The present invention relates to formulations for oral use in various pharmaceutical forms (capsules, single-dose vials, tablets and powder) containing probiotic strains, beta-glucans and/or plant extracts, vitamins and minerals, and the use thereof as immunomodulators, in particular for use in the prevention of viral and/or bacterial infections, in particular of the respiratory tract.

State of the art

The immune system is a complex integrated network of chemical and cell mediators, biological structures and processes, developed in the course of evolution to defend the body against all forms of chemical, trauma- or infection-related damage.

The immune system performs three major functions: it protects the body against pathogens; it removes damaged or dead cells and tissues and old red blood cells; it recognises and removes abnormal cells, such as neoplastic cells;

The set of immune reactions that do not retain any memory of the pathogenic attack is defined as innate, natural or aspecific immunity. Chemical and cell mediators responsible for the first line of defense are involved, such as

• neutrophils, monocytes and tissue macrophages, which phagocyte pathogens;

• complement proteins, cytokines and interferon, and major histocompatibility complex.

The set of immune reactions which, after producing a specific response to a given antigen, create memory cells, is called acquired or specific immunity; in this way, a subsequent encounter with the same antigen triggers a faster, stronger reaction than that produced at the first encounter. T and B lymphocytes, natural killer (NK) cells and antibodies are involved.

The cellular components of the immune system responsible for innate immunity are present in all tissues, whereas the cellular components of the immune system responsible for adaptive immunity are mainly present in lymphoid tissues.

The intestine produces two-thirds of the immune system cells, and is closely associated with the other elements of the defensive apparatus located throughout the body.

If the microbiota (i.e. the set of organisms that populate the intestine) is rich in beneficial bacteria, it can react better to aggression by undesirable agents such as germs, rhinoviruses, influenza and parainfluenza viruses.

In the market of products that improve general well-being and therefore also have a favourable impact on the immune function, four different types of product are available on the market in general:

• vitamins and minerals, either individually or combined, for the treatment of primary multivitamin deficiency (caused by an insufficient dietary intake of vitamins) and secondary multivitamin deficiency (caused by disorders that prevent correct use of vitamins by the body);

• botanical extracts, either individually or combined, food supplements to prepare the body in advance to deal with winter illnesses by supporting the normal function of the immune system;

• probiotic mixtures to balance the intestinal bacterial flora;

• and some combinations of the mentioned active ingredients.

Although mentioned products can be useful to support the immune defenses, they have the following limitations: they are based on individual types of active ingredients or combinations of some of the active ingredients listed, which act on some of the symptoms or aspects of the disorder; they possess low stability because probiotic mixtures have a high tendency to degrade; they are not effective in a single administration, but require multiple daily administrations; pre-clinical studies supporting safety and efficacy of the formulations already present on the market are limited or often absent; they have limited efficacy, and do not provide support to the whole immune system.

The pharmaceutical formulations according to the present invention overcome the mentioned limitations, providing a synergic combination of specific probiotic strains and functional ingredients for immune regulation.

The formulations according to the invention are useful as immune system stimulating and regulating agents, with a more complete action and greater efficacy than is already known on specific population targets, in particular adult and paediatric targets, on which they have been clinically tested.

An immunomodulator is a therapeutic (chemical or biological) agent designed to regulate alterations in the body’s immune responses, attenuating or activating them, by means of interaction on the effector cells (e.g. macrophages, dendritic cells) and on cells that stimulate or inhibit antibody production (B and T lymphocytes).

The probiotic strains present in the formulation according to the present invention are as follows:

* Bifidobacterium lactis Bl-04®. Bifidobacteria are one of the main groups of probiotics that form the intestinal microflora of adults. Bifidobacterium lactis, due to its particular resistance to the action of bile and its ability to tolerate oxygen, is able to colonise the upper part of the small intestine.

• Lactobacillus rhamnosus CRL1505, which exhibits a specific action on the immune function of adults and children and adheres to the gastrointestinal mucosa; it is also resistant to gastric acidity and bile salts, due to its ability to produce anti-stress proteins that provide a greater chance to surviveduring intestinal transit after oral administration. Description of the invention

The object of the present invention is therefore a pharmaceutical formulation containing: a) at least 10 ⁹ CFU per daily dose of a probiotic microorganism selected from Bifidobacterium lactis and Lactobacillus rhamnosus b) 10% to 90% by weight of the total weight of the formulation of a yeast extract containing beta-glucans ranging from 20% to 40% by weight of the total weight of the extract, and/or 1% to 8% by weight of the total weight of the formulation of a plant extract selected among Sambucus nigra L., Malpighia glabra L., Astragalus membranaceus Moench, Uncaria tomentosa and a mixture of extracts of the mentioned plants; c) one or more vitamins selected among vitamin B complex, vitamin D and vitamin C, and d) a mineral selected among selenium and zinc or a mixture thereof.

The probiotic microorganism is preferably present in the formulation in an amount ranging between 10 ⁹ and 10 ¹⁰ CFU per daily dose.

The beta-glucans are isolated from cereals such as oats, barley, wheat, rye, com, quinoa, millet, buckwheat, rice, wild rice and spelt, or from yeasts or fungi. A yeast extract, preferably isolated from Saccharomyces cerevisiae, containing 20% to 40% of beta-glucans compared with the total weight of the extract, is used in the present invention.

The formulations according to the invention can contain, in addition to or as an alternative to beta-glucans, extracts of one or more plants selected among Sambucus nigra L., Malpighia glabra L., Astragalus membranaceus Moench and Uncaria tomentosa (Willd. ex Schult.) DC or mixtures of extracts of the mentioned plants.

In particular, extracts of Sambucus nigra L. and Malpighia glabra L. (also called acerola or Barbados cherry) are preferred.

The extracts can be dried extracts or liquids, such as alcoholic, hydroalcoholic, or glycerin extracts.

Sambucus nigra L. extract is preferably as glycerin extract obtained by cold maceration in a solution of glycerin and water in percentages ranging from 1 to 80%, with a drug:extract ratio (DER) ranging from 1:1 to 1:5.

The Malpighia glabra L. extract is preferably as dried extract obtained by aqueous extraction with a drug: extract ratio (DER) ranging from 1 : 1 to 1 : 10.

The plant extract or mixture of plant extracts is preferably present in an amount ranging between 1.5% and 6% by weight of the total weight of the formulation.

The pharmaceutical formulation according to the invention contains a vitamin selected among the vitamin B complex, such as vitamin B6 or B12 or a mixture thereof, a D group vitamin, such as vitamin D3, and vitamin C or a mixture of the mentioned vitamins.

The formulation also contains a mineral selected among zinc and selenium, preferably with a minimum content of 15% of the Nutrient Reference Values (NRV), i.e. 1.5 mg for zinc and 8.25 pg for selenium.

The mentioned formulation can contain only zinc, only selenium, or a mixture of both.

A prebiotic agent selected among inulin, fructooligosaccharides, galacto- oligosaccharides or a mixture thereof can also be present in the formulation.

The pharmaceutical formulation according to the invention can also contain a carrier, diluent, anticaking agent, preservative, antioxidant, buffer, pH adjuster or flavouring agent, or mixtures thereof.

The pharmaceutical formulation can have the delivery form of capsules, powder, tablets, or single-dose vials for oral use.

The pharmaceutical formulations according to the invention are useful as immunomodulators .

“Immunomodulators” here means medicaments that stimulate or reinforce the immune response. The formulations according to the invention can be used as immunomodulators in the prevention of diseases or disorders such as viral and/or bacterial infections, in particular of the respiratory tract, in both paediatric and adult populations.

A preferred embodiment according to the invention (Formulation A) contains: a) 10 ⁹ CFU to 10 ¹⁰ CFU of Bifidobacterium lactis; b) 100 mg to 800 mg, preferably 130 mg to 500 mg, of Saccharomyces cerevisiae extract containing 20-40% by weight of beta-glucans compared with the total weight of the extract; c) 0.001 mg to 10 mg of one or more B complex vitamins d) 1.5 mg to 15 mg of zinc; e) 8.25 pg to 100 pg of selenium;

A further preferred embodiment of the invention (Formulation B) is a formulation containing: a) 10 ⁹ CFU to 10 ¹⁰ CFU of Lactobacillus rhamnosus ; b) 100 to 1000 mg of glycerin extract of Sambucus nigra L. c) 0.001 mg to 10 mg of one or more B complex vitamins d) 0.75 pg to 50 pg of vitamin D e) dried extract of Malpighia glabra L containing 12 mg to 1000 mg of vitamin C; f) 1.5 mg to 15 mg of zinc.

The formulations according to the invention exhibit a synergic action on components of the different cells responsible for activation of the immune system. The formulations are designed to modulate the innate and adaptive immune response in different population targets (adult and paediatric).

Some examples of formulations according to the invention are shown below. Example 1 - capsule or powder or tablet

Example 2 - capsule or powder or tablet

Example 3 - capsule or powder or tablet

In vitro evaluation of immunomodulating activity on human cells

Experimental protocol

Screening tests were conducted on samples to establish their activity on both the innate monocyte component and the adaptive component based on the lymphocyte component, using a comparative analysis of the formulations according to the invention compared with the isolated ingredients of the formulation and with products already present on the market.

The effectiveness to modulate cytokine release in mixed populations of human Peripheral Blood Mononuclear Cells (PBMC) ex vivo was evaluated by determining the downstream activity of PMBC activation, using the ELISA assay of IL-6, TNF-a, IL-10 and IFN-g.

In particular, tests were conducted on: individual active ingredients according to the invention (selected probiotics,

Sambucus nigra extract, beta-glucan) to demonstrate the synergic effect of the formulation according to the invention; and

• formulation A according to the invention or

• formulation B according to the invention by comparison with products present on the market having a similar formulation (mixtures of alive probiotics, combined with vitamins and minerals and botanical extracts and/or beta-glucans), demonstrating the superiority and innovativenessof the formulation compared with already known products.

The experimental procedure is described in detail below.

Isolation of peripheral blood lympho-monocyte cells (PBMCs)

The material required for the separation and count of the PBMCs was purchased from Sarstedt, Verona. The RPMI 1640 medium and the antibiotics streptomycin and penicillin were purchased from Merck, Milan.

The following operating steps were performed to isolate the PBMCs:

• Fresh blood was drawn from healthy volunteers and placed in blood count test tubes (sol. EDTA 80 pl/3 ml of blood).

• The blood was centrifuged with Histopaque 1.077 (Merck, Milan) in the proportion of 5 (blood):3 (Histopaque) for 15 minutes at 2700 rpm.

The PBMC ring was recovered, dissolved and made up to volume with saline solution (15 ml) in a sterile test tube. • Two washes were performed with sterile saline solution, followed by centrifugation at 2300 rpm for 10 minutes and re- suspension, after which the PBMCs were inoculated into 1 ml of RPMI 1640 medium.

• The cells were counted with Trypan Blue in a Biirker cytometric chamber.

• Finally, the cells were re-suspended in a medium consisting of 99% RPMI, 0.5% of solution containing the antibiotics penicillin and streptomycin; and 0.5% glutamine 200 nM.

Treatment of PBMC with the samples under study

PBMCs, at the density of lxlO ⁶ in 1 ml in RPMI, were placed in 24-well plates. The PBMCs were treated immediately. RPMI was inserted in triplicate into control wells (CTRL), while samples at different concentrations were inserted into the sets of stimulated cells. The experiments were conducted at two different stimulation times: 6 and 24 hours. Salmonella enteriditis (Merck, Milan), 100 ng/ml, was used as positive control.

Cytokine assay

ELISA kits sold by AbCam were used for the TNF-a, IL-2, IL-6, IL-10 and IFN-g assays. The Perkin Elmer Nivo 3s was used as plate reader.

The assays were conducted in duplicate on independent triplicates of the cell groups (n=6).

“Non-competitive sandwich” ELISA tests were used for the assays.

The samples, coincubated with the test substances, were subjected to three freezing and thawing cycles: -80°C to +20°C.

The samples were analysed in strict accordance with the protocols illustrated in the data sheets supplied with the kits:

• 100 mΐ of capture antibody was added to the coating buffer in each well and left under incubation overnight at 2-8°C.

• The wells were washed 4 times with the washing liquid. was added to all wells.

• 100 mΐ of solution containing the standard protein was added to two columns of wells (1 and 2), and progressive dilutions were performed (wells A to G) for the calibration curve; the standards at concentration 0 were added to wells 1H and 2H.

• 100 mΐ of the various test samples was added to all other wells.

• 100 mΐ of biotinylated detection antibody was added in assay diluent to all wells, and incubated at room temperature for two hours.

• The wells were washed 4 times with the washing liquid.

• 100 mΐ of peroxidase conjugate in assay diluent was added to each well, and incubated at room temperature for 1 hour.

• The wells were washed 5 times, immersed for 30 seconds to 1 minute per well.

• 100 mΐ of TMB substrate (tetramethylbenzidine and hydrogen peroxide) was added to all wells, and incubated in the dark for 15 minutes. e 100 mΐ of stop solution consisting of 2N sulphuric acid was added to each well.

• The plate was inserted in the microplate reader and the absorbance read at 450 nm.

The statistical difference in the test results among the samples was determined by analysis of variance (ANOVA) followed by Student’s T test. Values with p<0.05 vs. the reference were considered statistically significant. The graphs and calculations were performed with the GraphPrism® program and Microsoft Excel.

Experimental results for the solid pharmaceutical form of Example 1:

The solid formulation demonstrated to induce a major immune response by activating IL6 and IL10 release on the PBMC cells (25 mcg/mL). The results are shown in Figures 1 and 2.

The probiotic component showed to have a high immunogenic activity, as demonstrated by the induction of TNF-a, IL-10 and IL-6 release (PBMC, 25 mcg/mL).

The formulation of Example 1 showed to perform an important synergic action among its ingredients, as demonstrated by the activation of IL-2 and IFN-g release (PBMC, 25 mcg/mL, 24h) compared with beta-glucan alone (yeast extract containing 20- 40% by weight of beta-glucans, contained in the formulation of Example 1 , tested as single, pure ingredient), which represents the majority ingredient by weight (>75%). The results are shown in Figure 3.

The comparative test conducted by comparison with formulations present on the market having similar compositions demonstrated that the formulation according to the invention was more effective in modulating immune parameters in the testconducted on PBMC cells, thus proving that it is an innovative product compared with the following competitors.

Competitor Composition 1: Lactobacillus rhamnosus GG-ATCC 53103, fructooligosaccharides (FOS), vitamin C, dried extract of shiitake mushroom (including beta-glucan (lentinan)).

Competitor Composition 2: Boswellia ( Boswellia serrata roxb.) dried gum resin extract, grapefruit ( Citrus x paradisi macfad.) dried seed extract, Biosterine®: holy basil (Ocimum sanctum /.) herb and sage (Salvia officinalis /.) dried leaf extract 40% rosmarinic acid, cat’s claw ( Uncaria tomentosa Wild dc.) dried bark extract.

Competitor Composition 3: Acacia honey, water, elder (Sambucus nigra ) fruit juice concentrate, mallow (Malva sylvestris ) freeze-dried leaf mucilage, pale purple coneflower (Echinacea pallida) multifraction freeze-dried root extract (Echina2-LMF).

The results of the test are shown in Figures 4 and 5.

Summarizing, none of the competitor products exhibited the same ability to overregulate the cytokines produced by the PBMCs as the formulation to which the present patent relates (25 mcg/mL). Only Competitor 3 exhibits cytokine release greater than that of the control and similar to that of the formulation of Example 1, but with the following quantitative differences: TNF-a: +12% vs +51% for the formulation of Example 1; IL-6: +25% vs +248% for the formulation of Example 1; IL-10: +46% vs +116% for the formulation of Example 1 (pO.OOl Competitor 3 vs. the formulation of Example 1).

Concluding, therefore, the formulation of Example 1 exhibited an interesting effectiveness to regulate the production and release of pro-inflammatory cytokines like TNF-a and IL-6, and of the regulatory cytokine IL-10, on the ex vivo population of human PBMCs. This action showed superiority related to the activity of the products currently present on the market used as comparators.

The formulation of Example 1 therefore proved to modulate the innate and adaptive components of the immune response, demonstrating that it is a suitable agent for preventive use, to strengthen the immune defenses.

Experimental results for the pharmaceutical form of Example 4 in vials:

The product of Example 4 proved to activate the immune response, inducing increased cytokine release (TNFa, IL-10, IL-6 and IL-2, 1 mcg/mL). The results are shown in Figure 6.

Moreover, the composition of Example 4 at 25 mcg/ml produced an increased release of 11-6 amounting to +39% vs. Ctrl and of IL-10 amounting to +45% vs. Ctrl (data not shown in Figure 6).

The synergy expressed by the product according to the invention is demonstrated by the improved response induced by the formulation according to the invention compared with the response of the glycerin extract of elder alone (as present in the formulation of Example 4, tested as pure single ingredient). The results are shown in Figure 7.

The product of Example 4 was also compared with Competitor 3, the composition whereof is described above, and with Competitor 1 and Competitor 2, having the following compositions:

Competitor 1: Lactobacillus rhamnosus GG-ATCC 53103 with vitamin D, zinc, Echinacea extract and beta-glucan. Competitor 2: Resveratrol from Polygonum cuspidatum titrated to 98%, beta 1.3/1.6 glucan to 75%, L-arginine, zinc and folic acid.

The results are shown in Figure 8.

A similar modulation of IL-10 is observed, but there was a greater effect of the formulation of Example 4 on TNF-a and IL-6. Competitor 2 did not produce any significant modulation of cytokine expression.