Pharmaceutical composition comprising metabolites from the bacterium Bacillus subtilis Gozen GZN-08 isolated from Camellia sinensis and modulating the immune response.

Abstract

The invention relates to a pharmaceutical composition comprising a combination of bioactives including theanine and polysaccharides extracted from Camellia sinensis, and at least two metabolites typically produced by the bacterium Bacillus subtilis, preferably Bacillus subtilis Gozen GZN-08 isolated from Camellia sinensis. The invention also relates to the pharmaceutical composition for its use in modulating the pulmonary microbiota while modulating the intensity of the immune system response by restoring normal white blood cell counts and increasing antibody production while reducing the production of pro-inflammatory cytokines.

Description

Title of the invention: Pharmaceutical composition comprising metabolites from the bacterium Bacillus subtilis Gozen GZN-08 isolated from Camellia sinensis and modulating the immune response.

[0001] The present invention relates to a pharmaceutical composition comprising an association of theanine and polysaccharides extracted from Camellia sinensis, and at least 2 metabolites produced by the bacterium Bacillus subtilis Gozen GZN-08 from the group: gallic acid, lobelin, biotin, catechin, pyridoxine, syringic acid, butyrate, propionate, zinc, linoleic acid, saikosaponin, acacetin, saponarin, butyric acid, characterized in that said composition modulates the pulmonary microbiota and increases the intensity of the immune system response.

[0002] The microbiota is known to be the collection of microorganisms, including bacteria, yeasts, fungi, and viruses, living in a specific environment called the microbiome within a host. Humans possess several microbiomes: intestinal, pulmonary, vaginal, skin, and those of the nose / mouth / pharynx area. An individual's microbiota is formed from the mother's vaginal microbiota at birth, which immediately colonizes the skin, while the microbiota from breast milk colonizes the intestines. Exposure to environmental bacteria, antibiotics, diet, and genetic factors then influences the evolution of the microbiota during the first few years. The presence of the microbiota helps limit the risk of colonization by pathogenic bacteria.Before the discovery of the microbiota, bacteria were mostly considered harmful or pathogenic, but today its role is better understood. It is now known to be essential for the proper functioning of digestion, metabolism, the immune system, and even the neurological system. However, the balance of the microbiota is fragile. An imbalance in this microbiota is called dysbiosis. This imbalance can be caused by a disruptive event such as a change in environment, diet, or medical treatment. Dysbiosis can contribute to numerous diseases such as obesity, diabetes, allergies, and even depression.

[0003] The inventor was particularly interested in the lung microbiota. It was long thought that the lungs were sterile; however, recent studies have shown that the lungs harbor a wide range of different microbial species. It has been observed that the airway microbiota is altered in the context of many respiratory disorders. Modulating the lung microbiota is therefore a valuable tool for maintaining lung health.

[0004] The gut microbiota, along with that of the skin, is the most important microbiota in the body. It can ferment indigestible food residues, promote the absorption of certain nutrients, hydrolyze several substances, participate in the synthesis of numerous vitamins, and regulate various metabolic pathways. The gut flora is also involved in the functioning of the intestinal immune system, which is essential for the barrier function of the intestinal wall. Indeed, bacteria such as Escherichia coli directly combat the colonization of the digestive tract by pathogenic bacteria through competition and the production of bactericidal substances. The gut flora is also very important from a very young age, as it allows the intestinal immune system to learn to distinguish between beneficial bacteria and pathogenic bacteria.

[0005] The influence of the microbiota on the formation of the immune response is known to be an increasingly studied subject today. These studies indicate the importance of the microbiota's influence on its host and demonstrate the key role of certain microbiota bacteria in activating the immune system, particularly in the gut. Dysbiosis can therefore lead to dysfunction in the activation of this system, an insufficient immune response, or even colonization by inflammatory pathogenic bacteria in the gut.

[0006] Treatments based on polysaccharides derived from Camellia sinensis (WO2011069781Al) have been proposed to stimulate the immune response, but this solution has a limited duration of action since it does not address an imbalance in the intestinal or pulmonary microbiota and therefore does not prevent a future insufficient immune response in cases of dysbiosis. Other probiotic-based solutions have been proposed, such as a strain of Lactobacillus reuteri to prevent or treat microbiota dysbiosis and thus prevent various types of diseases related to this dysbiosis (WO2016066763A1), or a strain of Lactobacillus rhamnosus to promote an anti-infectious and / or anti-inflammatory immune response (EP2983685A1). However, these solutions use live strains, which are not stable over time and may therefore lead to a decrease in efficacy over time.Furthermore, live strains have a limited effect, due not only to their low stability over time, but also because they depend on the bacterial composition of the subject, which can limit the implantation of new bacteria and consequently their effectiveness.

[0007] It was while studying the effect of fermentation on the efficacy of tea extracts that the Applicant unexpectedly discovered that these problems can be easily solved by using a pharmaceutical composition comprising a combination of theanine and polysaccharides extracted from Camellia sinensis, and at least 2 metabolites, typically produced by a bacterium Bacillus subtilis, typically Bacillus subtilis Gozen GZN-08, selected from the group: gallic acid, lobelin, biotin, catechin, pyridoxine, syringic acid, butyrate, propionate, zinc, linoleic acid, saikosaponin, acacetin, saponarin, butyric acid, characterized in that said composition modulates the pulmonary microbiota and modulates the intensity of the immune system response.

[0008] SUMMARY

[0009] In a first aspect, the invention relates to a pharmaceutical composition comprising a combination of bioactives including theanine and polysaccharides extracted from the leaves of Camellia sinensis matcha, and at least two metabolites produced by the bacterium Bacillus subtilis, selected from the group: gallic acid, lobelin, biotin, catechin, pyridoxine, syringic acid, butyrate, propionate, zinc, linoleic acid, saikosaponin, acacetin, and saponarin. Typically, the pharmaceutical composition is intended to modulate the pulmonary microbiota and the intensity of the immune system response.

[0010] In one embodiment the pharmaceutical composition comprises zinc at a concentration of at least 0.5% and preferably at least 2% by weight relative to the total weight of bioactives in said composition and polysaccharides at a concentration of at least 0.5% and preferably at least 5%.

[0011] The pharmaceutical composition of the invention may be in powder form or freeze-dried emulsion. It can be in the form of creams, liquids, pastes, inhalers, lotions, emulsions, gels, syrups, solids, powders, masks, sticks, tablets, sprays, aerosols, capsules, oils, or ready-to-use drinks.

[0012] The invention also relates to a method for preparing a pharmaceutical composition according to the invention. The method comprises at least one slow fermentation step of Camellia sinensis in the presence of the bacterium Bacillus subtilis Gozen GZN-08.

[0013] The invention further relates to the pharmaceutical composition as described above, for its use in:

[0014] - the treatment or prevention of dysbiosis, preferably dysbiosis pulmonary and / or intestinal.

[0015] - the reduction of signs of inflammation and in the reduction of production of pro-inflammatory cytokines; in the restoration of a normal white blood cell count and the increase in antibody production by lymphocytes;

[0016] - the restoration of a normal white blood cell count and the increase in production of antibodies by lymphocytes;

[0017] - the increase in nitric oxide production by endothelial cells and in the modulation of blood vessel vasodilation; and / or

[0018] - the increase in hemoglobin stability and in the increase in the rate of circulating hemoglobin. DETAILED DESCRIPTION

[0019] The object of the present invention therefore relates to a pharmaceutical composition comprising an association of theanine and polysaccharides extracted from Camellia sinensis, and at least 2 metabolites, typically produced by a Bacillus subtilis bacterium, typically Bacillus subtilis Gozen GZN-08, selected from the group: gallic acid, lobelin, biotin, catechin, pyridoxine, syringic acid, butyrate, propionate, zinc, linoleic acid, saikosaponin, acacetin, saponarin, butyric acid, characterized in that said composition modulates the intestinal microbiota and increases the intensity of the immune system response.

[0020] In one embodiment, the at least 2 metabolites are produced by Bacillus subtilis Gozen GZN-08.

[0021] It should be noted that the pharmaceutical composition is characterized in that it comprises zinc at a concentration of at least 0.5% and preferably at least 2% by weight relative to the total weight of bioactives in said composition and polysaccharides at a concentration of at least 0.5% and preferably at least 5%.

[0022] The invention also relates to a pharmaceutical composition characterized in that it is used in the treatment or prevention of dysbiosis, preferably pulmonary and / or intestinal dysbiosis. Furthermore, the object of the present invention also relates to a pharmaceutical composition characterized in that it is used to reduce signs of inflammation and to decrease the production of inflammatory cytokines. The invention also relates to a pharmaceutical composition characterized in that it is used to restore a normal white blood cell count and to increase antibody production by lymphocytes.

[0023] It should be noted that the pharmaceutical composition is characterized in that it is used to increase the production of nitric oxide by endothelial cells and to modulate the vasodilation of blood vessels. Therefore, the object of the present invention also relates to a pharmaceutical composition characterized in that it is used to increase the stability of hemoglobin and to increase the circulating hemoglobin level.

[0024] In one embodiment, the pharmaceutical composition is in the form of a powder or a lyophilized emulsion. It may be in the form of, but not limited to, creams, liquids, pastes, inhalers, lotions, emulsions, gels, syrups, solids, powders, masks, sticks, tablets, sprays, aerosols, capsules, oils, or a ready-to-use beverage.

[0025] The present invention will be better understood by reading the definitions below which allow for a better understanding of the action of the invention.

[0026] “Fermentation”: refers to a transformation undergone by certain materials organic matter under the action of enzymes secreted by microorganisms.

[0027] For the sake of clarity in the explanations that follow, we decide to call BIOMATCHA a pharmaceutical composition according to the present invention.

[0028] The applicant describes below a pharmaceutical composition comprising an association of theanine and polysaccharides extracted from Camellia sinensis, and at least 2 metabolites produced by a Bacillus subtilis bacterium, typically Bacillus subtilis Gozen GZN-08, selected from the group: gallic acid, lobelin, biotin, catechin, pyridoxine, syringic acid, butyrate, propionate, zinc, linoleic acid, saikosaponin, acacetin, saponarin, butyric acid, characterized in that said composition modulates the pulmonary microbiota and modulates the intensity of the immune system response.

[0029] The present invention also relates to its composition in that it is made up of various metabolites, and comprises at least zinc at a minimum concentration. The object of the present invention is therefore described as a pharmaceutical composition characterized in that it comprises zinc at a concentration of at least 0.5% and preferably at least 2% by weight relative to the total weight of bioactives in said composition and polysaccharides at a concentration of at least 0.5% and preferably at least 5%.

[0030] The object of the present invention also relates to a method for manufacturing the composition of metabolites produced by the bacterium Bacillus subtilis, typically Bacillus subtilis Gozen GZN-08. These metabolites are prepared by the fermentation of Bacillus subtilis, typically Bacillus subtilis Gozen GZN-08. The applicant describes below a slow fermentation process of extracts of Camellia sinensis and Bacillus subtilis, typically Bacillus subtilis Gozen GZN-08, in several steps. The Camellia sinensis plant is first collected. The bacterium Bacillus subtilis Gozen GZN-08 is then inoculated in the presence of the Camellia sinensis plant, which is stored at high density in a humid environment at a temperature between 30°C and 45°C. This step lasts between 2 and 36 hours and activates the production of metabolites by the bacteria, at least one of which is of the genus Bacillus.Indeed, the applicant discovered that the Bacillus subtilis Gozen GZN-08 strain produces metabolites that can modulate the microbiota and increase the intensity of the immune response. The fermentation step therefore primarily allows for the fermentation of this bacterium of interest. The fermentation product can then be extracted and filtered to select the molecules of interest. Extraction can be performed by aqueous extraction. Alcoholic extraction, extraction by another type of solvent, distillation, extraction by a supercritical fluid. Filtration can be carried out by a liquid chromatography step or a membrane filter step. The object of the present invention therefore relates to a process for preparing the composition comprising at least one slow fermentation step.

[0031] It is now known that the gut microbiota plays a key role in initiating and adapting the immune response, not only in the digestive tract but also at a distance in the lungs. A balanced gut and / or lung microbiota helps limit the proliferation of harmful bacteria through competition. A balanced lung microbiota can prevent or reduce lung infections, control inflammatory reactions, and support the development of an immune system. For example, in the case of Covid-19, the lung microbiota plays an important role in the development and severity of the disease. Indeed, the disease begins with a lung infection that resolves on its own in the majority of cases; however, some develop severe respiratory distress and organ failure.The worsening of COVID-19 symptoms is caused by an excessive inflammatory response that leads to the destruction of lung tissue. A study conducted by Zijie Shen et al. (2020) demonstrated that COVID-19 patients had a different lung microbiota composition than healthy individuals. The consumption of metabolites from BIOMATCHA modulates the lung microbiota. BIOMATCHA is therefore described as a pharmaceutical composition for use in the treatment or prevention of dysbiosis, preferably pulmonary dysbiosis.

[0032] The importance of a balanced gut microbiota in human health is increasingly recognized. Chronic inflammatory bowel diseases are characterized by an imbalance in the gut flora. Dysbiosis can also promote colonization of the digestive system by various pathogenic microorganisms, which can exacerbate intestinal inflammation. In cases of inflammation, cells secrete pro-inflammatory cytokines. Pro-inflammatory cytokines are proteins that enable communication between immune system cells when a signal is detected, thus directing the immune response. Oral administration of theanine reduces the expression of these inflammatory cytokines. Pure theanine is easily absorbed through intestinal cells; however, its combination with other molecules impairs its absorption.The Applicant noted that combining theanine with a green tea polysaccharide complex increases theanine absorption in the intestine and blood theanine levels. Therefore, consuming BIOMATCHA increases blood theanine levels and reduces the production of inflammatory cytokines. more effectively than a conventional green tea extract. The object of the present invention is therefore described as a pharmaceutical composition for use in reducing the signs of inflammation and in decreasing the production of inflammatory cytokines.

[0033] Up to 70% of the immune system's response originates in the intestines. Indeed, the majority of substances foreign to the body enter the body via the digestive system. Intestinal cells are therefore rich in immune cells, such as lymphocytes located beneath the intestinal mucosa. Lymphocytes are white blood cells responsible, among other things, for the production of antibodies. The presence of certain molecules foreign to the body, called antigens, activates the immune response, allowing the proliferation of lymphocytes and increasing antibody production. The metabolites contained in BIOMATCHA stimulate white blood cells and promote their antibody production. BIOMATCHA is therefore described as a pharmaceutical composition for its use in restoring normal white blood cell counts and increasing antibody production by lymphocytes.

[0034] Nitric oxide is a molecule that primarily acts as a messenger within the body. Proper nitric oxide secretion provides numerous benefits to the body, for example, by enhancing memory, strength, and endurance, as well as reducing the risk of inflammation and regulating blood pressure. Indeed, nitric oxide is an excellent vasodilator and allows for improved blood flow throughout the body. Vasodilation is particularly important in cases of lung injury. BIOMATCHA promotes the production of nitric oxide by endothelial cells. The object of the present invention is therefore described as a pharmaceutical composition for use in increasing nitric oxide production by endothelial cells and in modulating vasodilation of blood vessels.

[0035] The increased vasodilation, induced by the production of nitric oxide, will allow for better blood flow. The composition also acts directly on hemoglobin, a protein found inside red blood cells. Hemoglobin is essential for transporting oxygen in the blood and transferring it from the lungs to the tissues. Iron or vitamin deficiencies, as well as dysbiosis of the gut microbiota, can cause anemia, i.e., a low hemoglobin level. Conventional green tea is known to inhibit hemoglobin. Unlike conventional green tea, consuming BIOMATCHA increases the number of red blood cells as well as the hemoglobin concentration. BIOMATCHA is therefore described as a pharmaceutical composition, for its use in increasing hemoglobin stability and in increasing circulating hemoglobin levels.

[0036] The object of the present invention is described as a pharmaceutical composition characterized in that the composition is in the form of a suspension, a foaming or non-foaming solution, an oil-in-water (O / W) or water-in-oil (W / O) emulsion, a multiple emulsion (oil-in-water-in-oil O / W / O, water-in-oil-in-water W / O / W), a microemulsion, a twin-phase emulsion, a PIT emulsion (emulsion obtained by phase inversion at temperature), a nanoemulsion, a pseudoemulsion (stable dispersion of two immiscible phases by means of a gelling agent), an aqueous gel, a fatty gel, a hydroalcoholic gel, a fatty phase, a gel, a powder, or a lyophilized emulsion, and may be in the form of, but not limited to, creams, liquids, pastes, inhalers, lotions, emulsions, gels, syrups, solids, syrups, powders, masks, sticks, tablets, sprays, aerosols, capsules, oils, or a ready-to-drink beverage. to employment.

[0037] We will now present, by way of example, some beneficial effects of the composition, clearly demonstrating the advantage of combining theanine with several types of polysaccharides and metabolites, without these examples being exhaustive. For the sake of clarity in the explanations that follow, it has been decided to call BIOMATCHA a pharmaceutical composition according to the invention intended to modulate the pulmonary microbiota and the intensity of the immune system response. EXAMPLES

[0038] Example 1: Modulation of the intestinal and pulmonary microbiota and modulation of inflammation

[0039] A double-blind clinical study was conducted to evaluate the effects of ingesting a dietary supplement containing BIOMATCHA on the composition of the intestinal and pulmonary microbiota. Forty healthy volunteers of both sexes, with no known medical conditions, were randomly assigned to two groups of 20 people. The first group ingested a placebo, while the second group ingested 600 mg of BIOMATCHA daily for four weeks.

[0040] Samples of the lung microbiota were collected from the nasopharyngeal tract by swab collection at the beginning and end of the study. Stool samples were also collected at the beginning and end of the study.

[0041] The diversity of microbial strains is analyzed in the aforementioned samples.

[0042] Blood samples were also taken at the beginning and end of the study. The levels of the inflammatory markers interleukin IL-6 and C-reactive protein Hs-CRP, as well as the white blood cell count, were measured.

[0043] The results of the lung microbiota are shown in Table 1 below.

[0044] [Tables 1] PLACEBO GROUP BIOMATCH GROUP A Strain Diversity +0.4% +8.9% Porphyromonas spp +0.1% +13.3% Pseudomonas spp +0.2% -5.5% Staphylococcus -0.1% -7.1%

[0045] The results of the intestinal microbiota sampled from the feces are shown in Table 2 below.

[0046] [Tables2] PLACEBO GROUP BIOMATCH GROUP A Diversity of strains +0.5% +10.1% B. longum +0.2% +21.7% L.acidophilus +0.3% +19.4% L.plantarum +0.2% +22.4% Roseburia intestinalis -0.1% +7.9% Faecalibacterium prausnitzii +0.0% +16.6% Akkermansia muciniphila +0.2% +8.2%

[0047] The results of the inflammatory marker analysis are shown in the table below.

[0048] [Tables3] PLACEBO GROUP BIOMATCH GROUP A IL-6 (pg / mL) +0.03 -0.26 Hs-CRP (mg / L) +0.02 -0.93 White blood cells (10³ / mm³) -0.3% +21.4%

[0049] It is observed that in the BIOMATCHA group, there is a modulation of the pulmonary microbiota, a decrease in the inflammatory markers IL-6 and Hs-CRp, as well as an increase in the white blood cell count. No significant change is observed in the placebo group. This demonstrates that the BIOMATCHA group is effective in modulating the lung microbiota, as well as in modulating inflammation and white blood cell count.

[0050] Example 2: Enhancement of nitric oxide production by endothelial cells

[0051] Cultured bovine aortic endothelial cells were treated with 25, 50 or 100 ql of BIOMATCHA. The activity of nitric oxide synthase eNOS, which is the enzyme that synthesizes nitric oxide (NO), is measured by the formation of L-[3H]citrulline.

[0052] The results are shown in Table 4 below.

[0053] [Tables4] Control Conventional Green Tea Extract BIOMATCHA 25 50 100 25 50 100 eNOS Activity (% of control) 100 105.1 138.5 271.8 112.8 156.6 350.8

[0054] The results show that conventional green tea extract and BIOMATCHA stimulate eNOS activity and thus stimulate nitric oxide (NO) production. It is noted that BIOMATCHA has significantly greater activity compared to conventional green tea extract. This demonstrates that the BIOMATCHA group is effective in increasing nitric oxide production.

[0055] Example 3: Improvement of hemoglobin stability and circulating hemoglobin levels

[0056] A double-blind clinical study was conducted to evaluate the effects of ingesting a dietary supplement containing BIOMATCHA on circulating hemoglobin levels. Forty hemodialysis patients with functional iron deficiency were randomly assigned to two groups of 20 people. The first group ingested a placebo, while the second group ingested a dietary supplement containing 600 mg of BIOMATCHA daily for four weeks.

[0057] The results are shown in Table 5 below.

[0058] [Tables5] PLACEBO BIOMATCHA Week 0 Week 4 Week 0 Week 4 Hb (g / dL) 10.4 ± 0.3 10.4 ± 0.5 10.5 ± 0.5 11.0 + 0.6

[0059] The results show that the consumption of BIOMATCHA increases the circulating hemoglobin level. The group consuming a placebo showed no change in the hemoglobin level.

[0060] Example 4: Improvement of hemoglobin stability

[0061] An in-vitro study on the stability of hemoglobin was carried out.

[0062] A solution of human hemoglobin is poured into a container containing a buffer solution heated to 60°C, with or without BIOMATCHA. The hemoglobin concentration and half-life (the time taken for the hemoglobin in the container to decrease by half in concentration) are analyzed regularly.

[0063] The results are presented in Table 6 below.

[0064] [Tableauxô] BIOMATCHA concentration 1 5 10 tBIOMATCHA / to 2.5 12.8 40.2

[0065] The results show that incubation with BIOMATCHA increases the half-life of hemoglobin. At the maximum concentration, the half-life of hemoglobin in the presence of BIOMATCHA (tBiOMA t cha) is 40 times greater than the half-life of hemoglobin in the absence of BIOMATCHA (t0).

[0066] Example 5: Dietary supplement to modulate the intestinal microbiota

[0067] -BIOMATCHA........................................................150 mg

[0068] - Maltitol, microcrystalline cellulose, silicon dioxide, stearate calcium........................................................150 mg

[0069] This food supplement should be taken twice a day. Users have noticed an improvement in intestinal problems, diarrhea, constipation, or digestive discomfort.

Claims

Demands

1. Pharmaceutical composition intended to modulate the pulmonary microbiota comprising a combination of bioactives including theanine and polysaccharides extracted from matcha leaves Camellia sinensis, and at least 2 metabolites selected from the group: lobelin, biotin, catechin, pyridoxine, syringic acid, butyrate, propionate, zinc, saikosaponin, acacetin, saponarin, said at least 2 metabolites being obtained from the fermentation of matcha leaves Camellia sinensis by a bacterium Bacillus subtilis at a temperature between 30°C and 45°C and between 2 and 36 hours followed by a liquid chromatography filtration step; characterized as comprising zinc at a concentration of at least 0.5% and preferably at least 2% and by weight relative to the total weight of bioactives in said composition of polysaccharides at a concentration of at least 0.5% and preferably at least 5%.

2. Pharmaceutical composition according to claim 1, characterized in that the composition is in the form of a lyophilized powder or emulsion, and preferably in the form of creams, liquids, pastes, inhalers, lotions, emulsions, gels, syrups, solids, syrup, powders, masks, sticks, tablets, sprays, aerosols, capsules, oils, or ready-to-use drink.

3. Pharmaceutical composition according to claim 1 or claim 2, for use in the treatment or prevention of dysbiosis, preferably pulmonary dysbiosis.

4. Pharmaceutical composition according to claim 1 or claim 2, for its use in increasing the stability of hemoglobin in the blood.