27. The method according to claim 18, wherein the Saccharomyces cerevisiae var. boulardii yeast strain, or its combination with the inactivated dry form of a Saccharomyces cerevisiae yeast strain, is comprised in a pharmaceutical composition, which further comprises at least one physiologically acceptable excipient.
28. The method according to claim 27, wherein the pharmaceutical composition is intended for topical administration or for oral administration.
29. The method according to claim 27, wherein the pharmaceutical composition further comprises at least one additional pharmaceutical active principle having soothing, anti-irritant, analgesic, antalgic, anti-inflammatory, healing, antibiotic, antipyretic, or antifungal activity.
30. The method according to claim 18, wherein the Saccharomyces cerevisiae var. boulardii yeast strain, or its combination with the inactivated dry form of a Saccharomyces cerevisiae yeast strain, is comprised in a dental medical device
31. The method according to claim 30, wherein the Saccharomyces cerevisiae var. boulardii yeast strain, or its combination with the inactivated dry form of a Saccharomyces cerevisiae yeast strain, is comprised in a dental medical device
32. The method according to claim 18, wherein the infectious disease is dental caries, gingivitis or periodontitis.
33. The method according to claim 18, wherein the infectious disease is induced by periodontopathogenic bacteria and/or cariopathogenic bacteria.
34. The method according to claim 33, wherein the periodontopathogenic bacteria are selected from Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, Porphyromonas gingivalis, Prevotella intermedia, and/or the cariopathogenic bacterium Streptococcus mutans.
35. The method according to claim 18, wherein the infectious disease is a side effect of a medical treatment, or the infectious disease is present or likely to develop or recur in a patient suffering from immune vulnerability, or the subject is a pregnant woman, an elderly person, a child or a patient with a hyperinflammatory phenotype.
Description
FIGURE LEGENDS
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Effects of Different Probiotics on Periodontopathogenic Bacteria and Cariogenic Bacteria
A. Probiotics Tested and Periodontopathogenic or Cariogenic Bacteria
[0081] Yeast Products. In the studies described below, two live dry yeasts and one inactivated dry yeast were evaluated.
[0082] The two live yeasts are: [0083] Saccharomyces cerevisiae strain number CNCM I-3856 (deposited by the Applicant at the CNCM on Oct. 17, 2007), and [0084] Saccharomyces cerevisiae var. boulardii strain number CNCM I-3799 (already described above).
[0085] The inactivated dry yeast is: [0086] The inactivated dry form of Saccharomyces cerevisiae strain number I-3856 (already described above).
[0087] Composition of a 14-species complex biofilm:
[0088] Periodontopathogenic Bacteria and Cariogenic Bacteria. The periodontopathogenic bacteria used include: [0089] Prevotella intermedia (formerly known as Bacteroides intermedius) which is an obligate anaerobic, Gram-negative pathogenic bacterium involved in periodontal infections, including gingivitis and periodontitis, and often found in acute necrotizing ulcerative gingivitis; [0090] Porphyromonas gingivalis, which is an anerobic, non-motile, Gram-negative, rod-shaped pathogenic bacterium found in the oral cavity, where it is involved in some forms of periodontal disease; [0091] Fusobacterium nucleatum, which is an anerobic, invasive, adherent and inflammatory bacterium, foreign to the human oral cavity, and which plays a role in periodontal disease by being a key component of periodontal plaque due to its abundance and ability to associate with other species in the oral cavity; and [0092] Aggregatibacter actinomycetemcomitans, which is a small, slow-growing, facultative aero-anaerobic, Gram-negative bacterium belonging to the physiological oral flora of humans and playing a role in periodontal disease.
[0093] The cariogenic bacteria used include: [0094] Streptococcus mutans, which is a Gram-positive cocci bacterium, part of the commensal flora of the oral cavity where it is responsible for dental caries in combination with dietary sugar, which it transforms into lactic acid; and [0095] Streptococcus sobrinus, which is a Gram-negative, non-motile, anaerobic bacterium, present in large quantities in the dental plaque of patients with caries.
[0096] Commensal Bacteria of the Oral cavity. The commensal bacteria of the oral cavity used in the present study include: Streptococcus sanguinis; Streptococcus gordonii; Streptococcus salivarius; Streptococcus mitis; Streptococcus oralis; Actinomyces viscosus; Actinomyces naeslundii; and Veillonella parvula.
B. Antibacterial Activity of Yeasts Against 6 Pathogenic Species
[0097] Protocol. The halo technique was used to detect and quantify the inhibition rate of commensal and pathogenic bacteria on agar plates. This technique is a competitive test, which consists in inoculating two spots, close to each other and each containing a different bacterial species, on an agar plate. Then, the ability of one to inhibit the growth of the other can be evaluated.
[0098] The agar plates were seeded with the culture of one of the 2 yeasts (live) adjusted to an optical density at 600 nm (OD.sub.600) of 0.5, which corresponds to a concentration of approximately 10.sup.8 CFU/mL—the BHI-2 agar plates did not contain hemin, menadione or blood. Each spot contained 7 μL of the culture of one of the 2 live yeasts of concentration 10.sup.8 CFU/mL.
[0099] After 24 hours of aerobic or anaerobic incubation, the agar plates were again inoculated for 24 h with 7 μL of a culture of a pathogenic species (Fusobacterium nucleatum (Fn), Prevotella intermedia (Pi), Aggregatibacter actinomycetemcomitans (Aa), and Porphyromonas gingivalis (Pg)), in close proximity to the spot containing the yeast species.
[0100] In total, the agar plates were incubated for 48 hours under anaerobic or aerobic conditions. After the 48-hour incubation, the inhibition surface was inspected and calibrated (with a ruler) (Table 1), and a standardized photograph (distance between the agar plate and the camera) was taken of each of the agar plates (
[0101] The pathogens tested with the 2 live yeasts were the following: Fusobacterium nucleatum (Fn), Prevotella intermedia (Pi), Aggregatibacter actinomycetemcomitans (Aa), and Porphyromonas gingivalis (Pg),
[0102] Results. The results obtained are presented in
TABLE-US-00001 TABLE 1 Rate of inhibition (mean ± standard deviation, N = 3) induced by the yeast species Saccharomyces cerevisiae var. boulardii I-3799 on the growth of certain oral pathogenic bacteria. inhibition distance Bacterial Competition mean ± SD* (mm) S. boulardii (24 h anaerobic) + P. intermedia 6.19 ± 0.09 (24 h anaerobic) S. boulardii (24 h anaerobic) + P. gingivalis 3.11 ± 0.14 (24 h anaerobic) S. boulardii (24 h anaerobic) + 4.48 ± 0.14 A. actinomycetemcomitans (24 h aerobic) S. boulardii (24 h aerobic) + 0.52 ± 0.07 A. actinomycetemcomitans (24 h aerobic) *SD = standard deviation
[0103] Table 1 provides the measured inhibition distances for cases where the growth of certain oral pathogens was significantly inhibited.
[0104] The results obtained show that the Saccharomyces cerevisiae var. boulardii strain induces a significant inhibition of the growth of the pathogenic species Prevotella intermedia, Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis under anaerobic conditions.
[0105] In addition, the growth of Aggregatibacter actinomycetemcomitans was also inhibited by the Saccharomyces cerevisiae var. boulardii strain under aerobic conditions.
[0106] Table 1. shows that the greatest order of magnitude of inhibition was observed on the growth of Prevotella intermedia under anaerobic conditions compared with other bacteria and pathogens.
[0107] It is also observed in
[0108] Conclusion. Only the Saccharomyces cerevisiae var. boulardii strain inhibited the growth of oral pathogens on agar plates.
C. Evaluation of the Effect of Yeast Products on the Composition of a Complex Biofilm Comprising 14 Species.
[0109] 1) Effects of Live Yeast Studied
[0110] Bioreactor Protocol. A multispecies community was established in a BIOSTAT B TWIN® reactor (Sartorius, Germany) A medium containing 750 mL of BHI-2 (Brain Heart Infusion (BHI) supplemented with 2.5 g/L mucin, 1.0 g/L yeast extract, 0.1 g/L cysteine, 2.0 g/L sodium bicarbonate and 0.25% (v/v) glutamic acid) was added to the bioreactor along with 5.0 mg/mL hemin, 1.0 mg/mL menadione and 200 μL/L Antifoam Y-30 (Sigma, St. Louis, USA). The mixture was pre-reduced for 24 hours at 37° C. by bubbling 100% nitrogen (N.sub.2) and 5% carbon dioxide (CO.sub.2) into the mixture while stirring continuously at 300 rpm, at a pH set at 6.7±0.1. After 24 hours, cultures of Streptococcus mutans (E), Streptococcus sobrinus (F), Prevotella intermedia (D), Porphyromonas gingivalis (C), Fusobacterium nucleatum (B), and Aggregatibacter actinomycetemcomitans (A) were added at an optical density of 1.4 and added to the bioreactor mixture. The medium was not replaced for the first 48 hours and then was replaced at a rate of 200 mL/24 hours.
[0111] Culture Protocol. Cultures of Saccharomyces cerevisiae strain number I-3856 (I-3856) and Saccharomyces cerevisiae var. boulardii strain number I-3799 (boulardii) (performed overnight) were adjusted to an optical density at 600 nm (OD.sub.600) of 0.5 (1.Math.10.sup.8 CFU/mL) in BHI-2 (see composition above). Aliquots of 1800 μL of these adjusted cultures were inoculated onto 24-well plates containing hydroxyapatite discs at the bottom of each well. 200 μL of the 14-species biofilm mixture from the bioreactor was added to each well. The controls contained 200 μL of the 14-species samples and 1800 μL of BHI-2. The 24-well plates were incubated under micro-aerobic conditions (6% oxygen) for 24 hours. Then, the supernatants were removed and the biofilms attached to the hydroxyapatite discs were washed with phosphate buffered saline (PBS). The biofilms were detached with 1500 μL of 0.05% Trypsin-EDTA for 45 minutes at 37° C. and 230 rpm, transferred to Eppendorf tubes, and centrifuged (6010×g, for 5 minutes). After removing the trypsin, the biofilm pellet was re-suspended in 500 μL of PBS, and DNA extraction was performed according to Qiagen instructions and analyzed by qPCR.
[0112] Results Obtained on Live Yeast. The results obtained are presented in
[0113] It is observed that the Saccharomyces cerevisiae var. boulardii strain is able to significantly inhibit the following periodontopathogens: Aggregatibacter actinomycetemcomitans (A), Fusobacterium nucleatum (B), Porphyromonas gingivalis (C) and Prevotella intermedia (D), as well as the cariopathogen Streptococcus mutans (E).
[0114] Conclusion. The biofilm experiments confirmed, in multispecies communities, the inhibitory activity of the Saccharomyces cerevisiae var. boulardii strain against oral pathogens, which had been demonstrated above in the agar plate experiments.
[0115] 2) Effects of Strain Saccharomyces cerevisiae var. boulardii I-3799 (Boulardii Yeast), Inactivated Dry Form of Strain Saccharomyces cerevisiae I-3856 (IY Yeast), and their Combination
[0116] The yeast products (boulardii and IY) were each dissolved in 100 mL of PBS at 37° C. and vortexed for 3 minutes. 10 mL of these solutions was centrifuged at 6010×g for 10 minutes. The supernatants were removed and the “boulardii” yeast pellet was suspended in BHI-2 by adjusting the concentration to an average of 5.Math.10.sup.8 CFU/mL. The “IY” yeast pellet was suspended in a solution containing a sample of the bioreactor mixture diluted 1:5. [0117] The following were placed in a first well: 1 mL of the solution of the “boulardii” yeast suspended in BHI-2 and 1 mL of the bioreactor mixture diluted 1:5 without “IY” (“boulardii” alone condition). [0118] The following were placed in a second well: 1 mL of the yeast solution “IY” and of the bioreactor mixture diluted 1:5 and 1 mL of BHI-2 without “boulardii” (IY alone condition). [0119] The following were placed in a third well: 1 mL of the “boulardii” yeast solution suspended in BHI-2 and 1 mL of the “IY” yeast solution and of the bioreactor mixture diluted ⅕ (boulardii+IY combination condition). [0120] The following were placed in a fourth well: 1 mL of the bioreactor mixture diluted 1:5 and 1 mL of BHI-2. This well constitutes a control well.
[0121] The 24-well plates were incubated under microaerobic conditions (6% oxygen) for 24 hours. Then, the supernatants were removed and the biofilms attached to the hydroxyapatite discs were washed with PBS. The biofilms were detached with 1500 μL of 0.05% Trypsin-EDTA for 45 minutes at 37° C. and 230 rpm, transferred to Eppendorf tubes and centrifuged (6010×g, for 5 minutes). After removal of the trypsin, the biofilm pellet was re-suspended in 500 μL of PBS, and DNA extraction was performed according to Qiagen instructions and analyzed by qPCR.
[0122] Results. The results obtained are presented in
TABLE-US-00002 TABLE 2 Difference between control values and pathogenic species values of the biofilm (hereafter δ) in the presence of Saccharomyces cerevisiae var. boulardii strain I-3799 (Boulardii), the inactivated dry form of Saccharomyces cerevisiae strain I-3856 (IY), and their combination. Pathogenic δ δ IY + Boulardii species δ IY Boulardii Sum δ IY + δ Boulardii combination Aa 0.46 0.38 0.84 1.1 Fn 0.65 0.59 1.24 1.47 Pg 0.32 0.2 0.52 0.77 Pi 0.3 0.6 0.9 1.38 Sm 0.51 0.43 0.94 1.4 Note: The values presented in Table 2 are not absolute values. They are the difference between the control values and the pathogen values.
[0123] These results show that each of the two strains individually (Saccharomyces cerevisiae var. boulardii (boulardii) and inactivated Saccharomyces cerevisiae (IY) and their combination are able to significantly inhibit the growth of two periodontal pathogens Aggregatibacter actinomycetemcomitans or Aa (A) and Fusobacterium nucleatum or Fn (B) and the cariogenic bacterium Streptococcus mutans or Sm (E). Furthermore, Table 2 shows that the δ of the IY+boulardii combination is greater than the sum of the IY δ and boulardii δ taken individually.
[0124] According to
[0125] According to
[0126] Conclusion. Thus, it has been demonstrated that the combination of the Saccharomyces cerevisiae var. boulardii strain and the inactivated dry Saccharomyces cerevisiae strain was shown to induce growth inhibition of most pathogens in the multispecies model used, in contrast to the Saccharomyces cerevisiae var. boulardii strain and the inactivated Saccharomyces cerevisiae yeast taken individually which have a lesser spectrum of inhibition of oral pathogen growth.
[0127] Anti-Inflammatory Effects of Saccharomyces cerevisiae var. boulardii I-3799
[0128] The aim of the study presented below is to evaluate the potential anti-inflammatory effects of the yeast Saccharomyces cerevisiae var. boulardii CNCM I-3799 on the inflammatory parameters of primary human monocytes and gingival fibroblasts.
A. Evaluation of Anti-Inflammatory Potential in Primary Human Monocytes Stimulation and Determination of Inflammatory Parameters in Primary Human
[0129] Monocytes. Primary human monocytes were isolated and enriched from buffy coats of healthy human blood donors. The cells were seeded in 24-well plates (roughly 500000 cells/mL in 1 mL) for ELISA experiments. The cells were incubated with LPS (10 ng/mL) for 24 hours. Yeast samples (5 doses ranging from 4.3.sup.E6 CFU to 4.3.sup.E8 CFU/mL, i.e.: 0.1 mg/mL; 1 mg/mL; 2.5 mg/mL; 5 mg/mL; 10 mg/mL) were added 30 minutes prior to LPS treatment directly into the cell culture. Dexamethasone was used as a positive control (2 doses, 1 μM and 10 μM). After 24 hours, the supernatants were removed, centrifuged, and the concentrations of MCP-1, IL-8, IL-6, IL-10, isoprostane, and PGE2 were assessed. Concentrations in EIAs (PGE2 and isoprostane, from Cayman) or ELISAs (MCP-1, IL-6, and IL-8, from eBioscience) were assessed according to the manufacturer's protocols. Each dose was studied 6 times using buffy coats from 3 different donors (n=2 per buffy coat, n=6 total).
[0130] Statistical Analysis. One-way ANOVA with Dunnett's multiple comparison tests at a significance level of 0.5.
[0131] Results. The results obtained are presented in
B. Evaluation of the Anti-Inflammatory Potential on Primary Human Gingival Fibroblasts
[0132] Measurement of IL-8, IL-6, Isoprostane and PGE2 in Primary Human Gingival Fibroblasts. Cultures of primary human gingival fibroblasts supplied by Provitro (Berlin, Germany) were maintained according to the supplier's protocol. Prior to stimulation, cells were seeded in 24-well plates for ELISA experiments. The cells were incubated without (unstimulated control) or with IL-113 (10 U/mL) for 24 hours. Yeast samples were added 30 minutes before IL-1β treatment in 5 doses (0.1 mg/mL; 1 mg/mL; 2.5 mg/mL; 5 mg/mL; 10 mg/mL) (n=3). Dexamethasone was used as a positive control (1 dose, 1 μM). After 24 hours, the supernatants were collected, centrifuged, and IL-6, IL-8, isoprostane, and PGE2 concentrations were assessed by EIA (PGE2 and isoprostane, from Cayman) or ELISA (IL-6 and IL-8, from eBioscience) following the manufacturer's protocol. Each dose was studied at least 3 times.
[0133] Results. The results obtained are presented in