Use of superoxide dismutase for the treatment and / or prevention of allergic asthma - Patent Application 20070233633

Abstract

Use of superoxide dismutase for the treatment and / or prevention of allergic asthma in a mammal, wherein the superoxide dismutase is administered orally alone or, optionally, in combination with administration of an aerosol preparation comprising at least one beta-2 adrenergic receptor agonist.

Description

[Technical field]

[0001] The present invention relates to the use of superoxide dismutase in the treatment and / or prevention of allergic asthma. [Background technology]

[0002] Superoxide dismutase (E.Cl.15.1.1.), also called SOD for short, comprises a family of universal metalloenzymes involved in the dismutation of the superoxide (02-) anion. Accumulation, or excessive production, of superoxide radical species in vivo is detrimental to most organisms. The physiological state in which such a condition occurs is known as oxidative stress, which is associated with many metabolic disorders and is itself involved in various pathological processes, such as carcinogenesis, atherosclerosis, aging, and inflammatory diseases such as celiac disease. Apart from the degree of evolution or subcellular localization, SOD exists in three main forms, differentiated by the metal ion contained within the molecule: copper-zinc or CuZn-SOD, manganese or Mn-SOD, and iron or Fe-SOD. CuZn-SOD and Mn-SOD are also found in more specialized cellular structures, such as peroxisomes or chloroplasts in plants, while in mammals, extracellular CuZn-SOD or Ec-SOD is specifically present in the extracellular compartment. Many experiments have shown that even if all SODs have the same basic antiradical activity, their effective biological activity is not identical with respect to a particular therapeutic indication and target organism. This has also been shown to be true with respect to the difference in activity between homologous and heterologous SODs. In this specification, the expression "homologous SOD" refers to SOD that is naturally produced by cells of the species under investigation. The expression "heterologous SOD" refers to SOD of foreign origin, produced by or derived from a species different from the species under investigation. For example, the anti-inflammatory activity of homologous SOD in carrageenan- or adriamycin-induced paw edema in rats is not functional compared to the anti-inflammatory activity of SOD of heterologous origin. The effectiveness of homologous SOD appears to depend not on the type of metal present in the active site or the molecular weight of the entire enzyme, but on changes, even subtle changes, in the amino acids of the enzyme.

[0003] A common method of administering SOD is orally, where it is often prepared as a solid dosage preparation and provided as a tablet or ingestible capsule. However, oral administration of SOD can be problematic in that SOD is rapidly degraded in the digestive tract, thus reducing its bioavailability and efficacy. This problem is further complicated by the difficulty of delivering SOD to the specific cellular location where it is most effective. One known commercial development that addresses the issue of in vivo digestive stability of orally administered SOD is a solid dosage product called Glisodin® (Isocell, France), which contains plant-derived SOD combined with prolamins obtained from wheat, specifically the prolamines known as gliadin. Glisodin® is currently known and marketed as a dietary supplement for people suffering from skin depigmentation problems such as vitiligo, and as a skin treatment and cosmetic agent for overall human health, as well as anti-aging. Many other studies have also reported the following effects of oral administration of Glisodin®:

[0004] Suppression of oxidative stress: Vouldoukis et al., “Supplementation with gliadin-combined plant superoxide dismutase extract promotes antioxidant defences and protects against oxidative stress”, Phytotherapy Research, 2004, 18(12), 957-962;

[0005] Nakajima et al., “Oral supplementation with melon superoxide dismutase extract promotes antioxidant defences in the brain and prevents stress-induced impairment of spatial memory”, published in Behavioral Brain Research, 2009, 200(1), 15-21;

[0006] C. Muth et al., “Influence of an orally effective SOD on hyperbaric, oxygen related cell damage” University Medical School, Ulm ,Free Radical Research, 2004;

[0007] Suppression of UV-induced oxidative stress: Mac-Mary et al, “Could a photobiological test be a suitable method to assess the anti-oxidant effect of a nutritional supplement (GliSODin)?”, European Journal of Dermatology, 2007, 17(3), 254-255);

[0008] Promotes immune regulation: Okada et al., “Prevention of inflammation-mediated acquisition of metastatic properties of benign mouse fibrosarcoma cells by administration of an orally available superoxide dismutase”, British Journal of Cancer, 2006, 94(6), 854-862);

[0009] Suppressing vascular inflammation: M. Cloarec, and al., “GliSODin, a vegetal SOD with Gliadin, as a preventative agent vs. atherosclerosis, as confirmed with carotid Ultrasound-B Imaging” American Hospital, Paris, European Annuals of Allergy & Clinical Immunology, 2007;

[0010] Asthma is a chronic medical condition affecting the lower airways and manifested as inflammation, bronchoconstriction, and bronchial hypersecretion. Over 300 million people worldwide are thought to have asthma, with approximately 70% of these people estimated to have allergic asthma.

[0011] Allergic asthma is characterized by an abnormal response of the immune system to inhaled particles such as those formed by dust mites, animal debris and detritus, fungal spores, and plant particles such as pollen. For the majority of people, these particles are recognized and tolerated by the immune system and do not cause any unfavorable reaction. However, in infected subjects, these particles induce a T helper 2 cell (Th2) immune response, followed by the production of IgE immunoglobulins, which in turn lead to the degranulation of basophils and mast cells, thereby releasing many inflammatory response mediators such as histamine and leukotrienes. These mediators cause bronchoconstriction and are responsible for asthma attacks. However, other T helper cell populations, such as T helper 17 cells or T regulators, are also known to be involved in asthma. These various immune mechanisms are accompanied by an overproduction of mucus and contraction of smooth muscle structures in the respiratory pathways, causing tissue remodeling of the human airways.

[0012] It is known that there is not just one type of asthma, but that there are many phenotypes in humans, but two main phenotypes can be distinguished: Th2-high and Th2-low. The Th2-high phenotype is characterized by high serum IgE levels and a high proportion of eosinophils. The Th2-low phenotype involves other mediators, such as Th17 cells. Current treatments, for example the use of corticosteroids for less severe forms of asthma, or anti-IgE treatments, such as the monoclonal antibody omalizumab, available for about a decade, or, more recently, the anti-IL-5 monoclonal antibody reslizumab, are by and large able to manage these conditions. However, these products must be administered intravenously and have a number of potentially problematic side effects, including coughing, difficulty swallowing, dizziness, fainting, feeling lightheaded when standing up quickly from a lying or sitting position, fast heart rate, hives, itching, or skin rash, swelling or puffiness of the eyelids or around the eyes, face, lips, or tongue, redness of the skin, chest tightness, and unusual tiredness or weakness.

[0013] Thus, one aspect of the present invention is to provide a less invasive and less problematic alternative treatment for allergic asthma that does not suffer from the drawbacks associated with intravenous administration of other modern therapeutic treatments and that can be optionally and additionally combined, if necessary, with more classical treatment regimens for managing the asthmatic condition.

[0014] Thus, according to one aspect, the present invention relates to the use of superoxide dismutase for the treatment and / or prevention of allergic asthma in a mammal, wherein the superoxide dismutase is administered orally alone or, optionally, in combination with the administration of an aerosol preparation comprising at least one β2 adrenergic receptor agonist.

[0015] According to yet another embodiment, the use of superoxide dismutase for the treatment and / or prevention of allergic asthma relates to the use, wherein the mammal is a human.

[0016] The superoxide dismutase can be selected from those generally known to those skilled in the art and can be selected from the group consisting of human superoxide dismutase, animal superoxide dismutase, bacterial superoxide dismutase, yeast superoxide dismutase and plant superoxide dismutase.However, advantageously, at least one superoxide dismutase is selected from the group consisting of CuZn superoxide dismutase, Mn superoxide dismutase, extracellular superoxide dismutase, Ni superoxide dismutase and Fe superoxide dismutase.According to one embodiment, the superoxide dismutase is a homologous or heterologous superoxide dismutase.

[0017] According to yet another advantageous embodiment, heterologous superoxide dismutases are preferred.

[0018] According to a particularly advantageous embodiment, the superoxide dismutase is a plant-derived superoxide dismutase, and more advantageously a heterologous plant superoxide dismutase. Such superoxide dismutase can be obtained or produced in various ways. For example, at least one superoxide dismutase can be extracted from a plant. When a plant is used for extracting or producing at least one superoxide dismutase, such a plant is advantageously a member selected from the group consisting of plant species of the Cucurbitaceae, Solanaceae, and Tritaceae families, and / or their various cultivars, and is advantageously selected from the group consisting of melon (e.g., Cucumis melo), tomato (e.g., Lycopersicum esculentum), and wheat (e.g., Triticum aestivum, Triticum vulgare, and Triticum durum), etc.

[0019] Among the various plant superoxide dismutases available, according to another embodiment, the superoxide dismutase is selected from the group of plant superoxide dismutases consisting of peroxisomal, chloroplast and cytoplasmic superoxide dismutases.

[0020] According to yet another embodiment, the superoxide dismutase is a plant-derived superoxide dismutase selected from the group consisting of melon superoxide dismutase and wheat superoxide dismutase.

[0021] Such extraction techniques for plant-derived superoxide dismutase are known per se, and corresponding dry extracts, for example from melon or wheat, are commercially available. For example, for superoxide dismutase obtained from a variety of vegetable melon (Cucumis melo L), the superoxide dismutase is usually provided in powder form as a freeze-dried extract of melon juice and is commercially available under the trade name "SOD B®" from Bionov, France. Such powders are generally light orange in color and have a measured SOD enzyme activity of 90,000 U(NBT) / g to 150,000 U(NBT) / g. Similarly, a dry extract of wheat SOD from Triticum vulgare plants is commercially available from Silab under the INCI name "Triticum Vulgare (Wheat) Germ Extract", CAS N° 84012-44-2, said extract being present as a beige lyophilized powder with an SOD enzymatic activity of 1,500,000 U(NBT) / g to 3,000,000 U(NBT) / g. In the present specification, the enzymatic activity of SOD is expressed in units of U(NBT) / g, and the activity is measured according to known NBT analysis protocols, such as those described in Zhou et al, in J. Pharm. Biomed. Anal. 2006, Mar 18; 40(5):1143-8, in Sections 2.1, 2.2.1, 2.3.1, 2.4.2 and 2.4.2.1, the contents of which are incorporated herein by reference.

[0022] According to yet another embodiment, the oral administration of superoxide dismutase consists of a solid preparation comprising plant-derived superoxide dismutase in combination with prolamin. Advantageously, the prolamin can comprise at least one prolamin-based peptide selected from the group consisting of gliadin fragments or derivatives, analogues, salts or metabolites thereof. More advantageously, the prolamin-based peptide fragment is a non-immunogenic analogue of gliadin. In a particularly advantageous embodiment, the prolamin-based peptide fragment is a non-immunogenic analogue of gliadin having competitive inhibitory activity against immunogenic prolamin-based peptides. The at least one prolamin-based peptide fragment can also be suitably selected from the group consisting of fully hydrolyzed, substantially hydrolyzed or slightly hydrolyzed prolamin-based peptide fragments. However, generally, the prolamin is a prolamin fragment selected from the group consisting of those fragments obtained from PTC (pancreatin, trypsin, chymotrypsin) hydrolyzed prolamins that mimic the process of gastrointestinal hydrolysis. According to one particularly advantageous embodiment, the prolamin is a prolamin fragment that has been hydrolyzed to such an extent that it functions as a targeting signal in the intestinal tract.

[0023] Advantageously, according to another embodiment, the oral administration of superoxide dismutase consists in administering a solid preparation comprising plant-derived superoxide dismutase in combination with gliadin, such a product being commercially known as the solid dosage preparation sold under the trade name Glisodin® by Isocell, France.

[0024] According to yet another embodiment, the oral administration of superoxide dismutase consists of a solid preparation comprising: An extract from dried melon juice containing superoxide dismutase at a standardized potency of 0.50% to 5.00% based on the total weight of the solid preparation; a) an extract from dried melon juice containing superoxide dismutase at a standardized potency of 0.50% to 5.00% based on the total weight of the solid preparation; b) 1.50% to 5.00% gliadin, based on the total weight of the preparation; c) qsp maltodextrin; d) The superoxide dismutase has an enzyme activity of 1 to 5 U(NBT) / mg.

[0025] According to another advantageous embodiment, the oral administration of superoxide dismutase consists of a solid preparation consisting of: a) an extract from dried melon juice containing a standardized potency of superoxide dismutase of 1.33% based on the total weight of the solid preparation; b) 3.33% gliadin, relative to the total weight of the preparation; c) qsp maltodextrin; d) Superoxide dismutase has an enzyme activity of 1.5 U(NBT) / mg.

[0026] According to another embodiment, the oral administration of superoxide dismutase consists of a solid preparation comprising: a) 0.04% to 0.40% wheat superoxide dismutase based on the total weight of the preparation; b) 1.50% to 5.00% gliadin, based on the total weight of the preparation; c) qsp maltodextrin; d) The superoxide dismutase has an enzyme activity of 1 to 10 U(NBT) / mg.

[0027] According to yet another embodiment, the oral administration of superoxide dismutase consists of a solid preparation comprising: a) 0.18% superoxide dismutase from wheat based on the total weight of the preparation; b) 3.50% gliadin, based on the total weight of the preparation; c) qsp maltodextrin; d) Superoxide dismutase has an enzyme activity of 4.5 U(NBT) / mg.

[0028] According to yet a further embodiment, the superoxide dismutase is orally administered to a human in an amount of between 100 mg and 2000 mg per day for a minimum of 15 consecutive days.

[0029] According to yet another advantageous embodiment, superoxide dismutase is orally administered to a human in an amount of 500 mg per day for a minimum of 15 days and a maximum of 180 consecutive days.

[0030] As indicated above, according to another embodiment, superoxide dismutase is administered orally, but in combination with the administration of an aerosol preparation that contains at least one β2-adrenergic receptor agonist.Short-term treatment of allergic asthma with β2-adrenergic receptor agonists is known per se.Accordingly, according to another embodiment, the at least one β2-adrenergic receptor agonist is a short-acting β2-adrenergic receptor agonist (also known as SABA for short), selected from the group consisting of bitolterol, fenoterol, isoprenaline, levosalbutamol, orciprenaline, pirbuterol, procaterol, ritodrine, salbutamol and terbutaline.According to another advantageous embodiment, the at least one β2-adrenergic receptor agonist is salbutamol. [Brief description of the drawings]

[0031] The present invention will now be described with reference to the following detailed examples, which serve for non-limiting illustrative purposes of the various aspects presented herein, and with reference to the accompanying drawings, in which: [Figure 1] 1 is a series of graphs plotting the results of an in vitro mixed lymphocyte reaction assay in naive control and asthmatic mice with and without a Glisodin formulation as described herein. [Diagram 2]1 is a series of graphs plotting the results of lymphocyte reactivation and proliferation in an adoptive transfer model of allergic asthma with or without a Glisodin formulation as described herein. [Diagram 3] 3 is a series of graphs plotting the results of an analysis of subpopulations of CD4+ T lymphocytes expanded in the adoptive transfer study whose results are shown in FIG. 2. [Figure 4] 1 is a series of graphs plotting total immunoglobulin production induced by house dust mite (HDM) allergic conditions in mice with and without a Glisodin formulation described herein. [Diagram 5] 1 is a series of graphs plotting allergy-specific immunoglobulin production induced by house dust mite (HDM) allergic conditions in mice with and without a Glisodin formulation described herein. [Figure 6] 1 is a series of graphs plotting chemoattractant assays in house dust mite (HDM) allergy-challenged mice with and without a Glisodin formulation as described herein. [Figure 7] 1 is a series of graphs plotting respiratory function in house dust mite (HDM) allergy-challenged mice with and without a Glisodin formulation as described herein. [Figure 8] 1 is a series of graphs plotting respiratory function in house dust mite (HDM) allergy-challenged mice with and without co-administration of salbutamol, with and without a Glisodin formulation as described herein. [Figure 9] 1 is a series of graphs plotting cell populations analyzed using flow cytometry from bronchoalveolar lavage fluid (BAL) of house dust mite (HDM) allergy-induced mice with or without Glisodin formulations described herein, with or without co-administration of salbutamol. [Figure 10] 1 is a series of graphs plotting the immune response of house dust mite (HDM) allergy-challenged mice with and without the co-administration of salbutamol in the presence or absence of a Glisodin formulation described herein. [Figure 11] 1 is a series of graphs plotting serum IgE levels in house dust mite (HDM) allergy-challenged mice with and without co-administration of salbutamol in the presence or absence of a Glisodin formulation described herein. [Figure 12] 1 is a series of graphs plotting cytokine responses in house dust mite (HDM) allergy-challenged mice with and without the co-administration of salbutamol in the presence or absence of a Glisodin formulation described herein. [Figure 13] 1 is a series of histological stainings of lung samples taken from house dust mite (HDM) allergy-challenged mice with or without the Glisodin formulation described herein, with or without co-administration of salbutamol. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032] Several experiments were conducted to demonstrate the effects of administering plant-derived SOD to mammals. For purposes of experimental evaluation, the following Glisodin® formulations were prepared: [Table 1]

[0033] Experimental protocol The experimental evaluation focused on the following areas: 1) The in vitro effect of Glisodin® formulations on lymphocyte reactivation in a model of allergic asthma, also known as the mixed lymphocyte reaction or MLR; 2) the in vivo effect of Glisodin® formulations on lymphocyte reactivation in an allergic asthma model, also known as the adoptive transfer model or AT; 3) the effect of Glisodin® formulations on humoral responses and the production of chemoattractant molecules at the molecular scale; 4) analysis of respiratory function in mice administered Glisodin® formulations and / or treated with salbutamol; 5) Analysis of the anti-inflammatory effect on mice treated with either Glisodin® and / or salbutamol. 6) Analysis of the comparative immune responses of mice treated with either Glisodin® and / or Salbutamol; 7) Comparative analysis of humoral responses between mice treated with either Glisodin® and / or Salbutamol; 8) Comparative analysis of cytokine responses between mice treated with either Glisodin® and / or Salbutamol; 9) Comparative histological analysis between mice treated with either Glisodin® and / or Salbutamol.

[0034] A mouse model of allergic asthma involving exposure to house dust mites was used. This model consisted of a preliminary sensitization challenge phase in which Balb / cj mice were administered whole house dust mite extracts percutaneously (days 0, 7, 14, and 21), followed by two intranasal provocations (days 27 and 34), which induce allergy-induced asthma symptoms in the test subjects. The efficacy of the Glisodin formulation was evaluated by force-feeding the mice with 5 mg of the Glisodin formulation diluted in water daily for 5 consecutive days each week for 5 weeks, starting 24 hours after the first intranasal sensitization challenge. For comparison, the same protocol was used to evaluate the effect of combined administration of Glisodin and salbutamol, in which salbutamol was administered as a nebulized aerosol in a solution concentration of 5 mg / ml for 30 seconds, 1 hour before each sensitization challenge.

[0035] Part 1 - In vitro mixed lymphocyte reaction (MLR) In part 1 of the analysis, i.e. in vitro MLR, dendritic cells from 6-week-old Balb / cj mice were differentiated from tibial bone marrow stem cells, i.e. bone marrow derived cells (also known as BMDCs). Similarly, CD4+ and CD8+ (LT) lymphocytes were isolated from the spleens of the same mice and enriched using negative selection. Lymphocytes from asthmatic mice were reactivated using dendritic cells loaded with an allergen, i.e. house dust mite. To measure the effect of Glisodin® formulations on the interaction of T lymphocytes with BMDCs, co-cultures of BMDCs and LTs were performed with and without Glisodin® formulations. After coculture, T lymphocytes were subjected to phenotypic analysis by flow cytometry using the following markers: IFN-FITC, Foxp3-Pe, CD4-PercP5.5, IL-4-APC, CD8-APC-H7, IL-17-Brilliant Violet 421, and CD25-Brilliant Violet 510.

[0036] The results are shown in Figure 1. Lymphocyte differentiation was measured in naive mice (CTL = control, white and dark grey) or asthmatic mice (FA, light grey and black histograms) from autologous dendritic cells loaded with human dust mites in the presence or absence of melon Glisodin (see Figures 1A and 1D) or wheat Glisodin (see Figures 1E and 1H). CD4+ lymphocyte differentiation was also measured by population discrimination: Th1 (Figures 1A and 1E), Th2 (Figures 1B and 1F), Th17 (Figures 1C and 1G), and Treg (Figures 1D and 1H). Applying ANOVA analysis with Tukey's test, confidence intervals were obtained as shown in the figures: *=p<0.05, **=p<0.01, ***=p<0.001 (mouse populations n=6-8 per group).

[0037] Regarding Th1 lymphocytes, characterized by the secretion of gamma-interferon (γ-IFN), no changes in differentiation were observed when comparing cells from naive mice with those from asthmatic mice, nor was there any notable effect of the presence or absence of the Glisodin formulation (see Figures 1A and 1E), and the profile remained ostensibly the same. These results were expected, given that Th1 responses are primarily involved in antibacterial and antiviral immune responses and are hardly involved in allergic responses. In contrast, Th2 lymphocytes are primarily involved in antiparasitic and antiallergic responses. A marked differentiation was observed for Th2 lymphocytes from asthmatic mice compared to naive mice, with differentiation increasing on the order of 100-700% (see Figures 1B and 1F, white and light gray histograms). What is particularly interesting here is that no differentiation was observed in the presence of the Glisodin formulation, suggesting that allergy-induced Th2 differentiation is inhibited by the Glisodin formulation (see Figures 1B and 1F, dark gray and black histograms). Moreover, the presence of Glisodin preparations does not affect the differentiation of Th2 lymphocytes in naive mice. Similarly, the differentiation of Th17 lymphocytes, involved in inflammatory responses, is increased 3-5 times in asthmatic mice compared to naive mice (see Figures 1C and 1G). Of particular note here is the fact that the presence of either wheat Glisodin or melon Glisodin also inhibits the differentiation of Th17 lymphocytes, although not as much as in naive mice (see Figures 1C and 1G). It should also be noted that these lymphocyte responses are controlled by T regulatory lymphocytes (Tregs), whose differentiation is characterized by high expression of CD25 and the transcription factor FoxP3 (see Figures 1D and 1H). As expected, a reduction in the number of differentiated Tregs is observed in asthmatic mice compared to naive mice. Nevertheless, here again, the presence of Glisodin increases the frequency of Treg differentiation in both naive and asthmatic mice compared to asthmatic mice (see Figures 1D and 1H). In summary, these results indicate that both wheat and melon Glisodin do not appear to affect Th1 differentiation, but inhibit the differentiation of Th2 and Th17 lymphocytes in an allergic environment.In contrast, the presence of Glisodin can be seen to increase the differentiation of Treg lymphocytes, regardless of the presence or absence of an allergic environment.

[0038] Further analysis included determining the activation of CD8+ lymphocytes by measuring their ability to produce y-IFN, known to be capable of maintaining lung inflammation in allergy-induced asthma. The results obtained showed that there was no statistically significant difference between the two types of mice, nor in the type of plant-derived SOD used in the Glisodin formulation. These results suggest that, in contrast to the effects observed in CD4+ T lymphocytes, there is no effect of Glisodin with respect to CD8+ T lymphocytes.

[0039] Part 2 - In vivo adoptive transfer For part 2 of the analysis, i.e. in vivo adoptive transfer (AT), T lymphocyte CD4+ and CD8+ cells are isolated from the spleens of naive or asthmatic Balb / cj Ly5.1 mice and enriched by negative selection. To determine the effect of Glisodin® on the reactivation of T lymphocytes during allergic reactions, the isolated T lymphocytes are labeled with carboxyfluorescein succinimidyl ester (CFSE) to measure their proliferation and then intravenously reinjected at the 2-3 million cell level into naive mice. Following this, the mice are exposed twice to house dust mites to reactivate the injected cells in vivo, and for comparison, the mice also receive or do not receive the Glisodin® formulation described above. After T lymphocyte reactivation, the latter is analyzed for T lymphocyte proliferation and phenotype in the lungs and spleens of mice using the following flow cytometry markers: CFSE, CD122-FITC, Ly5.1-Pe, CD4-PercP5.5, CCR4-Pe-Cy7, CCR6-Pe-Cy7, GATA3-APC, RORγt-APC, CD8-APC-H7, IL-17-Brilliant Violet 421, CD44-Brilliant Violet 421, CXCR3-Brilliant Violet 510, IL-17RA-Brilliant Violet 510.

[0040] Lymphocyte proliferation was measured after exposure to house dust mites and the results are shown in Figure 2. This measurement was obtained by analyzing progressive dilutions of CFSE and the results are shown in Figure 2A-2D for lung and spleen cell proliferation, while Figure 2E-2H show the proliferation rates in the lung (Figure 2E and 2F) and spleen (Figure 2G and 2H), respectively. Applying ANOVA analysis with Tukey's test, confidence intervals were obtained as shown in the figures: *=p<0.05, **=p<0.01 ***=p<0.001 (populations were n=6-8 mice per group).

[0041] Using the Ly5.2 marker, a marked increase in T lymphocyte proliferation was observed in T lymphocytes transferred to asthmatic mice (light grey histograms) compared to lymphocytes from naive mice, which showed no signs of proliferation (white histograms) (see Figures 2E-2H). The observed proliferation thus serves as a marker for the reactivation of lymphocytes sensitized after exposure to house dust mites. Similarly, an increased reactivation of lymphocytes transferred from asthmatic mice to mice also exposed to Glisodin (black histograms) was also observed compared to those lymphocytes from naive mice (dark grey histograms). Of particular interest, however, is the observation that lymphocyte reactivation was reduced by approximately 50% in mice that were also exposed to the Glisodin formulation compared to mice that were not exposed to the Glisodin formulation. No significant differences in activity were observed between wheat SOD and melon SOD Glisodin formulations.

[0042] Further analysis was now performed to identify subpopulations of expanded CD4+ T lymphocytes. The results are shown in Figure 3. This was achieved by performing population analysis among expanded cells obtained from donors, using the Ly5.2 marker and CFSE dilution. As shown in Figure 3A, it was observed that the frequency of Th1 lymphocytes was not affected, either by the allergy-inducing environment or by the application of Glisodin. Nevertheless, an increased proliferation of Th2 and Th17 lymphocytes was observed in allergic mice compared to control mice (see Figures 3B and 3C). Furthermore, as a confirmation of the in vitro results, the in vivo results show that the Glisodin formulation inhibits the cell proliferation of both Th2 and Th17 lymphocytes induced by allergy (see Figures 3B and 3C). These results confirm the anti-inflammatory and anti-allergic effects of the Glisodin formulation both in vitro and in vivo.

[0043] Part 3 - Molecular scale analysis In part 3 of the experiment, mouse sera from part (a) were used. ELISA was used to assess the total and specific amounts of the following antibodies against house dust mites: IgE, IgA, IgG1 and IgG2a. Similarly, bronchoalveolar lavage (BAL) in part (2) was used to measure the amount of the following chemoattractants and proinflammatory molecules: RANTES or CCL5, a known chemotactic molecule for T cells, eosinophils, and basophils, which plays an active role in leukocyte recruitment at inflammatory sites; Eotaxin or CCL11, a known chemotactic molecule for eosinophils; KC or keratinocyte-derived chemokine or CXCL1, a known chemotactic molecule for neutrophils; MCP-1 or CCL2, a known chemotactic molecule for monocytes, lymphocytes, and basophils, which cause degranulation; MIP1 or CCL3, which is involved in the recruitment and activation of polymorphonuclear leukocytes in acute inflammatory conditions; and TARC or CCL17, a potent chemokine that attracts regulatory T cells. These determinations were made across 42 samples as follows; seven controls of wheat Glisodin formulations; 8 controls of melon Glisodin formulations; 9 asthma test samples of wheat Glisodin preparations; 8 asthma test samples of melon Glisodin preparations; 10 asthma test samples as positive controls;

[0044] First, the total amount of antibodies produced from the serum of the mice was measured. This measurement was performed by ELISA and ANOVA analysis was applied, resulting in the confidence intervals marked: *=p<0.05, **=p<0.01***=p<0.001 and the population was n=6–8 mice per group.

[0045] An increase in the total number of IgE was observed in allergic mice compared to control mice (see Figure 4A). However, no significant effect on total IgE production was observed in either control or allergy-induced asthmatic mice, with or without the Glisodin preparation (see Figure 4A). Furthermore, no changes were observed in the production of IgA, IgG1, and IgG2a, regardless of the type of mice investigated and with or without the Glisodin preparation (see Figures 4B-4D). Similar results were obtained with the melon Glisodin preparation (results not shown). To try and better understand these observations, house dust mite (HDM)-specific Ig was measured. The results are shown in Figure 5, applying an ANOVA analysis with Tukey's test, with confidence intervals as follows: *=p<0.05, **=p<0.01***=p<0.001 (populations were n=6-8 mice per group).

[0046] In contrast to what was observed for total Ig, an increase in IgE specific to house dust mite (HDM) was observed in asthmatic mice compared to control mice (see Figure 5A). The presence of the Glisodin preparation was found to inhibit the increase in specific IgE induced in the allergic asthma protocol, while it had no noticeable effect on naive control mice (see Figure 5A). In contrast, HDM-specific IgA was reduced in allergic subjects, and the Glisodin preparation also inhibited this decrease compared to control mice (see Figure 5B). Glisodin was not effective in reducing the production of HDM-specific Ig2a and IgG1 (see Figures 5C and 5D). Taken together, these results demonstrate that the Glisodin preparation exerts a protective effect against allergic reactions, mainly through a reduction in specific IgE and an increase in specific IgA. Similar results were obtained with melon Glisodin preparations (not shown). Thus, the SOD contained in these preparations is useful for the treatment and prevention of allergic asthma.

[0047] Similarly, bronchoalveolar lavage (BAL) from previous experiments of mice exposed to Glisodin formulations was further analyzed for the presence and amount of specific chemoattractants, namely RANTES, KC, MCP-1, MIP1, TARC, and Eotaxin, as shown in Figure 6, and ANOVA analysis with Tukey test was performed, with confidence intervals marked: *=p<0.05, **=p<0.01 et ***=p<0.001 (n=6-8 mice per group). The results show that, compared to control mice, the allergic mice have an increased production of RANTES, KC, Eotaxin, and TARC (see Figures 6A, 6B, 6D, and 6E), but this increase is not observed for MIP1 and MCP1 (see Figures 6C and 6F). Of particular interest here is that mice supplemented with the Glisodin formulation showed increased suppression of the production of these chemoattractants RANTES, eotaxin, and TARC, whose production is induced by allergic asthma (see Figures 6A, 6D, and 6E).Similarly, the Glisodin formulation had no significant effect on KC, MIP1, or MCP1 (see Figures 6B, 6C, and 6F).

[0048] Part 4 - Respiratory function Respiratory function of the mice was analyzed using the flexiVent™ system available from SciReq, and the results are shown in Figures 7 and 8. The results show that the Glisodin formulation alone had no effect on healthy mice, while the respiratory function of asthmatic mice was significantly improved. This effect is seen as a decrease in airway resistance, as shown in Figures 7A and 7D, which reflects the resistance that air experiences as it passes through the lungs, and this resistance increases in asthmatic lungs due to the narrowing of the bronchial diameter. The improvement in respiratory function brought about by Glisodin can also be seen in the compliance plots shown in Figures 7B and 7E, where compliance is the ability of the lungs to adapt their volume in response to changes in pressure, but also in response to changes in elasticity, which is the inverse of compliance and indicates the elasticity of the lungs, as shown in Figures 7C and 7F. In the chart in Figure 7, naive untreated mice are shown as controls or CTL, naive mice receiving wheat Glisodin as CTLwGli, naive mice receiving melon Glisodin as CTLmGli, asthmatic mice as HDM, and asthmatic mice receiving either wheat Glisodin (HDMwGli) or melon Glisodin (HDMmGli). Respiratory function in response to increasing doses of the bronchoconstrictor methacholine was measured using the flexiVent™ system. ANOVA analysis of the data using Tukey's test was performed to establish the confidence intervals shown on the graph: *=p<0.05, **=p<0.01***p<0.001 (populations were n=6-8 mice per group).

[0049] The efficacy of Glisodin was also analyzed in combination with salbutamol, a β2-adrenergic receptor agonist with bronchodilatory and anti-inflammatory properties used to treat asthma attacks, and the results are shown in Figure 8. Here, a significant effect on improving respiratory function with the administration of salbutamol was revealed in terms of resistance (see Figures 8A and 8D), compliance (see Figures 8B and 8E), and airway elasticity (see Figures 8C and 8F). However, no synergistic or additive effects were observed when combining both Glisodin and salbutamol administration. In the graph, CTLs or controls are shown in untreated, untreated asthmatic mice (HDM), asthmatic mice treated with salbutamol (Salbutamol), asthmatic mice administered salbutamol and wheat Glisodin (Salbu wheat), or asthmatic mice administered salbutamol and melon Glisodin (Salbu melon), all measured by flexiVent as a response to increasing doses of the bronchoconstrictor methacholine. ANOVA analysis of the data using Tukey's test was performed to establish confidence intervals shown in the graphs: *=p<0.05, **=p<0.01***p<0.001 (populations were n=6–8 mice per group).

[0050] Part 5 - Inflammatory Response At the end of the mouse study protocol, bronchoalveolar lavage (BAL) was collected and cell populations were analyzed using flow cytometry. The results of this analysis are shown in Figure 9, which shows the total number of cells (Total), the number of Ly6C+ neutrophils (Neu), the number of CCR3+ neutrophils (Eo), the number of Ly6G+ monocytes (Mo), and the number of CD3+ lymphocytes (Ly), where CTL are naive untreated mice, CTLwGli are naive mice administered wheat Glisodin, CTLmGli are naive mice administered melon Glisodin, HDM are untreated asthmatic mice, HDMwGli are asthmatic mice administered wheat Glisodin, HDMmGli are asthmatic mice administered melon Glisodin, Salbutamol are asthmatic mice treated with Salbutamol, Salbwheat are asthmatic mice receiving a combination of Salbutamol / Wheat Glisodin treatment, and SalbMelon are asthmatic mice receiving a combination of Salbutamol / Melon Glisodin treatment. ANOVA analysis of the data using Tukey's test was performed to establish confidence intervals shown in the graphs: *=p<0.05, **=p<0.01***p<0.001 (populations were n=6–8 mice per group).

[0051] In asthmatic mice, the total cell count in the BAL and lungs was significantly increased compared to control mice. Nevertheless, in these mice administered wheat or melon Glisodin, this number was significantly reduced compared to untreated asthmatic mice (see Figures 9A and 9C). The reduction in cell count was most pronounced in eosinophils and lymphocytes, and to a lesser extent in monocytes, but not in neutrophils. As with the respiratory function analysis, no significant differences in the reduction in cell count were observed when wheat and melon Glisodin were compared side-by-side. A test of the cumulative or synergistic effect of combining Glisodin and salbutamol administration was also performed, and the results are shown in Figures 9B and 9D. Salbutamol showed a characteristic anti-inflammatory effect, reducing the number of cells in the BAL, especially the number of eosinophils and lymphocytes. No complementary effect was observed with combined therapeutic administration of wheat or melon Glisodin (see Figures 9B and 9D).

[0052] Part 6 - Immune Response At the end of the treatment protocol, mouse lungs were harvested, crushed, restimulated with whole dust mite extract, and the resulting cell populations were analyzed using flow cytometry. The results are shown in Figure 10, where the following cell types were counted: Th1 (CD3+, CD4+, IFNγ+), Th2 (CD3+, CD4+, IL-13+), and Th17 (CD3+, CD4+, IL-17+). ANOVA analysis of the data using Tukey's test was performed to establish the confidence intervals shown on the graph: *=p<0.05, **=p<0.01***p<0.001 (populations were n=6–8 mice per group).

[0053] T lymphocyte differentiation is the phenomenon whereby naive T lymphocytes, with a certain specificity for an antigen, also acquire the ability to exert a specific effect. This process is triggered in lymph nodes during the interaction of naive T lymphocytes with cells presenting a specific antigen. These antigen-presenting cells produce various cytokines that can induce various different differentiation programs of T lymphocytes depending on the environment in which they are activated before migrating to lymphoid organs. As a result, it can be said that there are several pathways for T lymphocyte differentiation, which are mutually exclusive. In this way, various different T lymphocyte populations can be distinguished by the type and distribution of the cytokines produced. In summary, these can be presented as follows: Th1 lymphocytes, which produce γ-IFN and are involved in antiviral and antibacterial responses; Th2 lymphocytes, which mainly produce IL-4 and IL-13, involved in antiparasitic and allergic responses; Th17 lymphocytes produce IL-17 and are involved in inflammatory responses.

[0054] From Figure 10, it can be seen that Th1, Th2, and Th17 lymphocyte cell counts were all significantly higher in asthmatic mice compared to control mice. In contrast, asthmatic mice administered Glisodin showed a marked decrease in Th1 lymphocyte counts (see Figures 10A and 10D). Similarly, mice treated with salbutamol, with or without co-treatment with Glisodin, showed a decrease in Th1 lymphocyte counts, but no cumulative effect was observed (see Figures 10A and 10D). No significant differences were observed between the two Glisodin formulations. Similar results were observed for Th2 lymphocyte responses (see Figures 10B and 10E) and Th17 lymphocyte responses (see Figures 10C and 10F), with a trend toward a cumulative effect between Glisodin and salbutamol.

[0055] Part 7 - Humoral reactions In allergic asthma, the recognition of an antigen leads to clonal selection and proliferation, followed by differentiation of B lymphocytes into plasma cells, formation of immune complexes between IgE and allergens, degranulation of effector cells and release of chemical mediators such as histamine and MCP-1. To evaluate the effect of Glisodin treatment on the expression of asthma attacks, serum levels of IgE specific for house dust mites were measured by ELISA and levels of MCP-1 production were measured. The results of these measurements are shown in Figure 11, applying an ANOVA analysis of the data using Tukey's test to derive the confidence intervals shown in the graphs produced. *=p<0.05, **=p<0.01 et ***p<0.001 (populations were n=6-8 mice per group).

[0056] It was observed that the level of HDM-specific IgE was higher in asthmatic mice than in control mice, and furthermore, both salbutamol and Glisodin were able to reduce its level without any signs of cumulative effects (see Figures 11A and 11C). Similarly, at the beginning of an asthmatic attack, characterized by the production of MCP-1, it is observed that asthmatic mice have higher levels of this molecule than control mice (see Figures 11B and 11D). However, contrary to the results shown in other analyses, in this case salbutamol reduced the levels of MCP-1 produced, whereas Glisodin seemed less effective (see Figures 11B and 11D).

[0057] Part 8 - Cytokine Responses Cytokines are produced in response to antigens present on the surface of foreign bodies or molecules that are considered foreign by the immune system. When an antigen response is initiated, cells responsible for the development of immune defenses are activated, stimulating the proliferation and differentiation of lymphocytes in particular. In the present case, Th2 inflammatory pathway cytokines (e.g. IL-5, IL-13) and Th17 cytokines (e.g. IL-17) were measured in BAL supernatants of different mouse groups, the results of which are shown in Figure 12, applying an ANOVA analysis of the data using Tukey's test, resulting in the confidence intervals shown in the established graph. *=p<0.05, **=p<0.01***p<0.001 (populations were n=6-8 mice per group).

[0058] As shown in Figure 12, an increase in the number of cytokines was observed in asthmatic mice compared to control mice. Glisodin formulations and salbutamol were effective in reducing IL-5 numbers and tended to reduce IL-17 numbers. However, it was also observed that neither salbutamol nor the various Glisodin formulations tested were able to reduce the levels of IL-13 (Figures 12A-12D).

[0059] Part 9 - Tissue Deterioration In situ evaluation of inflammation, bronchoconstriction, and asthma severity was performed by histological analysis, where lung samples from the various mouse groups were stained with eosin and hematoxylin, with a population of 6 mice per group, and images are presented at a scale of 100 μm, as shown in Figure 13. The degree of tissue deterioration was evaluated in a double-blind protocol by two independent assessors using a scoring system. A reduction in asthma severity was observed in mice treated with Glisodin or salbutamol, but no significant cumulative effect of combining both treatments was observed. Moreover, no notable differences were observed between the two Glisodin products.

[0060] The conclusion drawn from the various analyses performed and described above is that, interestingly, Glisodin prevented the development of airway hyperresponsiveness in asthma and inhibited airway eosinophilia in allergic mice. Furthermore, it prevented the increase in IL-5 in the lungs of allergic mice and reduced HDM-induced Th2 and Th17 responses. Administration of Glisodin leads to an improvement in the respiratory function of asthmatic mice. This effect is associated with an anti-inflammatory effect on the immune response and an associated reduction in humoral responses. Administration of Glisodin did not seem to prevent the exacerbation of asthma symptoms, but had a positive effect on allergic sensitization. These conclusions were further confirmed by histological analysis.

Claims

1. 2. Use of a superoxide dismutase for the treatment and / or prevention of allergic asthma in a mammal, said superoxide dismutase being administered orally alone or optionally in combination with at least one β ２ 20. The use of a medicament for treating atopic dermatitis in combination with the administration of an aerosol preparation comprising an adrenergic receptor agonist.

2. The use according to claim 1 , wherein the mammal is a human.

3. 3. The use according to claim 1 or claim 2, wherein the superoxide dismutase is a heterologous superoxide dismutase, preferably a plant-derived superoxide dismutase.

4. The use according to any one of claims 1 to 3, wherein the plant-derived superoxide dismutase is selected from the group consisting of melon superoxide dismutase and wheat superoxide dismutase.

5. The use according to any one of claims 1 to 4, wherein the oral administration of superoxide dismutase consists of a solid preparation comprising plant-derived superoxide dismutase in combination with a prolamine.

6. The use according to any one of claims 1 to 5, wherein the oral administration of superoxide dismutase consists of a solid preparation comprising superoxide dismutase of plant origin in combination with gliadin.

7. The use according to any one of claims 1 to 6, wherein the oral administration of superoxide dismutase consists of a solid preparation comprising: a) an extract from dried melon juice containing a standardized potency of superoxide dismutase of 0.50% to 5.00% based on the total weight of said solid preparation; b) from 1.50% to 5.00% gliadin, relative to the total weight of the preparation; c) qsp maltodextrin; d) the superoxide dismutase has an enzymatic activity of 1 to 5 IU / mg;

8. 8. The use according to claim 7, wherein the oral administration of superoxide dismutase consists of a solid preparation consisting of: a) an extract from dried melon juice containing a standardized potency of superoxide dismutase of 1.33% based on the total weight of said solid preparation; b) 3.33% of gliadin relative to the total weight of the preparation; c) qsp maltodextrin; d) the superoxide dismutase has an enzyme activity of 1.5 U(NBT) / mg;

9. The use according to any one of claims 1 to 6, wherein the oral administration of superoxide dismutase consists of a solid preparation comprising: a) 0.04% to 0.40% of wheat-derived superoxide dismutase based on the total weight of the preparation; b) from 1.50% to 5.00% gliadin, relative to the total weight of the preparation; c) qsp maltodextrin; d) The superoxide dismutase has an enzyme activity of 1 to 10 U(NBT) / mg.

10. 10. The use according to claim 9, wherein the oral administration of superoxide dismutase consists of a solid preparation comprising: a) 0.18% wheat-derived superoxide dismutase based on the total weight of the preparation; b) 3.50% gliadin relative to the total weight of the preparation; c) qsp maltodextrin; d) the superoxide dismutase has an enzyme activity of 4.5 U(NBT) / mg;

11. The use according to any one of claims 1 to 10, wherein the superoxide dismutase is orally administered to a human in an amount of 100 mg to 2000 mg per day for a minimum of 15 consecutive days.

12. 12. The use according to claim 11, wherein the superoxide dismutase is orally administered to a human in an amount of 500 mg per day for a minimum of 15 days and a maximum of 180 consecutive days.

13. The at least one β ２ 2. The use according to claim 1, wherein the adrenergic receptor agonist is selected from the group consisting of bitolterol, fenoterol, isoprenaline, levosalbutamol, orciprenaline, pirbuterol, procaterol, ritodrine, salbutamol, and terbutaline.

14. The at least one β ２ 2. The use according to claim 1, wherein the adrenergic receptor agonist is salbutamol.

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