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    • 专利摘要:
    COMPOSITION WITH PROBIOTIC BACTERIA FOR USE IN TREATING IMMUNOLOGICAL DISORDERS The present invention relates to a composition comprising bacteria for the treatment of pathologies associated with changes in the immune system. More specifically, the present invention relates to the use of selected probiotic bacteria to prepare a composition for treating allergies, such as atopic dermatitis.
    公开号:BR112012022755B1
    申请号:R112012022755-6
    申请日:2011-03-07
    公开日:2021-03-02
    发明作者:Giovanni Mogna；Gian Paolo Strozzi；Luca Mogna；Lorenzo Drago
    申请人:Probiotical S.P.A.；
    IPC主号:
    专利说明:

    DESCRIPTION
    The present invention relates to a composition comprising bacteria for the treatment of pathologies associated with changes in the immune system. More specifically, the present invention relates to the use of selected probiotic bacteria to prepare a composition for treating allergies, such as atopic dermatitis.
    It is well known that atopic dermatitis (henceforth AD, for the sake of brevity) is a skin disorder of chronic recurrence, which begins in the neonatal period or childhood and can persist into adulthood. Thus, the phases of AD are divided into neonatal, prepubertal and adult. AD is an inflammatory skin disease that, like asthma and allergic rhinitis, is related to the local infiltration of T lymphocytes that produce interleukin 4 (IL-4) and interleukin 5 (IL-5).
    IL-4, as is well known, regulates the development of the auxiliary phenotype T 2 (Th2), with the consequent overproduction of immunoglobulin (lg) and eosinophilia. Elevated serum IgE levels and positive skin tests for food and inhalant allergens occur in 80 to 90% of patients with atopic dermatitis.
    AD is associated with both genetic and immunological factors, although a large number of external factors modify its expression. In 60% of AD cases, there is a family history of atopy; in other words, if a parent has atopic diathesis, there is a 60% risk that the children are atopic.
    Atopy is the genetic predisposition to the development of anaphylactic reactions located after contact, ingestion, inoculation or inhalation of allergens.
    The immunological factors taken into account in the etiology of AD are: food allergy, allergy induced by contact with allergens and irritating agents, allergy induced by aeroallergens and immunoregulatory abnormalities.
    Among the various treatments aimed at curing and / or preventing allergies or inflammations in the skin, especially in the treatment of atopic dermatitis, there are many drug-based treatments.
    Drug-based treatments have several contraindications that limit their use. In addition, such drugs do not have good levels of tolerance in some categories of patients.
    Even so, at the moment, there is still no treatment with absolute or definitive effectiveness. It is only possible to take certain precautions against the principle of allergies or, as necessary, to use medications to reduce uncomfortable symptoms, such as itching and flushing of the skin.
    With this in mind, there remains a need for treatment that does not present the limitations of drug-based treatments and that can be administered to all categories of patients.
    In particular, there remains a need for a treatment that serves to combat the principle of allergies and the development of inflammations in the skin and / or to reduce uncomfortable symptoms, such as redness of the skin tissue, itching and eczema.
    The applicant provides an answer to the aforementioned needs since he was successful, after intense research activity, in the selection of specific probiotic bacterial strains.
    The material of the present invention is a composition comprising at least one culture of probiotic bacteria according to the attached independent claim.
    Furthermore, the subject of the present invention relates to the use of at least one culture of probiotic bacteria according to the attached independent claim. We define other preferred embodiments of the present invention in the detailed description below by way of example and, therefore, without the intention of limiting its scope.
    The applicant stipulated for himself the objective of selecting specific bacterial strains in order to assess their effect on the clinical course and on the quality of life of patients with moderate or severe AD, as well as in order to determine their influence on clinical and immunological parameters.
    In addition, the applicant has set itself the objective of selecting bacterial strains with probiotic activity that are tolerated by patients and capable of colonizing the intestinal tract.
    The diagnosis of AD was assessed as moderate or severe by adopting a standardized severity index for Atopic Dermatitis, hereinafter referred to as AD (SCORAD index - SCORing Atopic Dermatitis', that is, “graduating atopic dermatitis”). This index is known to those skilled in the art.
    The bacterial strains of the present invention have been found to have a high immunomodulatory effect and are therefore useful in the treatment of pathologies associated with changes in the immune system.
    After intense research activity, the applicant selected, from among the several strains tested, the following probiotic bacterial strains: 1) Lactobacillus salivarius (LS01) DSM 22775, deposited on July 23, 2009 by the company Probiotical SpA from Novara, Italy; 2) Bifidobacterium breve (BR03) DSM 16604, deposited on July 20, 2004 by the company Probiotical SpA from Novara, Italy; 3) Lactobacillus pentosus (LPS01) DSM 21980, deposited on November 14, 2008 by the company Probiotical SpA from Novara, Italy; 4) Streptococcus thermophilus (FP4) DSM 18616, deposited on September 13, 2006 by the company Mofin Sri of Novara, Italy; 5) Lactobacillus casei ssp. rhamnosus (LR04) DSM 16605, deposited on 20 July 2004 by the company Probiotical SpA from Novara, Italy; and 6) Lactobacillus acidophilus (LA02) DSM 21717, deposited on August 6, 2008 by the company Probiotical SpA from Novara, Italy.
    The crops listed above were all deposited with the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), in Germany, in accordance with the Budapest Treaty.
    In the context of the present invention, the cultures of probiotic bacteria listed above can be in the form of live bacteria, dead bacteria or cell components, cell extracts or lysates thereof.
    In a preferred embodiment, the composition comprises at least one bacterial culture for use as an immunomodulatory composition capable of modulating the immune system.
    The expression “immunomodulatory composition” means that it is able to modulate the immune system in the sense that it is able to stimulate / provoke various responses from it, thus making it more reactive, for example, intervening in the production of specific cytokines.
    For greater advantage, said immunomodulatory composition induces the immune system to produce type 1 cytokines.
    For greater advantage, the bacterial strains and the corresponding bacterial cultures were shown to be able to improve clinical and immunological parameters. In particular, it was observed that they are able to: 1) significantly improve the SCORAD and IQVD indices; 2) decrease microbial translocation (plasma LPS levels) and activate CD8 + T lymphocytes; 3) increase the percentage of both total regulatory T lymphocytes (Treg) and those that express the TLR2 and TLR4 markers; 4) improve the Th1 / Th2 and Th17 / Treg ratios.
    Therefore, the composition of the present invention (immunomodulatory composition) is capable of preventing, decreasing and / or curing pathologies associated with changes in the immune system, such as allergies, atopy, allergic rhinitis, food hypersensitivity, atopic dermatitis, eczema, asthma and immunodeficiencies.
    According to one embodiment, the present invention relates to a composition comprising or, alternatively, consisting of at least one culture of probiotic bacteria selected from the group comprising or, alternatively, consisting of cultures of Lactobacillus salivaríus (LS01 ) DSM 22775, Bifidobacterium breve (BR03) DSM 16604 and Lactobacillus pentosus (LPS01) DSM 21980.
    In a preferred embodiment, the composition comprises or, alternatively, consists of cultures of Lactobacillus salivaríus (LS01) DSM 22775 and Bifidobacterium breve (BR03).
    In a preferred embodiment, the composition comprises or, alternatively, consists of said culture of Lactobacillus salivaríus (LS01) DSM 22775 and said culture of Bifidobacterium breve (BR03) DSM 16604 together with at least one more culture selected from the group comprising or, alternatively, consists of: Lactobacillus pentosus (LPS01) DSM 21980, Streptococcus thermophilus (FP4) DSM 18616, Lactobacillus casei ssp. rhamnosus (LR04) DSM 16605 and Lactobacillus acidophilus (LA02) DSM 21717.
    In a preferred embodiment, the composition comprises or, alternatively, consists of cultures of Lactobacillus salivaríus (LS01) DSM 22775, Bifidobacterium breve (BR03) DSM 16604 and Lactobacillus pentosus (LPS01) DSM 21980.
    According to another preferred embodiment, the present invention relates to the use of at least one culture of probiotic bacteria selected from the group comprising or, alternatively, consisting of cultures of Lactobacillus salivaríus (LS01) DSM 22775, Bifidobacterium breve (BR03 ) DSM 16604 and Lactobacillus pentosus (LPS01) DSM 21980 for the preparation of a composition capable of preventing, decreasing and / or curing pathologies associated with changes in the immune system, such as allergies, atopy, allergic rhinitis, food hypersensitivity, atopic dermatitis, eczema , asthma and immunodeficiencies.
    According to another preferred embodiment, the present invention relates to the use of a composition comprising or, alternatively, consisting of said cultures of Lactobacillus salivarius (LS01) DSM 22775 and Bifidobacterium breve (BR03) DSM 16604.
    Preferably, the present invention relates to the use of said culture of Lactobacillus salivarius (LS01) DSM 22775 and said culture of Bifidobacterium breve (BR03) DSM 16604 together with at least one more culture selected from the group comprising or, as alternative, consists of: Lactobacillus pentosus (LPS01) DSM 21980, Streptococcus thermophilus (FP4) DSM 18616, Lactobacillus casei ssp. rhamnosus (LR04) DSM 16605 and Lactobacillus acidophilus (LA02) DSM 21717, for the preparation of a composition for the preventive and / or curative treatment of atopic dermatitis.
    According to another preferred embodiment, the present invention relates to the use of a composition comprising or, alternatively, consisting of said cultures of Lactobacillus salivarius (LS01) DSM 22775, Bifidobacterium breve (BR03) DSM 16604 and Lactobacillus pentosus (LPS01 ) DSM 21980.
    For greater advantage, the composition comprises or, alternatively, consists of Lactobacillus salivarius (LS01) DSM 22775 and Bifidobacterium breve (BR03) DSM 16604 at a weight ratio between 1: 3 and 3: 1; preferably 1: 1.
    Alternatively, the composition comprises or, alternatively, consists of Lactobacillus salivarius (LS01) DSM 22775, Bifidobacterium breve (BR03) DSM 16604 and Lactobacillus pentosus (LPS01) DSM 21980, preferably at a weight ratio of 1: 1 :1.
    The bacterial cultures of the present invention can be considered as "active ingredients" that can be mixed, in appropriate proportions, to physiologically and / or pharmaceutically acceptable excipients.
    In order to improve its activity, the active ingredients can be administered systemically, for greater advantage, orally, in the form of solid state compositions prepared according to techniques known to those skilled in the art. In a preferred embodiment, the compositions are in the form of lyophilizates, powders, granules, tablets, soft gel capsules or suspensions.
    The compositions of the present invention can be pharmaceutical, dietetic, nutritional or nutraceutical. The compositions of the present invention may further comprise dietary fibers with prebiotic activity, such as fructo-oligosaccharides (FOS), inulin and partially hydrolyzed guar gum (GGPH).
    The pharmaceutical, dietary, nutritional or nutraceutical compositions of the present invention comprise at least one culture of probiotic bacteria as indicated above. The final concentration of the composition is between 1x107 and 1x1011 CFU / g of composition, preferably between 1x108 and 1x101 ° CFU / g of composition.
    The compositions of the present invention, which comprise selected probiotic bacteria, are useful as immunomodulatory compositions capable of modulating the immune system.
    Said compositions are capable of curing and / or preventing pathologies associated with changes in the functions of the following cytokines: Th1 (IFN-y and IL-12) and Th2 (IL-4, IL-5 and IL-10).
    In addition, said compositions are capable of inducing the immune system to produce type 1 cytokines.
    Therefore, the compositions of the present invention have valid application in the preventive and / or curative treatment of pathologies associated with alterations in the immune system, in particular, in the preventive and / or curative treatment of allergies, atopy, allergic rhinitis, food hypersensitivity, atopic dermatitis, eczema, asthma and immunodeficiencies.
    For greater advantage, the compositions of the present invention have valid application in the preventive and / or curative treatment of atopic dermatitis.
    For greater advantage, the administration, in patients suffering from atopic dermatitis DA, of a probiotic composition comprising the culture of Lactobacillus salivaríus (LS01) DSM 22775 and the culture of Bifidobacterium breve (BR03) DSM 16604 in a concentration between 1x107 and 1x1011 CFU, preferably 1x109 CFU / g, leads to a reduction in the Scorad index (lower Scorad values indicate better quality of life).
    Furthermore, the administration of said probiotic composition for a period of 12 to 24 weeks, preferably 16 weeks, is able to decrease / block the secretion of the cytokine IL-4.
    Experimental session (I) The applicant tested all six strains mentioned above. A first clinical study was conducted to test a composition that with the culture of Lactobacillus salivaríus (LS01) DSM 22775 and the culture of Bifidobacterium breve (BR03) DSM 16604.
    A group of 40 patients (male and female, aged 18 to 55 years) with moderate / severe AD was randomly divided (1: 1) into two groups.
    The diagnosis of AD was assessed as moderate or severe by adopting a standardized severity index for AD (SCORAD index - SCORing Atopic Dermatitis', literally, “grading atopic dermatitis”). This index is known to those skilled in the art.
    To represents time zero as the starting point of treatment, while T16 represents the period of 16 weeks of treatment. In T16, an examination was performed, with Scorad measurement and sample collection.
    Group A was treated with a composition with the culture of Lactobacillus salivaríus (LS01) DSM 22775 and the culture of Bifidobacterium breve (BR03) DSM 16604, at a concentration of 1x109 CFU (which corresponds to 0.01 g). The two cultures were mixed with 1 g of corn starch to obtain a final composition of 1.01 in sachets. The 20 patients in group A received 2 sachets a day for 16 weeks.
    Group B was treated with a placebo represented by 1 g of corn starch (exclusively). The 20 patients in group A received 2 placebo sachets a day for 16 weeks.
    The following cytokines were tested: Th1 (IFN-y and IL-12) and Th2 (IL-4, IL-5 and IL-10). We carefully collected and analyzed all data from the 40 patients. Figure 1 illustrates a histogram referring to the Scorad index at T0eT16.
    The results obtained demonstrate that the administration, in the 20 patients of group A, of the probiotic composition with the culture of Lactobacillus salivaríus (LS01) DSM 22775 and the culture of Bifidobacterium breve (BR03) DSM 16604 in a concentration of 1x109 determined the reduction in the index Scorad in AD victims (lower Scorad values indicate better quality of life).
    In addition, the administration of the said probiotic composition for 16 weeks prevented the secretion of the cytokine under consideration, IL-4, whose average value was comparable to that obtained at the time of registration. The administration of placebo, on the other hand, was unable to prevent the release of the cytokine IL-4, which increased significantly after 16 weeks of treatment (disease progression).
    We performed a series of preclinical studies in vitro on a group of bacteria comprising, among others, the culture of Lactobacillus salivarius (LS01) DSM 22775 and the culture of Bifidobacterium breve (BR03) DSM 16604.
    Pre-clinical in vitro studies, comparing the immunological faculties of the tested bacterial strains, demonstrated that the culture of Lactobacillus salivarius (LS01) DSM 22775 and the culture of Bifidobacterium breve (BR03) DSM 16604 have superior capacity in inducing the production of type 1 cytokines.
    We also tested cultures of the bacteria Lactobacillus pentosus (LPS01) DSM 21980, Streptococcus thermophilus (FP4) DSM 18616, Lactobacillus casei ssp. rhamnosus (LR04) DSM 16605 and Lactobacillus acidophilus (LA02) DSM 21717.
    The in vitro culture of the L. pentosus (LPS01) DSM 22775 strain has been shown to induce a very strong pro-cytokine Th1 profile.
    In addition, we tested all cultures of the Lactobacillus pentosus (LPS01) DSM 21980, Streptococcus thermophilus (FP4) DSM 18616, Lactobacillus casei ssp. rhamnosus (LR04) DSM 16605 and Lactobacillus acidophilus (LA02) DSM 21717 as to their ability to induce synthesis of IL-12 by PBMCs in an in vitro study. IL-12 is a Th1-type cytokine and represents a key element in the treatment of allergies and other atopic pathologies.
    As an example, the data referring to L. pentosus (LPS01) DSM 21980 tested in PBMCs are disclosed as: Th-1 (pg / ml) = 1.754; Th-2 (pg / ml) = 326; Th1 / Th2 = 5.38.
    Experimental session (II) The applicant conducted a first clinical study using a composition with cultures of Lactobacillus salivarius (LS01) DSM 22775 and Bifidobacterium breve (BR03) DSM 16604, as well as a second clinical study using a composition with cultures of Lactobacillus salivarius (LS01) DSM 22775, Bifidobacterium breve (BR03) and Lactobacillus pentosus (LPS01) DSM 21980. For the sake of simplicity, we will report only the details of the first clinical study, since the second clinical study was conducted using the same methods . Both clinical studies (first and second) produced comparable results.
    The applicant performed the first clinical test (randomly and double-blind in patients with severe or moderate dermatitis) in order to assess the clinical efficiency of the administration of two probiotic bacterial strains, namely, Lactobacillus salivaríus (LS01), deposited in the DSMZ under deposit number DSM 22775, and Bifidobacterium breve (BR03), deposited at DSMZ under deposit number DSM 16604. Materials and methods 5 We recruited 48 patients with atopic dermatitis (hereinafter AD) for the study; the characteristics of which are given in Table 1. Table 1
    The present clinical study was conducted from April to September 2010. 10 Thirty-two patients (treatment group) received a preparation with two probiotic bacterial strains, namely, L. salivaríus (LS01) DSM 22775 and B. breve ( BR03) DSM 16604, in a dose of 1x109 CFU / g of each strain in maltodextrin.
    16 patients (placebo group) received a preparation 15 exclusively of maltodextrin. Both groups were treated twice daily for 12 weeks. The compositions in lyophilized form were dissolved in water and administered orally to patients.
    None of them changed their diet and all were instructed not to consume fermented products that contained live microorganisms during the 20 clinical study. The present study excluded patients who were victims of active allergic diseases (whether respiratory or skin) or contagious diseases during pregnancy or breastfeeding. The present study excluded patients treated with probiotics, antibiotics or immunomodulators in the six months preceding enrollment or treated with oral steroids one month before enrollment. All patients declared their consent at the time of registration.
    The clinical evaluation was based on the SCORAD index (SCORing Atopic Dermatitis; literally, “grading atopic dermatitis”), the IQVD (dermatological quality of life index) and immunological and microbial translocation parameters. In each specific patient, all evaluations were performed at enrollment (reference value), after three months of treatment (3 ms) and two months after the end of therapy (3 + 2 ms). Questionnaire on symptoms and quality of life
    The diagnostic criteria for AD were established following the recently published clinical guide. We assessed clinical severity using the SCORAD index, developed by the European Task Force for the treatment of atopic dermatitis. In the course of the study, in order to measure variations in symptoms related to the pathology, all enrolled patients completed the specific IQVD (dermatological quality of life index) questionnaire. The SCORAD evaluation was carried out at the beginning, at the end and two months after the suspension of treatment (either with probiotics or placebo) by an operator who did not know in which group the patient was enrolled. The IQVD was completed by the patients at the time of registration, as well as at 12 and 20 weeks.
    Separation of plasma and mononuclear cells from peripheral blood (PBMC)
    Regarding the separation of plasma and mononuclear cells from peripheral blood (PBMCs), we collected samples of peripheral venous blood from all patients at the time of enrollment, 3 months after treatment and 2 months after discontinuation of therapy. The plasma was kept until analysis time, while PBMCs were separated by density gradient centrifugation. Cell viability was determined using an Adam-MC cell counter.
    Stimulation of PBMCs We incubate PBMCs for 18 hours in the presence or absence of bacterial stimulus with lipopolysaccharides (LPS). We performed an analysis of the cytokines induced by the antigenic stimulus by means of flow cytometry, adding Brefeldina A as an inhibitor of cell transport in the last hours of culture.
    Immunophenotypic analysis The percentage of CD8 + T lymphocytes activated directly in peripheral venous blood samples was calculated using cytofluorometric analysis using monoclonal antibodies (mAb) conjugated to uorochromes (CD8, CD38, CD45RO). After incubation with the mAbs, the erythrocytes were lysed and the cells were fixed and analyzed with an FC500 flow cytometer (Beckman Coulter).
    Analysis of lymphocyte subpopulations The identification of different T lymphocyte subpopulations was performed in cultures of unstimulated PBMCs and cultures of PBMCs stimulated for 18 hours with LPS at all times (reference value, 3 ms and 3 + 2 ms). At the same time, we carry out cell labeling using combinations of fluorescent antibodies capable of recognizing molecules on both the cell surface and intracellular components. More specifically, type 1 (Th1) helper T lymphocytes have been identified as CD4 + / IFN-y + / Tbet +; helper T lymphocytes type 2 (Th2) such as CD4 + / IL-4 + / GATA3 +; helper T lymphocytes of type 17 (Th17) as CD4 + / IL-17 + / RORyT + and, finally, regulatory T helper lymphocytes (Treg) as CD4 + / CD2 5 + / FoxP3 / IL-10 + / TGF-β + .
    Evaluation of the expression of Toll-like receptors (TLR) in regulatory T lymphocytes
    We carry out the analysis in non-stimulated PBMCs in accordance with the protocol provided by the mAb distributor. More specifically, the following mAbs were needed: CD4, CD25, FoxP3, TLR-4, TLR-2.
    Plasma LPS assay In order to determine the LPS concentration present in plasma samples, we used the kit “LAL Chromogenic Endopoint Assa / (LAL Chromogenic Endpoint Assay) kit marketed by the company Hycult biotechnology according to the manufacturer's instructions . We performed the test on a 96-well plate. After 45 minutes of incubation at room temperature, we read the absorbance at a wavelength of 405 nm using a spectrophotometer and calculate the LPS concentration, expressed in pg / ml, by interpolation with a known concentration curve.
    Collection and storage of faecal samples We collect faecal samples at the time of registration, after 3 months of treatment and 2 months after the end of the study. Such samples were immediately placed at 4 ° C, divided into aliquots and stored at -80 ° C until the moment of analysis.
    Quantification of bacteria present in faecal samples
    We performed the quantification of all aerobic bacteria and the following bacterial groups: Enterobacteria, Staphylococci, Lactobacilli (more specifically, L. salivaríus LS01) and Bifidobacteria (more specifically, B. breve BR03). Fecal samples were diluted in physiological solution and, appropriate dilutions, incubated according to the following scheme:
    All AEROBIC bacteria: Triptych soy agar with 5% sheep blood (AS);
    Enterobacteria1. MacConkey Agar (MC); Staphylococci: salted mannitol agar (MSA); Lactobacilli: Agar de Man, Rogosa and Sharpe (MRS); Bifidobacteria: Selective medium for bifidobacterium (BSM).
    MC and BSM media were incubated at 37 ° C for 24 and 48 hours, respectively. AS, MRS and BSM media were incubated at 37 ° C in the presence of 10% carbon dioxide for 24, 48 and 72 hours, respectively.
    All colonies were identified by growth in selective media, Gram lineage, cell morphology and tests to determine catalase and oxidase.
    The composition of the intestinal microbiota was expressed as the count (mean ± standard deviation from the base 10 logarithm (Iog10) per gram of feces) of each bacterial group. The detection limits were 2 Iog10 CFU / g in all microorganisms, except for B. Breve, whose limit was 5 log 10 CFU / g. The changes in the counts of the different microbial groups were calculated by: [(Log 10 CFU / g in T12 or T16) - (Log 10 CFU / g in T0)]
    Molecular identification of L. salivaríus LS01 and B. breve BR03
    The two bacterial strains were initially identified based on their morphological peculiarities: L. salivaríus LS01 grown in agarized BSM form cream-colored colonies, with a round shape and 2 to 4 mm in diameter; a B. breve BR03 in agarized BSM medium forms fuchsia-colored colonies of rounded and elongated shape with 1 to 2 mm in diameter.
    In cultures in which it was possible to assume the presence of L. salivaríus and B. breve strains, ten random colonies were analyzed by Polymerase Chain Reaction (PCR) according to the guidelines published by the National Health Institute. All colonies classified as L. salivaríus or B. breve were also characterized by performing pulsed-field gel electrophoresis (PFGE) and comparing the profiles to suitable references.
    Statistical analysis We analyze the results using appropriate statistical tests. Student's t-tests were used to compare patients during treatments. We evaluated possible relationships using Pearson's correlation test. Variations in bacterial count were analyzed using the Wilcoxon-Mann-Whitney test. Statistical analysis was performed using the SPSS statistical package.
    Results a) Clinical effectiveness 46 patients with AD completed the clinical study; 2 patients (1 per group) were excluded. SCORAD (SCORAD index) and IQVD assessments were performed during follow-up examinations. Patients treated with probiotics showed a significant reduction in SCORAD at the end of treatment (p = 0.001), which also remained after the treatment was discontinued (p = 0.006) (Table 2). Similarly, IQVD showed improvement in patients treated with the probiotic mixture (registration x 3 ms of treatment: p = 0.024; registration x 2 ms after suspension: p = 0.001). In the group of patients to whom we administered the placebo, there was no significant difference in terms of SCORAD or IQVD (Table 2). b) LPS concentration in plasma The LPS concentration is an index of microbial translocation; the increase in LPS concentration is related to changes in intestinal permeability. The treatment with two probiotic strains caused a reduction in the concentration of LPS in the plasma, which was maintained after the interruption of the therapy (reference value x 3 months of treatment: p = 0.050; reference value x 2 months after suspension of treatment: p <0.001; 3 months of treatment x 2 months after suspension of treatment: p <0.001), as shown below (Table 3). Table 3

    In patients treated with placebo, a significant increase in plasma LPS levels was observed during the study period (reference value x 2 months after treatment discontinuation: p = 0.004; 3 months of treatment x 2 months after treatment discontinuation: p = 0.016) (Table 3). At the end of the study, the plasma LPS concentration was significantly higher in the control group (placebo) compared to the treatment group (p <0.001). c) Activated T lymphocytes We analyzed activated CD8 + T cells by evaluating the expression of the CD38 and CD45 molecules in them. A progressive reduction in these cells was observed during the study period in patients treated with both probiotics (reference value x 2 months after treatment discontinuation: p = 0.001; 3 months of treatment x 2 months after treatment discontinuation: p <0.001) (Table 4). There was no significant difference in the placebo-treated group. Table 4
    Regulatory T cells were evaluated both in the absence of stimulation and after stimulation with LPS. After treatment with the probiotic mixture, the percentage of unstimulated Treg lymphocytes increased significantly (reference value x 2 months after treatment discontinuation: p = 0.002; 3 months of treatment x 2 months after treatment discontinuation: p = 0.034) (Table 5A and Table 5B). Similar results were observed in the analysis after stimulation with LPS. Table 5A

    e) Expression of Toll-like receptors in regulatory T lymphocytes
    We evaluated the expression of Toll 2 (TLR-2) and 45 (TLR-4) type receptors directly in peripheral venous blood. As shown in Tables 5C and 5D, the expression of both molecules in Treg lymphocytes increased significantly after 3 months of probiotic administration; this result was also maintained 2 months after the suspension of treatment (TLR-2: reference value x 3 months of treatment: p <0.001; reference value x 2 months after 10 treatment suspension: p = 0.010; TLR-4: reference value x 3 months of treatment: p <0.001; reference value x 2 months after suspension of treatment: p = 0.011). In the control group (placebo), there was a significant drop in the expression of the TLR-2 molecule in Treg lymphocytes at the end of the study (3 months of treatment x 2 months after treatment suspension: p = 0.009). 15 Table 5C
    Table 5D
    f) Subpopulations of Th1, Th2 and Th 17 lymphocytes
    Th1, Th2 and Th 17 lymphocyte subpopulations were evaluated in the absence of stimulation and after stimulation with LPS in both groups under analysis: the percentage of Th1 subpopulation, identified based on the expression of the CD4 and Tbet molecules and secreting the IFN cytokine -y, increased significantly in patients treated with the probiotic mixture both in the absence of stimulation and after stimulation with LPS (without stimulation: reference value x 3 5 months of treatment: p = 0.003; LPS: reference value x 3 months of treatment : p = 0.025; reference value x 2 months after suspension of treatment: p = 0.019) (Tables 6A and 6B). In patients treated with probiotics, two months after the end of treatment, a further reduction in Th1 cells was observed (p <0.001). Table 6A
    Table 6B

    The percentage of the Th2 cell subpopulation, identified based on the expression of the CD4 and GATA3 molecules and secreting the IL-4 cytokine, dropped significantly during the administration of probiotics but, after discontinuing therapy, it increased again (without stimulation: reference value x 3 months of 15 treatment: p = 0.016; 3 months of treatment x 2 months after treatment discontinuation: p = 0.005; LPS: reference value x 3 months of treatment: p = ns; 3 months of treatment x 2 months after treatment suspension: p = 0.0045) (Table 6C and 6D). Table 6C


    The percentage of Th17 cell subpopulation, identified based on the expression of the CD4 and RORyT molecules and secreting the IL-17 cytokine, decreased in patients treated with probiotics after 3 months of treatment 5 (without stimulation: reference value x 3 months of treatment: p = 0.037; LPS: reference value x 3 months of treatment: p = 0.046) (Tables 6E and 6F). Two months after discontinuing treatment, significant differences were observed between the two groups under analysis with regard to the Th2 and Th17 subpopulations (p = 0.008 and p = 0.031, respectively). We did not find any significant difference in the 10 populations of Th1, Th2 and Th17 cells in the patients in the control group (placebo). Table 6E
    Table 6F

    After 3 months of probiotic administration, a significant increase in the Th1 / Th2 ratio was observed (p = 0.028) (Table 7A). This effect was lost after treatment interruption (p = 0.002). In contrast, in the group treated with the bacterial mixture, the Th17 / Treg ratio gradually decreased, a result that was also verified 2 months after the interruption of therapy (p = 0.029). During the study, patients enrolled in the control group (placebo) did not show significant variations in the Th1 / Th2 and Th17 / Treg ratios. The Th17 / Treg ratio was significantly higher in the placebo-treated group compared to the probiotic-treated group after 3 months of treatment (p = 0.037) (Table 7B). Table 7A
    Table 7B
    h) Correlations
    In patients who were given probiotics, at the end of treatment, a negative correlation was observed between SCORAD and the percentage of Treg lymphocytes after stimulation with LPS (p = 0.020) and between the percentage of activated CD8 + T lymphocytes and that of cells Th1 stimulated with LPS (p = 0.046).
    A significant positive correlation was observed between the concentration of LPS in plasma and the percentage of unstimulated Th2 cells (p = 0.016), between the concentration of LPS in plasma and the percentage of Th17 cells stimulated with LPS (p = 0, 04) and between the percentage of activated CD8 + T lymphocytes and that of Th17 cells stimulated with LPS (p = 0.037). i) Changes in the fecal microbiota and detection of L. salivaríus LS01 and B. breve BR03 strains Table 8 gives the cell count at the time of registration, after 3 months of treatment and 2 months after the end of the administration. Table 8
    * Decreased significance (p <0.05) compared to the reference value
    Significant difference (p <0.05) compared to placebo 5 at the same time. In the control group (placebo), there was no significant change in the fecal flora. In the group treated with probiotics, after 3 months, a significant drop in staphylococci was detected. This finding was also significant compared to the control group after 3 months of treatment. The 10 reduction remained statistically significant even two months after the probiotics were discontinued. We found no significant changes in any other microbial group analyzed. The findings regarding probiotic strains in the fecal samples of patients enrolled in the study are given in Table 9. All 15 colonies with morphology corresponding to that of the strains considered, identified by PCR and PFGE analysis, corresponded to those of the strains previously identified as L. salivary LS01 or B. breve BR03. After 3 months of treatment, the LS01 L salivary strain was found in all patients in an amount between 102 and 105 CFU / g. After three months of treatment, the B. breve BR03 strain was detected in 90% of patients in an amount between 106 and 108 CFU / g.
    In some patients, both strains were also isolated 2 months after stopping treatment, but in a smaller amount compared to that obtained shortly after the end of administration.
    The applicant was able to verify that the compositions of the present invention, in particular those of said first clinical study (Lactobacillus salivarius (LS01) DSM 22775 and Bifidobacterium breve (BR03) DSM 16604) and said second clinical study (Lactobacillus salivarius (LS01) DSM 22775 , Bifidobacterium breve (BR03) DSM16604 and Lactobacillus pentosus (LPS01) DSM 21980) given for 3 months in adult patients with severe or moderate atopic dermatitis proved to be able to improve their clinical and immunological parameters. More specifically, at the end of treatment with probiotics, the following was observed: 1) significant improvement in the SCORAD and IQVD indices; 2) reduction in microbial translocation (plasma LPS levels) and activation of CD8 + T lymphocytes; 3) increase in the percentage of both total regulatory T lymphocytes (Treg) and those that express the TLR2 and TLR4 markers; 4) improvement in the Th1 / Th2 and Th17 / Treg ratios.
    For greater advantage, it was demonstrated that the probiotic strains used were able to colonize the patients' gastrointestinal tract and decrease the load of staphylococci in the feces.
    For greater advantage, the clinical effect of the administration of probiotics, in terms of improving the symptoms of the pathology and the quality of life, was revealed after 3 months of treatment. This effect proved to persist for even 2 months after stopping treatment.
    For greater advantage, the persistence of the 3 probiotic bacterial strains (Lactobacillus salivarius (LS01) DSM 22775, Bifidobacterium breve (BR03) DSM16604 and Lactobacillus pentosus (LPS01) DSM 219809) was also observed in the gastrointestinal tract after the suspension of treatment, suggesting the faculty of specific bacterial combinations to colonize the intestine.
    Changes in intestinal permeability play a fundamental role in the pathogenesis of atopic dermatitis and in other diseases characterized by an inflammatory component. The results of the clinical study demonstrated an important aspect, which is represented by the presence of a negative correlation between plasma LPS levels and atopy. More specifically, the presence of a significant improvement in microbial translocation, both during and after the probiotic treatment, demonstrated the immunomodulatory effect of possession of the 3 bacterial strains, capable of improving the intestinal barrier acting both directly and indirectly.
    Studies on HIV infections have shown an association between microbial translocation and immunological activation with excessive T cell turnover. The evaluation of the percentage of activated CD8 + T lymphocytes, considered an important prognostic factor in HIV infections, is one of the most widely accepted brands. of LPS-mediated activation of the immune system. As expected, in patients with atopic dermatitis enrolled in the present study, a reduction in plasma LPS values was observed associated with a reduction in activated CD8 + T cells. These data demonstrate that the tested bacterial strains have strong immunomodulatory activity due to their ability to increase the intestinal barrier. In the placebo group, this correlation was not observed.
    Four different subpopulations of CD4 + T helper lymphocytes have been identified based on their ability to secrete cytokines: Th1 lymphocytes, major producers of IFN-y and IL-2, are involved in cell-mediated immune responses; the Th2 lymphocytes, which secrete IL-4, are responsible for activating the humoral response; the Th17 lymphocytes, which secrete IL-17, IL-21 and IL-22, are involved in immune responses directed against extracellular pathogens and in autoimmune diseases; regulatory T cells with immunoregulatory function control the overactivation of immune responses.
    A reduction in Treg cells has been reported in patients with atopic dermatitis and asthma, and their number is inversely correlated to IgE secretion, eosinophilia and IFN-y levels.
    The administration of probiotic strains caused a significant increase in the percentage of total Treg lymphocytes and a significant increase in Treg lymphocytes that express TLR2 and TLR4 receptors after treatment. These cell subpopulations play an immunosuppressive activity as the main function. Colonization of S. aureus is a crucial point for the development of atopic dermatitis, as is the positive correlation between the severity of the disease and colonization by S. aureus. The beneficial effects of probiotics may be due to a recovery, mediated by Treg cells, of immunological tolerance to S. aureus and gram-negative bacteria. In addition, a negative correlation was found between the SCORAD index and Treg cells, suggesting their positive role in the evolution of atopic dermatitis.
    The Th2 subtype plays a critical role in the pathogenesis of allergic reactions and is responsible for the acute phase of atopic dermatitis, while the Th1 subpopulation has a protective function associated with the chronic phase of the disease. In patients suffering from atopic dermatitis, Th17 cells increase in acute skin lesions, but not in chronic ones, and positively correlate with the severity of the disease. The data obtained in this study demonstrated that this new probiotic combination increases the functionality of Th1 cells at the expense of the Th2 and Th17 subpopulations, thus increasing the Th1 / Th2 and Th17 / Treg ratios.
    Subtypes Th2 and Th17 correlate positively with the level of LPS in the plasma, suggesting a strong interaction between microbial translocation and changes in the immune system. This discovery is further confirmed by the positive correlation between Th17 and activated CD8 +. The role played by microbial intestinal flora in the development of immunological tolerance and in the pathogenesis of allergic diseases has also been amply demonstrated. Alteration of the intestinal microflora plays a role in the onset of atopic dermatitis and in the worsening of skin symptoms. The same authors, when analyzing the composition of the faecal microbiota of sensitized children, panting and non-panting, observed that lactobacteria and bifidobacteria were similar in both groups.
    In the present study, a significant difference was observed in the microbial composition of the two groups analyzed in relation to Staphylococci. Therefore, it is presumed that this genus plays a crucial role both at the level of the skin and in the intestine of patients suffering from atopic dermatitis and that the administration of the present probiotic combination restores balance.
    The present clinical study demonstrated that the bacterial strains of the present invention are well tolerated and capable of colonizing the intestinal tract and provoking beneficial clinical effects in patients with atopic dermatitis thanks to the modulation of some of the most important immunopathological changes of this inflammatory skin disease.
    The subject of the present invention relates to the use of the bacterial strains tested above in an additional therapy that assists in the treatment of atopic dermatitis in adult patients.
    权利要求:
    Claims (6)
    [0001]
    1. Composition comprising a culture of probiotic bacteria, characterized by the fact that the culture of probiotic bacteria is Lactobacillus salivarius (LS01) DSM 22775, deposited on 7/23/2009 at Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH-DSMZ.
    [0002]
    2. Composition according to claim 1, characterized by the fact that said bacterial cultures are present in said compositions in the form of live bacteria, dead bacteria or cell components, cell extracts or lysates thereof.
    [0003]
    Composition according to claims 1 and 2, characterized in that it is in the solid state for oral administration.
    [0004]
    4. Composition according to any one of the preceding claims, characterized by the fact that said cultures of probiotic bacteria are present in a concentration between 1x107 and 1x1011 CFU / g of composition, preferably between 1x108 and 1x1010 CFU / g of composition.
    [0005]
    5. Composition, according to any one of the preceding claims, characterized by also comprising dietary fibers with prebiotic activity selected from fructo-oligosaccharides (FOS), inulin and partially hydrolyzed guar gum (GGPH).
    [0006]
    6. Composition according to any one of the preceding claims, characterized in that it is a pharmaceutical, dietetic, nutritional or nutraceutical composition.
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    同族专利:
    公开号 | 公开日
    EP2626076A1|2013-08-14|
    PT2544698T|2016-07-15|
    EP2544698A1|2013-01-16|
    RU2712751C9|2020-04-28|
    RU2016129186A3|2019-11-21|
    US9931363B2|2018-04-03|
    KR20130018758A|2013-02-25|
    ITMI20100375A1|2011-09-09|
    ES2603629T3|2017-02-28|
    DK2544698T3|2016-07-04|
    RU2712751C2|2020-01-31|
    BR112012022755A2|2016-07-19|
    UA111715C2|2016-06-10|
    EP2626076B1|2016-08-24|
    CN103037876B|2015-04-01|
    AU2011225798B2|2017-02-02|
    US20150017142A1|2015-01-15|
    CN103037876A|2013-04-10|
    EP2544698B1|2016-05-04|
    WO2011110918A1|2011-09-15|
    PL2626076T3|2017-08-31|
    JP2013521335A|2013-06-10|
    JP6182628B2|2017-08-16|
    CN104800247A|2015-07-29|
    DK2626076T3|2016-09-19|
    US20170071989A1|2017-03-16|
    ZA201207377B|2015-04-29|
    RU2605293C2|2016-12-20|
    US8734783B2|2014-05-27|
    RU2012140052A|2014-04-20|
    CA2792343C|2019-04-02|
    HUE029245T2|2017-02-28|
    IT1398553B1|2013-03-01|
    RU2016129186A|2018-12-10|
    CA2792343A1|2011-09-15|
    KR101872946B1|2018-06-29|
    WO2011110918A8|2011-11-10|
    HUE030888T2|2017-06-28|
    US9498503B2|2016-11-22|
    JP2016128502A|2016-07-14|
    PT2626076T|2016-10-04|
    US20130149342A1|2013-06-13|
    PL2544698T3|2017-08-31|
    ES2581803T3|2016-09-07|
    JP5931754B2|2016-06-08|
    ZA201409369B|2015-09-30|
    AU2011225798A1|2012-10-04|
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    法律状态:
    2017-06-06| B25C| Requirement related to requested transfer of rights|Owner name: PROBIOTICAL S.P.A. (IT) |
    2017-10-31| B25C| Requirement related to requested transfer of rights|Owner name: PROBIOTICAL S.P.A. (IT) |
    2018-01-16| B25B| Requested transfer of rights rejected|Owner name: PROBIOTICAL S.P.A. (IT) |
    2018-04-10| B06F| Objections, documents and/or translations needed after an examination request according art. 34 industrial property law|
    2018-04-17| B07D| Technical examination (opinion) related to article 229 of industrial property law|
    2019-08-20| B07E| Notice of approval relating to section 229 industrial property law|Free format text: NOTIFICACAO DE ANUENCIA RELACIONADA COM O ART 229 DA LPI |
    2020-03-10| B07A| Technical examination (opinion): publication of technical examination (opinion)|
    2020-09-15| B07A| Technical examination (opinion): publication of technical examination (opinion)|
    2021-01-12| B09A| Decision: intention to grant|
    2021-03-02| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 07/03/2011, OBSERVADAS AS CONDICOES LEGAIS. |
    优先权:
    申请号 | 申请日 | 专利标题
    ITMI2010A000375A|IT1398553B1|2010-03-08|2010-03-08|COMPOSITION INCLUDING PROBIOTIC BACTERIA FOR THE TREATMENT OF PATHOLOGIES ASSOCIATED WITH THE ALTERATION OF THE IMMUNE SYSTEM.|
    ITMI2010A000375|2010-03-08|
    PCT/IB2011/000490|WO2011110918A1|2010-03-08|2011-03-07|Composition comprising probiotic bacteria for use in the treatment of immune disorders|
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