Compositions for treating or preventing an allergy or allergic reaction

Abstract

The present invention relates to a composition comprising the supernatant of a cell culture of peripheral blood mononuclear cells (PBMCs) for use in the treatment or prevention of an allergy or allergic reaction caused by the administration of at least one food to the human body or a mammal and / or the inhalation of an allergen or the systemic administration of at least one drug.

Description

Technical Field

[0001] This invention relates to compositions and methods for treating and preventing disorders caused by the ingestion of allergens by humans or mammals. Background Technology

[0002] During the sensitization phase of an allergic reaction, professional antigen-presenting cells phagocytose antigens (referred to as allergens in the context of allergies) and cross-present the processed antigens to T cells. H 2. Lymphocytes. These cells respond to encountered allergens by releasing interleukin-4 and interact with B lymphocytes. Activated B cells, along with IL-4, produce immunoglobulin E (IgE) against the allergen. The secreted IgE binds to IgE-specific receptors present on, for example, mast cells. These immune cells are thus sensitized to the allergen. Re-exposure results in the allergen binding to IgE-coated immune cells, leading to activation of the sensitized cells after receptor cross-linking. Activated mast cells respond by releasing large amounts of immune mediators, such as histamine, cytokines, and pro-inflammatory lipids, from intracellular granules. The released mediators have multiple effects, including vasodilation, mucus secretion, and nerve stimulation, leading to itching and redness. These symptoms can be localized or systemic.

[0003] Mast cells are primarily found in tissues repeatedly exposed to pathogenic stimuli, such as the skin, respiratory tract, and gastrointestinal tract. Typically, mast cells are activated via immunological signals, such as Fcγ receptors cross-linked with antigen / IgE complexes, proteins of the complement system, or Toll-like receptor agonists. The response of mast cells to activation is the release of large amounts of immune mediators. Mast cells are known for their role in IgE-dependent allergic reactions and for targeting mast cell functions (such as degranulation), which may help alleviate most pain symptoms associated with allergic reactions.

[0004] The purpose of this invention is to provide methods and means for preventing or treating allergic reactions caused by the application of allergens to humans or mammals. Summary of the Invention

[0005] This invention relates to a composition comprising the supernatant of a cell culture of peripheral blood mononuclear cells (PBMCs) for the treatment or prevention of allergies or allergic reactions caused by administration of at least one food and / or inhalation of an allergen or systemic administration of at least one drug to a human or mammal, wherein the PBMCs are subjected to ionizing radiation before or during culture.

[0006] Surprisingly, the supernatant of PBMC cultures can be used to treat or prevent allergies and anaphylactic reactions caused by the administration of food or inhalation of allergens to humans or mammals. Furthermore, the compositions of this invention can also be used to treat or prevent allergies or anaphylactic reactions caused by systemic administration of at least one allergen.

[0007] Stem cell-based therapies have long been considered a promising means of regenerating various injured tissues and organs. This concept has been further fueled by numerous studies reporting observable regenerative effects on secreted factors, rather than the cells themselves. Peripheral blood mononuclear cells (PBMCs) are an attractive and readily available source of cellular secretory proteomes with a wide range of potential uses, compared to stem cells. While immunomodulatory effects have been attributed to the secretory proteome of preferred γ-irradiated PBMCs (Aposec), surprisingly, the supernatant of PBMC cultures has shown beneficial effects on allergic reactions and anaphylaxis, particularly when triggered by allergens.

[0008] Another aspect of the invention relates to a method for treating or preventing allergies or allergic reactions caused by the ingestion of at least one allergen by a human or mammal, the method comprising the step of administering a composition as defined herein.

[0009] Another aspect of the invention relates to a method for determining whether a composition, as defined herein, is suitable for treating or preventing an allergy or allergic reaction caused by administration of at least one food and / or inhalation of an allergen to a human or mammal, or systemic administration of at least one drug, the method comprising the steps of:

[0010] a) Expose at least two areas of a mammal’s skin to the allergen;

[0011] b) Applying the composition as defined in any one of claims 1 to 17 to at least one of the skin regions, wherein at least one of the skin regions is not treated with the composition;

[0012] c) Compare the skin areas that have been in contact with the allergen and the skin areas that have been in contact with both the allergen and the composition;

[0013] d) Identify the differences between the regions, and

[0014] e) Determine whether the composition is suitable for treating or preventing the disorder or disease. Attached Figure Description

[0015] Figure 1This demonstrates that MNCaposec prevents the release of compound 48 / 80 and IgE / anti-IgE-induced mediators from primary human mast cells. Mast cell degranulation was assessed by the release of beta-hexaminidase upon stimulation of primary human mast cells with (A) compound 48 / 80 and (B) IgE / anti-IgE. An asterisk indicates that Aposec showed a P < 0.05 compared to the control culture medium (CellGro). Mast cell culture medium refers to the medium routinely used for culturing mast cells (DMEM alone).

[0016] Figure 2 This study demonstrates that the PBMC-secreted proteome, specifically aposec, alleviates ear swelling in DNFB-induced hypersensitivity. Ear thickness was assessed using micrometer-assisted measurements 24 hours after DNFB re-excitation. P < 0.05 Aposec vs. culture medium control group. Detailed Implementation

[0017] This invention relates to a composition comprising a supernatant of PBMC cell culture for the treatment or prevention of allergies or allergic reactions caused by administration of at least one food and / or inhalation of an allergen to a human or mammal, or systemic administration of at least one drug.

[0018] Cells, particularly mammalian cells, are known to secrete numerous substances during culture into cell culture media. The conditioned media thus obtained can be used to treat and / or prevent various diseases and disorders. For example, WO 2010 / 070105 and WO2010 / 079086 disclose conditioned media (“supernatants”) obtained by culturing PBMCs, which can be used to treat various inflammatory conditions. Therefore, “supernatant of cell cultures of peripheral blood mononuclear cells (PBMCs)” as used herein refers to any supernatant obtained by culturing PBMCs in vitro in a culture medium. After the culture step, the cultured PBMCs are removed from the culture medium to obtain a substantially cell-free, preferably completely cell-free, supernatant. The PBMC culture supernatant contains, in addition to the components of the culture medium, substances produced and secreted by the PBMCs, and / or even lysed PBMCs. The “supernatant” is interchangeable with the conditioned media obtained by culturing PBMCs.

[0019] The supernatant of the present invention can be obtained by culturing PBMCs, which are subjected to ionizing radiation before or during culturing. Gamma radiation is preferred.

[0020] As used herein, "allergy or anaphylactic reaction caused by administration of at least one food and / or inhaled allergen or systemic administration of at least one drug to a human or mammal" refers to any adverse reaction caused by administration of food and / or inhaled allergens to a human or mammal. Food allergens are usually administered to humans or mammals orally. Inhaled allergens are usually administered by inhalation into the lungs and respiratory tract via the nose or mouth. Some drugs used to treat humans and mammals can cause anaphylactic reactions when administered systemically to humans and mammals. These drugs can be administered to humans or mammals orally, by inhalation, parenteral administration, or any other route, resulting in systemic distribution of the drug within the human or mammalian body. Parenteral administration of drugs can include intramuscular, intraperitoneal, intravenous, and other routes of administration. Topical application of a drug allergen or any other allergen to the skin does not result in systemic spread and therefore does not result in systemic administration to humans or mammals.

[0021] Allergens that cause allergies or allergic reactions when applied to humans or mammals include food allergens, drug allergens, inhaled allergens (such as pollen and chemicals), and all kinds of allergens applied to or introduced into humans or mammals. Therefore, typical allergic reactions to allergens applied to humans or mammals when introduced, such as through the bloodstream, include, among other things, runny nose, difficulty breathing, nausea, diarrhea, nosebleeds, ear infections, wheezing, coughing, and even allergic reactions.

[0022] As used herein, “allergen” refers to an antigen that can stimulate a hypersensitivity response in humans and / or mammals via an immunoglobulin E (IgE) reaction, typically leading to excessive histamine release from mast cells. The term “allergen” includes, if the allergen is of biological origin and is a protein or peptide, fragments of such naturally occurring allergens when these fragments exhibit similar effects in histamine release in humans or mammals as those of naturally occurring allergens.

[0023] As used herein, the terms "preventing" and "prevention" refer to the prevention or suppression of the recurrence, onset, and development of allergies or their symptoms in humans and mammals due to the application of the supernatant according to the invention. In some embodiments, "preventing" and "prevention" refer to reducing the risk of developing an allergic reaction in response to a specific allergen. The term "preventing" includes not only measures to prevent the occurrence of an allergic reaction but also, once an allergic reaction has occurred, suppressing its progression and reducing its consequences.

[0024] As used herein, the terms "treatment" and "treating" refer to reducing or inhibiting the progression and duration of an allergy, reducing or improving the severity of an allergy, and improving one or more of its symptoms. "Treatment" also includes improving and / or reversing the symptoms of an allergy or allergic reaction. The term "treatment" refers to both therapeutic treatment and preventative measures. For example, those who may benefit from treatment using the compositions and methods of the present invention include individuals who already have an allergy and those who wish to prevent an allergy.

[0025] According to another preferred embodiment of the present invention, at least one allergen is a biological or chemical allergen.

[0026] Biological allergens include allergens derived from biological systems, such as plants, animals (e.g., insects and arachnids), or microorganisms (e.g., molds and bacteria). These allergens are, in most cases, proteins, polypeptides, or peptides. Biological allergens are well-known in the field and are publicly available in various databases such as http: / / www.allergen.org / or http: / / www.allergome.org / .

[0027] According to a further preferred embodiment of the present invention, the biological allergen is selected from the group consisting of animal allergens, plant allergens, mold allergens, or bacterial allergens.

[0028] Animal allergens are allergens that include one or more compounds found in animals (including vertebrates and invertebrates). Vertebrate allergens that may be present in mixed allergen compositions include avian allergens, such as egg allergens, such as nGal d 1 ovalbumin, nGal d 2 ovalbumin, nGal d 3 conalbumin, and complete egg white allergens; mammalian allergens, such as milk allergens, such as nBos d 4 α-lactalbumin, nBos d 5 β-lactoglobulin, nBos d 8 casein, nBos d lactoferrin, and complete milk allergens; and fish allergens, such as rCyp c 1, rGadc 1, complete cod allergens, whitefish allergens, and pinkfish allergens. Invertebrate allergens that may be present in a mixed allergen composition include: crustacean allergens, such as shrimp allergens, such as rPen a 1 tropomyosin and complete shrimp allergens; insect allergens, such as bee sting venom allergens, wasp sting venom allergens, and mosquito bite allergens; and others. Inhaled animal allergens may include cat or dog hair and dander, cockroach calyxes, and dust mite excrement.

[0029] Plant allergens are allergens that include one or more compounds found in plants. Relevant plant allergens include: wheat allergens, such as rTria 19 Omega-5 prolysin, complete wheat allergens, prolysin wheat, and rTria 14 LTP; kiwifruit allergens, such as rAct d 8 PR-10 and complete kiwifruit allergens; celery allergens, such as rApi g 1.01 PR-10, rPhl p 12, complete celery allergens, and CCDMOUXF3 from bromelain; soybean allergens, such as rGly m 4 10 PR-10, complete soybean allergens, nGly m 5β-conglycinin, and nGly m 6glycinin; and stone fruit allergens, such as f419, f420, f421, f95, f242, o214 rPru p 1 PR-10, rPru p 3 LTP, primary complete stone fruit allergens, and CCD from bromelain. MUXF3, etc.; oat allergens, such as oat component allergens and oat complete allergens; sesame allergens, such as sesame seed component allergens and sesame seed complete allergens. Plant allergens also include inhaled allergens, such as pollen allergens. These allergens may include birch pollen allergens (e.g., Bet v 1), grass pollen allergens (e.g., Phl p 1), ryegrass, and timothy grass allergens.

[0030] Allergens include those isolated from natural sources or recombined or chemically produced.

[0031] According to another preferred embodiment of the present invention, the PBMC cell culture comprises monocytes, T cells, B cells and / or NK cells.

[0032] According to a further preferred embodiment of the present invention, PBMC cells are cultured in a cell culture medium selected from cell growth media, preferably CellGro medium, more preferably the group consisting of CellGro GMP DC medium, RPMI, DMEM, X-vivo and Ultraculture.

[0033] Before or during culture, PBMCs in a PBMC cell culture are subjected to ionizing radiation. In addition to these stress-inducing conditions, PBMCs can be subjected to further stress. Therefore, according to a preferred embodiment of the invention, PBMCs are subjected to one or more further stress-inducing conditions before or during culture.

[0034] As used in this article, the term "under stress-induced conditions" refers to the culture conditions that induce stress in cells. Conditions that induce cellular stress include, among others, heat, chemicals, radiation, hypoxia, and osmotic pressure.

[0035] Additional stress on the cells of this invention leads to a further increase in the expression and secretion of substances that are beneficial for the treatment of inflammatory skin conditions, particularly those related to local ischemia.

[0036] According to a preferred embodiment of the invention, the pressure-inducing conditions include low oxygen, ozone, heat (e.g., above 2°C, preferably above 5°C, more preferably above 10°C, above the optimal culture temperature of PBMC, i.e., 37°C), radiation (e.g., ultraviolet radiation and gamma rays), chemicals, osmotic pressure (i.e., osmotic conditions that are at least 10% higher than those commonly found in body fluids, especially blood) or combinations thereof.

[0037] Therefore, according to another preferred embodiment of the present invention, the stress-inducing conditions are selected from the group consisting of ultraviolet radiation, low oxygen, ozone, heat, osmotic pressure and pH changes.

[0038] According to another preferred embodiment of the invention, the PMC is subjected to ionizing radiation, preferably gamma radiation, at a dose of at least 10 Gy, preferably at least 20 Gy, more preferably at least 40 Gy, and even more preferably at least 50 Gy.

[0039] According to a preferred embodiment of the present invention, the PBMCs are cultured for at least 4 hours, preferably at least 6 hours, and more preferably at least 12 hours before the supernatant of the cell culture is separated.

[0040] According to another preferred embodiment of the invention, the composition of the invention is applied before, during, and / or after an allergic reaction and / or exposure to at least one allergen.

[0041] The compositions of the present invention can be administered at different stages of an allergic reaction or even before the reaction occurs. In a particularly preferred embodiment of the invention, the compositions can be administered to a human or mammal before exposure to an allergen. Surprisingly, the compositions of the present invention are able to prevent the uptake of an allergen or fragment thereof by antigen-presenting cells. Therefore, if such uptake can be prevented, the allergen or fragment thereof will not be presented to the immune system of a human or mammal.

[0042] According to a preferred embodiment of the present invention, the PCBMC cell culture contains 1×10 5 Up to 1×10 8 PBMCs / ml, preferably 1×10⁻⁶ 6 Up to 1×10 7 PBMCs / ml, more preferably 2×10 6 Up to 5×10 6 PBMC / ml.

[0043] It has been shown that, when a certain amount of PBMC / ml cell culture medium is cultured, the composition of the supernatant obtained by culturing PBMCs exhibits advantageous properties.

[0044] According to another preferred embodiment of the invention, 0.1 to 5 ml of supernatant / kg body weight, preferably 0.3 to 3 ml / kg body weight, more preferably 0.5 to 2 ml / kg body weight, and even more preferably 0.8 to 1.2 ml / kg body weight, is applied to a human or mammal.

[0045] The compositions of the present invention comprise a supernatant in an amount sufficient to treat or prevent allergies and allergic reactions. The volume / kg body weight of the supernatant applied to humans or mammals is a direct reference to the supernatant. If the volume applied to humans or mammals is too large, it can be reduced, for example, by lyophilization. Therefore, the volume of the composition of the present invention to be applied may be less than the indicated volume of the supernatant. Those skilled in the art will know which volumes can be applied using specific routes of administration.

[0046] According to a preferred embodiment of the invention, the composition is administered by inhalation, topical application, oral administration, sublingual administration, oral administration, subcutaneous administration, or intravenous injection.

[0047] The compositions of the present invention may contain pharmaceutically acceptable excipients, such as diluents, stabilizers, and carriers. Depending on the dosage form, the formulations of the present invention comprise their respective components. Methods for preparing formulations according to the present invention are well known to those skilled in the art.

[0048] To extend the shelf life of the compositions according to the invention, the supernatant or even the entire composition may be lyophilized. Methods of lyophilizing such formulations are well known to those skilled in the art.

[0049] Before use, the lyophilized formulation can be in contact with water or an aqueous solution containing buffers, stabilizers and salts.

[0050] According to another preferred embodiment of the invention, the mammal is a horse, dog, cat, or camel.

[0051] The compositions of the present invention can be used to treat any kind of mammal, but are most preferably the aforementioned mammals.

[0052] Another aspect of the invention relates to a method for treating or preventing allergies or allergic reactions caused by the ingestion of at least one allergen by a human or mammal, the method comprising the step of administering a composition as defined above.

[0053] Another aspect of the present invention relates to a method for determining whether a composition as defined above is suitable for treating or preventing an allergy or allergic reaction caused by the ingestion of at least one allergen by a human or mammal, the method comprising the steps of:

[0054] a) Expose at least two areas of a mammal’s skin to the allergen;

[0055] b) Apply the composition as defined above to at least one of the skin regions, wherein at least one of the skin regions has not been treated with the composition;

[0056] c) Compare the skin areas that have been in contact with the allergen and the skin areas that have been in contact with both the allergen and the composition;

[0057] d) Identify the differences between the regions; and

[0058] e) Determine whether the composition is suitable for treating or preventing the disorder or disease.

[0059] To test whether the compositions of the present invention can be used to treat or prevent allergies or allergic reactions caused by administration of at least one food and / or inhalation of an allergen or systemic administration of at least one drug to a human or mammal, a scratch assay can be used. If the compositions of the present invention reduce allergic reactions at scratch sites compared to untreated scratch sites or scratch sites treated with a negative control composition, the compositions of the present invention can be applied to humans or mammals in need.

[0060] Example

[0061] Example 1: Damage to mast cell degranulation caused by Aposec

[0062] Materials and methods

[0063] Aposec manufacturing

[0064] As described by Wagner T et al. (Sci Rep. 2018; 8(1):1801), the secretory proteome (“Aposec”) of PBMCs was used. Briefly, PBMCs were obtained by Ficoll-Paque PLUS (GE Healthcare, USA) assisted density gradient centrifugation and the concentration was adjusted to 2.5 × 10⁻⁶. 7Cells / mL. Subsequently, cells were exposed to 60 Gy of cesium-137 gamma rays (IBL 437C, Isotopen Diagnostik CIS GmbH, Germany) and cultured for 24 ± 2 hours in phenol red-free CellGenix GMPDC medium (CellGenix GmbH, Germany). Cells and cell debris were removed by centrifugation, and the supernatant was passed through a 0.2 μm filter. Virus removal was performed using the Theraflex methylene blue technique (MacoPharma, France), and the lyophilized powder was irradiated with gamma rays (25,000 Gy, Gammatro 1500, Mediscan, Austria) as previously described (Haider T et al. Exp Neurol. 2015; 267:230-42). Sterile lyophilized products were typically stored frozen at -80°C.

[0065] Isolation and in vitro maintenance of primary human mast cells

[0066] Skin and subcutaneous adipose tissue used for mast cell isolation were obtained from patients undergoing abdominoplasty. Subcutaneous tissue and reticular dermis were removed, and the remaining tissue was cut into small pieces and enzymatically digested overnight at 4°C (2.4 U / mL dispersin II from Bacillus polymyxa, Roche, Switzerland). After epidermal removal, the dermal tissue was digested for 2 hours at 37°C with collagenase I (Gibco, Thermo Fisher Scientific, USA). CD117+ mast cells were enriched using magnetic cell sorting technology (MACS system, Miltenyi Biotec, Germany) as recommended by the manufacturer. To improve the purity of the isolated cells, the isolation process was repeated once more using CD117+ cells from the first isolation. CD117+ mast cells were cultured in DMEM supplemented with 10% (vol / vol) heat-inactivated fetal bovine serum (all Gibco), 1% (vol / vol) penicillin / streptomycin (Biochrom, Germany), and 100 ng / mL recombinant human stem cell factor (PeproTech, USA).

[0067] Degranulation induced by compound 48 / 80 in primary human mast cells

[0068] Primary human mast cells were seeded at a density of 50,000 to 100,000 cells / well in 50 μL of DMEM without a color pH indicator in flat-bottomed 96-well plates supplemented with serum and SCF as described above. Cells were pretreated overnight with 50 μL of Aposec or 50 μL of medium (CellGenix) as a control. The next day, cells were carefully washed with 100 μL of HEPES (N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid; Thermo Fisher Scientific) and demyelinating was induced by adding 100 μL of HEPES containing 50 μg / mL of compound 48 / 80 (Sigma Aldrich, USA). For the non-stimulated control group, HEPES was added. Cells were cultured at 37°C and in normal ambient CO2 for 1 hour. Separate and preserve 50 μL of the supernatant, and lyse the cells with 100 μL of 0.1% Triton X-100 (Sigma Aldrich).

[0069] IgE / anti-IgE induced degranulation of primary human mast cells

[0070] Primary human mast cells were seeded at a density of 50,000 to 100,000 cells / well in 50 μL of DMEM without a color pH indicator in flat-bottomed 96-well plates, supplemented with serum and SCF as described above. Cells were pretreated with 50 μL of Aposec, 50 μL of medium control (CellGenix), or 50 μL of DMEM (mast cell medium), and additionally stimulated overnight with 100 ng / mL human IgE (myeloma, Merck KGaA, Germany). The next day, cells were carefully washed with 100 μL of HEPES (N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid; Thermo Fisher Scientific) and demyelinated by adding 100 μL of HEPES containing 5 μg / mL anti-IgE antibody (Jackson ImmunoResearch Laboratories, Inc., USA). For the unstimulated control group, HEPES was added. Cells were cultured at 37°C and in normal ambient CO2 for 1 hour. Separate and preserve 50 μL of the supernatant, and lyse the cells with 100 μL of 0.1% Triton X-100 (Sigma Aldrich).

[0071] Reagents for β-aminohexosidase test

[0072] The substrate solution for the hexosiminidase assay was prepared by dissolving 8.9 g of disodium hydrogen phosphate dihydrate (Na₂HPO₄·2H₂O) and 650 mg of p-nitro-N-acetyl-β-D-glucosamide in 400 mL of double-distilled water and adjusting the pH to 4.5 with 0.4 M citric acid (all reagents Sigma-Aldrich). The stop solution consisted of 15.02 g of glycine in 900 mL of aqua bidest and adjusted to pH 10.7 with 3 M sodium hydroxide.

[0073] β-Aminohexosidase test

[0074] 50 μL of substrate solution was added to 50 μL of supernatant and 50 μL of Triton-lysed cells, respectively, and the samples were incubated at 37 °C and ambient CO2 for 90 min. 75 μL of stop buffer was added, and the optical density at 405 nm was measured using LUMIstar OPTIMA Reader (BMGLABTECH, Ortenberg, Germany) with FluOstar OPTIMA software (version 1.20-0, BMG LABTECH).

[0075] Data and statistical analysis

[0076] The percentage of β-aminohexosidase released is calculated using the following formula:

[0077]

[0078] Data are expressed as the arithmetic mean of technical replicates and the standard error of the mean. Statistical analysis was performed using a one-tailed t-test. Comparisons between the two Aposec groups and the culture medium control group were considered statistically significant with P < 0.05.

[0079] result

[0080] Aposec abolishes forced mast cell degranulation.

[0081] To determine whether Aposec could prevent mast cell degranulation, mast cells were pretreated with either Aposec or a culture medium control group, and the release of β-aminohexosidase from primary human mast cells after stimulation with compound 48 / 80 and IgE / anti-IgE was assessed. Compared with the culture medium control group, Aposec significantly prevented the release of mediators induced by compound 48 / 80 and IgE / anti-IgE (after stimulation with compound 48 / 80 and IgE / anti-IgE, Aposec released 27.9±3.6% and 27.3±1.1% of β-aminohexosidase, respectively, while the control culture medium group released 35.7±3.4% and 36.7±3.1% of the mediator, respectively; P<0.05 Aposec vs. control group). Figure 1 Compared with mast cell culture medium, the control culture medium showed no effect on mast cell degranulation [after stimulation with compound 48 / 80 and IgE / anti-IgE, the mast cell culture medium released 36.7±2.3 and 36.9±1.9 media, respectively. In (A) and (B), P>0.05 mast cell culture medium vs. control culture medium].

[0082] in conclusion

[0083] These data demonstrate that Aposec effectively prevents the release of mediators from primary human mast cells when stimulated with compound 48 / 80 and IgE / anti-IgE. Following chemical stimulation, enzyme release was reduced by more than 20%, and after IgE / anti-IgE treatment, Aposec reduced β-aminohexosidase release by more than 25% compared to the culture medium control group. In conclusion, these data suggest that Aposec may be used to treat mast cell degranulation-mediated hypersensitivity reactions.

[0084] Example 2: Relieving symptoms of allergic hypersensitivity reactions by in vivo application of Aposec

[0085] background

[0086] Extensive research into the pathology of allergic hypersensitivity reactions over the past few decades has contributed to a better understanding of the immune response. However, the complex and multifaceted etiology of this disease remains a major obstacle to developing effective new therapeutic interventions, and clinical treatment options remain limited to date. Given the previously described potent anti-inflammatory effects of aposec, its potential to alleviate symptoms associated with allergic hypersensitivity reactions was investigated.

[0087] Materials and methods

[0088] mouse model

[0089] 1-Fluoro-2,4-dinitrobenzene (DNFB, Sigma-Aldrich) was used as an allergen to induce inflammatory symptoms in C57BL / 6 mice. 20 μL of a 0.25% (vol / vol) olive oil solution of DNFB was administered on days 0 and 1. Ears were treated with Aposec daily, while the contralateral ear received carrier culture medium for 6 consecutive days starting from day 0.

[0090] Micrometry

[0091] Twenty-four hours after DNFB re-excitation, ear thickness was assessed using an electronic digital micrometer (0-25mm, Marathon Management Inc., USA), measuring the thickness of the outer two-thirds of the ear. Measurements were taken in quadruplicate.

[0092] Statistical analysis

[0093] Statistical evaluation of the data was performed using GraphPad Prism 6 software (GraphPad Software Inc.). One-tailed paired t-tests were performed to compare Aposec with the control medium. A p-value less than 0.05 was considered statistically significant. Data are expressed as the arithmetic mean of biological replicates and the standard error of the mean.

[0094] result

[0095] Aposec reduces allergen-induced tissue swelling.

[0096] Using a mouse DNFB-induced hypersensitivity reaction as a model, the anti-inflammatory effect of Aposec on in vivo allergic reactions was studied. The degree of ear swelling reflected the severity of the immune response. Compared with the moderate control group, Aposec significantly reduced ear swelling 24 hours after re-exposure to DNFB (ear thickness in the carrier-treated group was 553.9±12.7 μm, while ear thickness in the Aposec-treated group was 472.4±47.3 μm, P<0.05; Aposec vs. moderate control group. Original ear thickness: 355±12.7 μm). Figure 2 ).

[0097] in conclusion

[0098] These data indicate that Aposec effectively prevents tissue swelling following re-exposure to allergens. These findings suggest that Aposec is a promising candidate for treating allergy-related symptoms.

Claims

1. The use of a composition as a medicament for treating or preventing allergies or allergic reactions caused by administration of at least one food and / or inhalation of an allergen to a human or mammal, or systemic administration of at least one drug, said composition comprising a supernatant of a cell culture of peripheral blood mononuclear cells (PBMCs), wherein, The PBMCs were subjected to an ionizing radiation dose of at least 10 Gy before or during incubation and were incubated for at least 6 hours.

2. The use according to claim 1, wherein, The at least one allergen is a biological allergen or a chemical allergen.

3. The use according to claim 2, wherein, The biological allergens are selected from the group consisting of animal allergens, plant allergens, mold allergens, or bacterial allergens.

4. The use according to any one of claims 1 to 3, wherein, The PBMC cell culture contains monocytes, T cells, B cells and / or NK cells.

5. The use according to any one of claims 1 to 3, wherein, The PBMCs are cultured in a cell culture medium selected from cell growth media.

6. The use according to claim 5, wherein, The cell culture medium is CellGro medium.

7. The use according to claim 5, wherein, The cell culture medium is selected from the group consisting of Cellgro GMP DC medium, RPMI, DMEM, X-vivo and Ultraculture.

8. The use according to any one of claims 1 to 3, wherein, The PBMC is subjected to a dose of ionizing radiation of at least 20 Gy.

9. The use according to claim 8, wherein, The PBMC is subjected to a dose of ionizing radiation of at least 40 Gy.

10. The use according to claim 8, wherein, The PBMC is subjected to a dose of ionizing radiation of at least 50 Gy.

11. The use according to any one of claims 1 to 3, wherein, The PBMCs were cultured for at least 12 hours before the supernatant of the cell culture was separated.

12. The use according to any one of claims 1 to 3, wherein, The composition is applied before, during, and / or after an allergic reaction and / or exposure to at least one allergen.

13. The use according to any one of claims 1 to 3, wherein, The cell culture of the PBMC contains 1×10 5 Up to 1×10 8 PBMC / ml.

14. The use according to claim 13, wherein, The cell culture of the PBMC contains 1×10 6 Up to 1×10 7 PBMC / ml.

15. The use according to claim 13, wherein, The cell culture of the PBMC contained 2 × 10 6 Up to 5×10 6 PBMC / ml.

16. The use according to any one of claims 1 to 3, wherein, Administer 0.1 to 5 ml of supernatant per kg of body weight to humans or mammals.

17. The use according to claim 16, wherein, Administer 0.3 to 3 ml of supernatant per kg of body weight to humans or mammals.

18. The use according to claim 16, wherein, Administer 0.5 to 2 ml of supernatant per kg of body weight to humans or mammals.

19. The use according to claim 16, wherein, Administer 0.8 to 1.2 ml of supernatant per kg of body weight to humans or mammals.

20. The use according to any one of claims 1 to 3, wherein, The composition is administered by inhalation, oral administration, sublingual administration, oral administration, subcutaneous administration, or intravenous administration.

21. The use according to any one of claims 1 to 3, wherein, The composition is applied topically.

22. The use according to any one of claims 1 to 3, wherein, The mammals mentioned are horses, dogs, cats, or camels.

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