Compositions for treating or preventing allergies or allergic reactions

Abstract

The present invention relates to a composition comprising the supernatant of a peripheral blood mononuclear cell (PBMC) cell culture for use in the treatment or prevention of allergies or allergic reactions caused by administration to the human or mammalian body of at least one food and / or inhalant allergen or by the systemic administration of at least one drug.

Description

[Technical field]

[0001] The present invention relates to compositions and methods for treating and preventing disorders caused by the incorporation of allergens into the human or mammalian body. [Background technology]

[0002] During the sensitization phase of allergy, professional antigen-presenting cells phagocytose antigens (in the context of allergy they are called allergens) and deliver the processed antigens to T cells. H 2 lymphocytes. These cells respond to allergen encounter by releasing interleukin 4 and interact with B lymphocytes. Activated B cells produce E-type immunoglobulin (IgE) against allergens along with IL-4. Secreted IgE is bound by IgE-specific receptors present, for example, on mast cells. This sensitizes these immune cells to the allergen. Re-exposure results in binding of the allergen to IgE-coated immune cells, which, after receptor cross-linking, causes activation of the sensitized cells. Activated mast cells respond by releasing a large amount of immune mediators, such as histamine, cytokines and pro-inflammatory lipid species from intracellular granules. The released mediators have pleiotropic effects, including vasodilation, mucus secretion and nerve stimulation, resulting in itching and redness. These symptoms can be local or systemic.

[0003] Mast cells reside primarily in tissues that repeatedly face pathogenic stimuli, such as the skin, airways, and gastrointestinal tract. In general, mast cells are activated by immunological signals, such as Fcγ receptors crosslinked by antigen / IgE complexes, proteins of the complement system, or Toll-like receptor agonists. Mast cells respond to activation with a massive release of immune mediators. Mast cells are best known for their role in IgE-dependent allergic responses, and targeting mast cell functions, such as degranulation, may help alleviate most painful symptoms associated with allergic responses.

[0004] It is an object of the present invention to provide methods and means for preventing or treating allergic reactions caused by allergens administered to the human or mammalian body. Summary of the Invention [Means for solving the problem]

[0005] The present invention relates to a composition comprising a supernatant of a peripheral blood mononuclear cell (PBMC) cell culture for use in the treatment or prevention of allergy or allergic reaction caused by administration to the human or mammalian body of at least one food and / or inhaled allergen or by systemic administration of at least one drug, wherein said PBMC are exposed to ionizing radiation before or during culture.

[0006] Surprisingly, it has been found that the supernatant of the PBMC culture can be used to treat or prevent allergies and allergic reactions caused by food or inhaled allergens administered to humans or mammals.Furthermore, the composition of the present invention can also be used to treat or prevent allergies or allergic reactions caused by systemic administration of at least one allergen.

[0007] For a long time, stem cell-based therapies have been considered a promising tool for the regeneration of various damaged tissues and organs. Since then, many studies have challenged this concept by reporting that secreted factors, and not the cells themselves, exerted the observed regenerative effects. Peripheral blood mononuclear cells (PBMCs) are an attractive and readily available source of cellular secretomes with diverse characteristics, in contrast to stem cells. Although immunomodulatory effects have been attributed to the secretomes of preferably γ-irradiated PBMCs (Aposec), it is surprising that the supernatants of PBMC cultures show beneficial effects regarding allergic responses and allergies, especially in the case of provoking allergens.

[0008] A further aspect of the present invention relates to a method for treating or preventing allergy or allergic reactions caused by the introduction of at least one allergen into the human or mammalian body, comprising the step of administering a composition as defined herein.

[0009] Another aspect of the present invention is a method for determining the suitability of a composition as defined herein for use in the treatment or prevention of an allergy or an allergic reaction caused by administration to the human or mammalian body of at least one food and / or inhaled allergen or by the systemic administration of at least one drug, comprising the steps of: a) contacting at least two skin areas of a mammal with an allergen; b) administering a composition as defined in any one of claims 1 to 17 to at least one of said skin areas, wherein at least one of said skin areas has not been treated with said composition; c) comparing the skin area contacted with the allergen to the skin area contacted with the allergen and the composition; d) identifying differences between said regions; e) determining whether said composition is suitable for treating or preventing said disorder or disease The present invention relates to a method comprising the steps of: [Brief description of the drawings]

[0010] [Figure 1] Figure 1 shows that MNCaposec prevents Compound 48 / 80 and IgE / anti-IgE induced mediator release by primary human mast cells. Mast cell degranulation assessed by β-hexaminidase released upon (A) Compound 48 / 80 and (B) IgE / anti-IgE stimulation of primary human mast cells. Asterisks indicate p<.05 for Aposec compared to media control (CellGro). Mast cell media refers to media routinely used to culture mast cells (DMEM alone).

[0011] [Diagram 2]Figure 1: PBMC secretome Aposec attenuates ear swelling in DNFB-induced hypersensitivity. Ear thickness assessed by micrometer-assisted measurements 24 hours after DNFB rechallenge. p<0.05 for Aposec vs. medium control. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] The present invention relates to a composition comprising a supernatant of a PBMC cell culture for use in the treatment or prevention of allergy or allergic reaction caused by administration to the human or mammalian body of at least one food and / or inhaled allergen or by the systemic administration of at least one drug.

[0013] It is known that cells, especially mammalian cells, secrete numerous substances during cultivation into cell culture medium. The conditioned culture medium so obtained can be used for the treatment and / or prevention of various diseases and disorders. For example, WO 2010 / 070105 and WO 2010 / 079086 disclose conditioned culture medium ("supernatant") obtained by culturing PBMCs and can be used for the treatment of various inflammatory conditions. Thus, as used herein, "supernatant of peripheral blood mononuclear cell (PBMC) cell culture" refers to any supernatant obtained by culturing PBMCs in vitro in a culture medium. After the culturing step, the cultured PBMCs are removed from the culture medium in order to obtain a substantially cell-free, preferably completely cell-free, supernatant. The supernatant of the PBMC culture contains substances produced and secreted by the PBMCs and / or lysed PBMCs next to the components of the culture medium. "Supernatant" can be used interchangeably with conditioned culture medium obtained by culturing PBMCs.

[0014] The supernatant of the present invention can be obtained by culturing PBMCs exposed to ionizing radiation prior to or during culture, the ionizing radiation being preferably gamma radiation.

[0015] As used herein, "allergy or allergic reaction caused by administration of at least one food and / or inhalation allergen to the human or mammalian body or by systemic administration of at least one drug" refers to any adverse reaction caused by administration of food and / or inhalation allergen to the human or mammalian body. Food allergens are usually administered orally to the human or mammalian body. Inhalation allergens are typically administered by inhalation through the nose or mouth to the lungs and airways. Some drugs used to treat the human and mammalian body may cause an allergic reaction when administered systemically to such body. Such drugs may be administered to the human or mammalian body orally, by inhalation, parenterally, or any other route of administration, resulting in systemic distribution of the drug within such body. Parenteral administration of drugs may include intramuscular, intraperitoneal, intravenous, and other routes of administration. Topical administration of drug allergens or any other allergens on the skin does not result in systemic spread and therefore does not result in systemic administration to the human or mammalian body.

[0016] Allergens that cause allergies or allergic reactions when administered to the human or mammalian body include food allergens, drug allergens, inhalant allergens (e.g., pollen, chemicals), and any kind of allergen administered / introduced to the human or mammalian body. Thus, typical allergic reactions caused by administration of an allergen to the human or mammalian body include, among others, runny nose, difficulty breathing, nausea, diarrhea, nosebleeds, ear problems, wheezing, coughing, or even anaphylaxis if the allergen is introduced, for example, into the bloodstream.

[0017] As used herein, "allergen" refers to an antigen capable of stimulating a hypersensitive reaction in the human and / or mammalian body, typically via an immunoglobulin E (IgE) response resulting in excessive release of histamine from mast cells. The term "allergen" also includes fragments of such naturally occurring allergens, when the allergen is of biological origin and is a protein or polypeptide, when such fragments show similar effects as the naturally occurring allergen with respect to the release of histamine in the human or mammalian body.

[0018] The terms "prevent" and "prevention" as used herein refer to the prevention or inhibition of the recurrence, onset and occurrence of allergy or its symptoms in the human and mammalian body resulting from administration of the supernatant according to the present invention. In some embodiments, "prevent" and "prevention" refer to the reduction of the risk of developing an allergy to a particular allergen. The term "prevention" encompasses not only preventing the onset of allergy, but also measures to arrest its progression and reduce its consequences once established.

[0019] The terms "treatment" and "treating" as used herein refer to the reduction or inhibition of the progression and duration of an allergy, the reduction or amelioration of the severity of an allergy, and the amelioration of one or more symptoms thereof. "Treatment" also encompasses the amelioration and / or reversal of 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 with the compositions and methods of the present invention include those who already have an allergy and those in whom an allergy is to be prevented.

[0020] According to another preferred embodiment of the invention, the at least one allergen is a biological or chemical allergen.

[0021] Biological allergens include allergens derived from living systems such as plants, animals (e.g. insects, arachnids) or microorganisms (e.g. molds, bacteria). Such allergens are most often proteins, polypeptides or peptides. Biological allergens are well known in the art and are disclosed in various databases such as http: / / www.allergen.org / or http: / / www.allergome.org / .

[0022] According to a further preferred embodiment of the invention, the biological allergen is selected from the group consisting of an animal allergen, a plant allergen, a mould allergen or a bacterial allergen.

[0023] Animal allergens are allergens that include one or more compounds found in animals, including both vertebrates and invertebrates.Vertebrate allergens that may be present in the mixed allergen composition include bird allergens, such as egg allergens, such as nGal d 1 ovomucoid, n Gal d 2 ovalbumin, nGal d 3 conalbumin, egg white complete allergen, 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, milk complete allergen, fish allergens, such as rCyp c 1, rGad c 1, cod complete allergen, white fish allergen, pink fish allergen, etc. Invertebrate allergens that may be present in the mixed allergen composition include crustacean allergens, such as shrimp allergens, e.g. rPen a 1 tropomyosin, shrimp complete allergen, etc., insect allergens, such as bee sting venom allergens, wasp sting venom allergens, mosquito bite allergens, etc. Inhalant animal allergens may include cat or dog hair and dander, cockroach mushroom body calyx and dust mite excreta.

[0024] Plant allergens are allergens that include one or more compounds found in plants. Plant allergens of interest include wheat allergens, e.g., rTri a 19ω-5 gliadin, wheat complete allergen, gliadin wheat, rTri a 14 LTP, etc., kiwi allergens, e.g., rAct d 8 PR-10, kiwi complete allergen, etc., celery allergens, e.g., rApi g 1.01 PR-10, rPhl p 12, celery complete allergen, bromelain derived CCD MUXF3, etc., soybean allergens, e.g., rGly m 4 10 PR-10, soybean complete allergen, nGly m 5β-conglycinin, nGly m 6 glycinin, etc., stone fruit allergens, e.g., f419, f420, f421, f95, f242, o214 rPru p 1 PR-10, rPru p 3 LTP, stone fruit primary complete allergen, bromelain derived CCD MUXF3, etc., oat allergens, e.g. oat component allergen, oat complete allergen, etc., sesame allergens, e.g. sesame seed component allergen, sesame seed complete allergen, etc. Plant allergens also include inhalant allergens, such as pollen allergens. Such allergens may include birch pollen allergens (e.g. Bet v 1), grass pollen allergens (e.g. Phl p 1), ryegrass and timothy grass allergens.

[0025] Allergens include allergens isolated from natural sources or recombinantly or chemically produced allergens.

[0026] According to another preferred embodiment of the invention, the PBMC cell culture comprises monocytes, T cells, B cells and / or NK cells.

[0027] According to a further preferred embodiment of the present invention, the PBMC cells are cultured in a cell growth medium, preferably a CellGro medium, more preferably a cell culture medium selected from the group consisting of Cellgro GMP DC medium, RPMI, DMEM, X-vivo and Ultraculture.

[0028] The PBMCs of the PBMC cell culture are exposed to ionizing radiation before or during culture. In addition to these stress-inducing conditions, the PBMCs may be exposed to further stresses. Thus, according to a preferred embodiment of the present invention, the PBMCs are exposed to one or more further stress-inducing conditions before or during culture.

[0029] As used herein, the term "under stress-inducing conditions" refers to culture conditions that result in stressed cells. Conditions that cause stress to cells include heat, chemicals, radiation, hypoxia, osmotic pressure, etc., among others.

[0030] Further stress on the cells of the present invention results in further increased expression and secretion of substances beneficial for treating inflammatory skin conditions, particularly skin conditions associated with ischemia.

[0031] According to a preferred embodiment of the invention, the stress-inducing conditions include hypoxia, ozone, heat (e.g., 2°C higher, preferably 5°C higher, more preferably 10°C higher than the optimal culture temperature for PBMCs, i.e., 37°C), radiation (e.g., UV radiation, gamma radiation), chemicals, osmolarity (i.e., osmolarity conditions that are at least 10% increased compared to the osmolarity conditions normally occurring in body fluids, in particular blood), or a combination thereof.

[0032] Therefore, according to a further preferred embodiment of the present invention, the stress-inducing conditions are selected from the group consisting of UV radiation, hypoxia, ozone, heat, osmotic pressure and pH shift.

[0033] According to another preferred embodiment of the present invention, the PMC is exposed 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, more preferably at least 50 Gy.

[0034] According to a preferred embodiment of the invention, the PBMCs are cultured for at least 4 hours, preferably at least 6 hours, more preferably at least 12 hours, before isolating the supernatant thereof.

[0035] According to another preferred embodiment of the invention, the composition of the invention is administered before, during and / or after the onset of an allergic reaction and / or exposure to at least one allergen.

[0036] The composition of the present invention can be administered at different stages of an allergic reaction or even before the reaction occurs. In a particular preferred embodiment of the present invention, the composition may be administered before the human or mammalian body is exposed to an allergen. Surprisingly, it has been found that the composition of the present invention can prevent the uptake of an allergen or a fragment thereof into antigen-presenting cells. Thus, if such uptake can be prevented, the allergen or a fragment thereof is not presented to the immune system of a human or mammalian.

[0037] According to a preferred embodiment of the present invention, the PCBMC cell culture is 1×10 5 ~1×10 8 PBMC / ml, preferably 1 x 10 6 ~1×10 7 PBMC / ml, more preferably 2×10 6 ~5×10 6 Contains PBMCs / ml.

[0038] It was found that the composition of the supernatant obtained by culturing PBMCs exhibits advantageous properties when culturing a certain amount of PBMCs per ml of cell culture medium.

[0039] According to another preferred embodiment of the present invention, 0.1-5 ml of supernatant / kg body weight, preferably 0.3-3 ml / kg body weight, more preferably 0.5-2 ml / kg body weight, more preferably 0.8-1.2 ml / kg body weight is administered to the human or mammalian body.

[0040] The composition of the present invention comprises a sufficient amount of supernatant to treat or prevent allergy and allergic reaction. The volume of the supernatant administered to the human or mammalian body per kg of body weight as described above refers directly to the supernatant. If the volume is too large to be administered to the human or mammalian body, this volume can be reduced, for example, by lyophilization. Therefore, the volume of the composition of the present invention administered can be lower than the volume indicated for the supernatant. Those skilled in the art know which volume can be administered using a specific administration route.

[0041] According to a preferred embodiment of the invention, the compositions are administered by inhalation, topically, orally, sublingually, bucally, subcutaneously or intravenously.

[0042] The composition of the present invention may contain pharma- ceutically acceptable excipients, such as diluents, stabilizers, carriers, etc. Depending on the dosage form, the preparation according to the present invention contains the respective components, the methods of preparing which are well known to those skilled in the art.

[0043] To extend the shelf life of the compositions according to the invention, the supernatant or even the complete composition may be lyophilized. Methods for lyophilizing such preparations are well known to those skilled in the art.

[0044] Prior to its use, the lyophilized preparation can be contacted with water or an aqueous solution containing buffers, stabilizers, salts, and the like.

[0045] According to another preferred embodiment of the invention, the mammal is a horse, a dog, a cat or a camel.

[0046] The compositions of the present invention can be used to treat any type of mammal, however the aforementioned mammals are most preferred.

[0047] 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 into the human or mammalian body, comprising the step of administering a composition as defined above.

[0048] A further aspect of the present invention relates to a method for determining the suitability of a composition as defined above for use in the treatment or prevention of an allergy or an allergic reaction caused by the introduction of at least one allergen into the human or mammalian body, comprising the steps of: a) contacting at least two skin areas of a mammal with an allergen; b) administering a composition as defined above to at least one of said skin areas, wherein at least one of said skin areas has not been treated with said composition; c) comparing the skin area contacted with the allergen to the skin area contacted with the allergen and the composition; d) identifying differences between said regions; e) determining whether said composition is suitable for treating or preventing said disorder or disease The present invention relates to a method comprising the steps of:

[0049] The scratch assay may be used to test whether the composition 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 inhalant allergen to the human or mammalian body, or by systemic administration of at least one drug. If the composition of the present invention can reduce the allergic reaction at the scratch site compared to an untreated scratch site or a scratch site treated with a negative control composition, the composition of the present invention can be administered to the human or mammalian body in need thereof.

[0050] example Example 1: Aposec-induced mast cell degranulation disorder

[0051] Materials and Methods

[0052] Aposec Manufacturing

[0053] PBMC secretomes ("Aposec") were generated as described in Wagner T et al. (Sci Rep. 2018;8(1):18016). Briefly, PBMCs were obtained by Ficoll-Paque PLUS (GE Healthcare, USA)-assisted density gradient centrifugation and 2.5 × 10 7 The cells were then exposed to 60 Gy of cesium-137 gamma irradiation (IBL 437C, Isotopen Diagnostik CIS GmbH, Germany) and cultured for 24 ± 2 h in phenol red-free CellGenix GMP DC 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 by gamma irradiation of the lyophilized powder (25,000 Gy, Gammatro 1500, Mediscan, Austria) using the Theraflex methylene blue technique (MacoPharma, France) as previously described (Haider T et al., Exp Neurol. 2015;267:230-42). Sterile lyophilizates were routinely stored frozen at -80 °C.

[0054] Isolation and in vitro maintenance of primary human mast cells

[0055] Skin and subcutaneous adipose tissue used for mast cell isolation were obtained from patients undergoing abdominoplasty. The subcutaneous tissue and reticular dermis were removed and the remaining tissue was cut into small pieces and subjected to enzymatic digestion (2.4 U / mL Dispase II from Bacillus polymyxa (Roche, Switzerland)) overnight at 4°C. After removing the epidermis, the skin tissue was digested in collagenase I (Gibco, Thermo Fisher Scientific, USA) for 2 h at 37°C. CD117 + Mast cells were enriched by magnetic cell sorting technology (MACS System, Miltenyi Biotec, Germany) as suggested by the manufacturer. To increase the purity of the isolated cells, CD117 +The isolation procedure was repeated once more using cells. CD117+ mast cells were cultured in DMEM (Gibco) supplemented with 10% (vol / vol) inactivated fetal bovine serum (Gibco), 1% (vol / vol) penicillin / streptomycin (Biochrom, Germany) and 100 ng / mL recombinant human stem cell factor (PeproTech, USA).

[0056] Compound 48 / 80-induced degranulation of primary human mast cells

[0057] Primary human mast cells were seeded at a density of 50,000–100,000 per well in flat-bottom 96-well plates in 50 μL of DMEM without serum and SCF supplemented with colored pH indicator as described above. Cells were pretreated overnight with 50 μL of Aposec or 50 μL of media control (CellGenix). The next day, cells were carefully washed with 100 μL of HEPES (N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid, Thermo Fisher Scientific) and degranulation was induced by the addition of 100 μL of HEPES containing 50 μg / mL of compound 48 / 80 (Sigma Aldrich, USA). For unstimulated controls, HEPES was added. Cells were incubated for 1 h at 37 °C with normal ambient CO2. 50 μL of supernatant was separated and saved, and cells were lysed in 100 μL of 0.1% Triton X-100 (Sigma Aldrich).

[0058] IgE / anti-IgE-induced degranulation of primary human mast cells

[0059] Primary human mast cells were seeded at a density of 50,000–100,000 per well in flat-bottom 96-well plates in 50 μL of DMEM without serum and SCF supplemented with colored pH indicator 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 further 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 degranulation was induced by the addition of 100 μL of HEPES containing 5 μg / mL of anti-IgE antibody (Jackson ImmunoResearch Laboratories, Inc., USA). For non-stimulated controls, HEPES was added. The cells were incubated for 1 hour at 37° C. with normal ambient CO 2. 50 μL of the supernatant was separated and saved, and the cells were lysed in 100 μL of 0.1% Triton X-100 (Sigma Aldrich).

[0060] Reagents for β-hexosaminidase assay

[0061] Substrate solution for the β-hexosiminidase assay was prepared by dissolving 8.9 g disodium hydrogen phosphate dihydrate (Na2HPO4·2H2O) and 650 mg p-nitro-N-acetyl-β-D-glucosamide in 400 mL double distilled water and adjusting the pH to 4.5 with 0.4 M citric acid (all reagents Sigma Aldrich). Stop solution consisted of 15.02 g glycine in 900 mL water with the pH adjusted to 10.7 using 3 M sodium hydroxide.

[0062] β-Hexosaminidase assay

[0063] 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 for 90 min at 37°C with ambient CO. 75 μL of stop buffer was added, and the optical density at 405 nm was determined by a plate-reading luminometer LUMIstar OPTIMA Reader (BMG LABTECH, Ortenberg, Germany) using the FlUOstar OPTIMA software (version 1.20-0, BMG LABTECH).

[0064] Data and statistical analysis

[0065] The percentage of β-hexosaminidase released is calculated using the following formula:

number

[0066] Data are presented as the arithmetic mean and standard error of the mean of technical replicates. Statistical analysis was performed by one-tailed t-test comparing the two groups, Aposec versus medium control, with p<.05 considered statistically significant.

[0067] result

[0068] Aposec inhibits forced mast cell degranulation

[0069] To determine whether Aposec can prevent mast cell degranulation, we pretreated mast cells with Aposec or medium control and assessed β-hexosaminidase release in primary human mast cells after stimulation with compound 48 / 80 and IgE / anti-IgE. Aposec significantly prevented both compound 48 / 80-induced and IgE / anti-IgE-induced mediator release compared to medium control (27.9±3.6% and 27.3±1.1% released β-hexaminidase with Aposec, compared with 35.7±3.4% and 36.7±3.1% released mediator with control medium after compound 48 / 80 and IgE / anti-IgE stimulation, respectively; both p<.05 for Aposec vs. control) (Figure 1). In comparison, control medium had no effect on mast cell degranulation compared to mast cell medium [36.7 ± 2.3 and 36.9 ± 1.9 released mediators in mast cell medium after compound 48 / 80 and IgE / anti-IgE stimulation, respectively; p > .05 for mast cell medium vs. medium control in (A) and (B)].

[0070] conclusion

[0071] The present data demonstrate that Aposec effectively prevents mediator release by primary human mast cells when stimulated with compound 48 / 80 and IgE / anti-IgE. While enzyme release was reduced by more than 20% after chemical stimulation, β-hexosaminidase release after IgE / anti-IgE treatment was reduced by more than 25% with Aposec compared to media control. Collectively, these data suggest the use of Aposec to treat mast cell degranulation-mediated allergic responses.

[0072] Example 2: Reduction of symptoms of allergic hypersensitivity by application of Aposec in vivo

[0073] background

[0074] Over the past decades, extensive research into the pathogenesis of allergic hypersensitivity has contributed to a better understanding of immunological responses. However, the complex and multifaceted disease pathogenesis represents a major obstacle to the development of effective novel therapeutic interventions, and clinical treatment options remain limited to date. As the potent anti-inflammatory effects of Aposec have been previously described, we investigated the possibility that Aposec may reduce allergic hypersensitivity-related symptoms.

[0075] Materials and Methods

[0076] Mouse model

[0077] 1-Fluoro-2,4-dinitrobenzene (DNFB, Sigma-Aldrich) served as an allergen to induce an inflammatory state in C57BL / 6 mice. 20 μL of 0.25% (vol / vol) DNFB in olive oil was administered on days 0 and 1. Ears were treated daily with Aposec, and the contralateral ear received vehicle medium from day 0 for 6 consecutive days.

[0078] Micrometer Measurement

[0079] Twenty-four hours after DNFB rechallenge, ear thickness was assessed using an electronic digital micrometer (0–25 mm, Marathon Management Inc., USA) to measure the thickness of the outer two-thirds of the ear. Measurements were performed in quadruplicate.

[0080] statistical analysis

[0081] Data were statistically evaluated using GraphPad Prism 6 software (GraphPad Software Inc.). One-tailed paired t-tests were performed to compare Aposec with control medium. A p-value of <0.05 was considered statistically significant. Data are presented as the arithmetic mean and standard error of the mean of biological replicates.

[0082] result

[0083] Aposec reduces allergen-induced tissue swelling

[0084] Murine DNFB-induced hypersensitivity was used as a model to study the anti-inflammatory effect of Aposec in allergic reactions in vivo. The degree of ear swelling, reflecting the severity of the immune response, was found to be significantly reduced by Aposec compared to medium control 24 hours after DNFB re-exposure (vehicle-treated ear thickness 553.9±12.7 μm vs. 472.4±47.3 μm with Aposec, p<.05 for Aposec vs. medium control; naïve ear: thickness 355±12.7 μm) (Figure 2).

[0085] conclusion

[0086] These data indicate that application of Aposec effectively prevented tissue swelling after allergen re-exposure. These findings indicate that Aposec is a promising candidate for treating symptoms associated with allergic reactions.

Claims

1. A composition comprising a supernatant of a peripheral blood mononuclear cell (PBMC) cell culture for use in the treatment or prevention of allergy or allergic reaction caused by administration of at least one food and / or inhaled allergen to the human or mammalian body or by systemic administration of at least one drug, wherein the PBMCs are exposed to ionizing radiation prior to or during culture.

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

3. The composition for use according to claim 2, wherein the biological allergen is selected from the group consisting of an animal allergen, a plant allergen, a mold allergen, and a bacterial allergen.

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

5. The composition for use according to any one of claims 1 to 4, wherein the PBMCs are cultured in a cell culture medium selected from the group consisting of cell growth medium, RPMI and DMEM.

6. The composition for use according to any one of claims 1 to 5, wherein the PBMCs are exposed to one or more further stress-inducing conditions before or during culture.

7. 7. The composition for use according to claim 6, wherein the stress-inducing conditions are selected from the group consisting of UV irradiation, hypoxia, ozone, heat, osmotic pressure and pH shift.

8. The composition for use according to any one of claims 1 to 7, wherein the PBMCs are exposed to ionising radiation at a dose of at least 10 Gy, at least 20 Gy, at least 40 Gy, or at least 50 Gy.

9. The composition for use according to any one of claims 1 to 8, wherein the PBMCs are cultured for at least 4 hours, at least 6 hours, or at least 12 hours before isolating the supernatant thereof.

10. The composition for use according to any one of claims 1 to 9, wherein said composition is administered before, during and / or after the onset of an allergic reaction and / or exposure to at least one allergen.

11. The PBMC cell culture is 1×10 ５ ~1×10 ８ PBMC / ml, 1 x 10 ６ ~1×10 ７ PBMC / ml, or 2 x 10 ６ ~5×10 ６ The composition for use according to any one of claims 1 to 10, comprising PBMCs / ml.

12. 12. The composition for use according to any one of claims 1 to 11, wherein 0.1 to 5 ml of supernatant / kg body weight, 0.3 to 3 ml / kg body weight, 0.5 to 2 ml / kg body weight, or 0.8 to 1.2 ml / kg body weight is administered to the human or mammalian body.

13. The composition for use according to any one of claims 1 to 12, wherein the composition is administered by inhalation, topically, orally, sublingually, bucally, subcutaneously or intravenously.

14. The composition for use according to any one of claims 1 to 13, wherein the mammal is a horse, a dog, a cat or a camel.

15. A method for determining the suitability of a composition as defined in any one of claims 1 to 13 for use in the treatment or prevention of allergies or allergic reactions caused by the administration to the human or mammalian body of at least one food and / or inhaled allergen or by the systemic administration of at least one drug, comprising the steps of: a) comparing an area of ​​mammalian skin contacted with an allergen to an area of ​​mammalian skin contacted with said allergen and said composition; b) identifying differences between said regions; c) determining whether said composition is suitable for treating or preventing said allergy or allergic reaction; and

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