Composition for the treatment or prevention of allergies or allergic reactions

Abstract

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

Description

Technology Field

[0001] The present invention relates to a composition and a method for treating and preventing disorders caused by the absorption of allergens into the human or mammalian body. Background Technology

[0002] During the sensitization phase of allergies, professional antigen-presenting cells phagocytize antigens (referring to allergens) and T process the treated antigens H 2. Cross-presentation to lymphocytes. These cells respond to allergen encounters via the release of interleukin 4 and interact with B lymphocytes. Activated B cells produce immunoglobulin E (IgE) against allergens in conjunction with IL-4. The secreted IgE binds to IgE-specific receptors present, for example, on mast cells. These immune cells are sensitive to allergens. Re-exposure results in the binding of allergens to IgE-coated immune cells, which triggers the activation of sensitized cells following receptor cross-linking. Activated mast cells respond with the massive release of immune mediators, such as histamine, cytokines, and pro-inflammatory lipids, from intracellular granules. The released mediators have pleiotropic effects, including vasodilation, mucus secretion, and neurostimulation, causing itching and redness. These symptoms can be localized or systemic.

[0003] Mast cells are primarily found in tissues that repeatedly face pathogenic stimuli, such as the skin, airways, and gastrointestinal tract. Generally, mast cells are activated by immune signals such as antigen / IgE complexes, proteins of the complement system, or Fcγ receptors cross-linked by toll-like receptor agonists. Mast cells respond to activation with the massive release of immune mediators. Mast cells are best known for their role in IgE-dependent allergic reactions, and targeting mast cell functions, such as degranulation, can help alleviate the most distressing symptoms associated with allergic reactions.

[0004] The object of the present invention is to provide a method and means for preventing or treating an allergic reaction caused by an allergen administered to the body of a human or mammal. The problem to be solved

[0005] 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 the administration of at least one food and / or inhaled allergen or the systemic administration of at least one drug to a human or mammalian body, wherein the PBMC is exposed to ionizing radiation before or during culture.

[0006] Surprisingly, it has been found that the supernatant of a 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 regarded as a promising tool for the regeneration of various damaged tissues and organs. Since then, numerous studies have challenged this concept through reports that secreted factors, rather than the cells themselves, exert the observed regenerative effects. Peripheral blood mononuclear cells (PBMCs) represent an attractive and easily accessible source of cell secretions with diverse functions, in contrast to stem cells. While immunomodulatory effects are preferably attributed to the secretions of γ-irradiated PBMCs (Aposec), it is surprising that the supernatant of PBMC cultures exhibits beneficial effects regarding allergic reactions and allergies, particularly when triggered by allergens.

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

[0009] Another aspect of the present invention relates to a method for determining the suitability of a composition as defined herein for use in the treatment or prevention of an allergic reaction or allergy induced by the administration of at least one food and / or inhaled allergen or the systemic administration of at least one drug to a human or mammalian body, comprising the following steps:

[0010] a) a step of bringing at least two or more skin areas of a mammal into contact with an allergen,

[0011] b) a step of administering a composition defined in any one of claims 1 to 17 to at least one of the skin areas, wherein the at least one of the skin areas is not treated with the composition, and

[0012] c) a step of comparing a skin area in contact with the allergen with a skin area in contact with the allergen and the composition,

[0013] d) a step of identifying differences between the above regions, and

[0014] e) A step of determining whether the above composition is suitable for treating or preventing the above disorder or disease. means of solving the problem

[0015] The present invention relates to a composition comprising the supernatant of a PBMC cell culture for use in the treatment or prevention of allergies or allergic reactions caused by the administration of at least one food and / or inhaled allergen or the systemic administration of at least one drug to the body of a human or mammal.

[0016] Cells, particularly mammalian cells, are known to secrete numerous substances while being cultured in cell culture media. The controlled culture media thus obtained can be used for the treatment and / or prevention of various diseases and disorders. For example, WO 2010 / 070105 and WO 2010 / 079086 describe controlled culture media ("supernatants") obtained by the culture of PBMCs and which can be used for the treatment of various inflammatory conditions. Accordingly, as used herein, "a supernatant of a peripheral blood mononuclear cell (PBMC) cell culture" refers to any supernatant obtained by culturing PBMCs in vitro in culture media. After the culture step, the cultured PBMCs are removed from the culture media to obtain a substantially cell-free, preferably completely cell-free, supernatant. The supernatant of the above PBMC culture contains substances produced and secreted by PBMC and / or even dissolved PBMC alongside the culture medium components. The "supernatant" can be used interchangeably with the controlled culture medium obtained by culturing PBMC.

[0017] The supernatant of the present invention can be obtained by culturing PBMCs that receive ionizing radiation before or during cultivation. The ionizing radiation is preferably gamma radiation.

[0018] As used herein, “An allergy or an allergic reaction caused by the administration of at least one food and / or inhalation allergen or by the systemic administration of at least one drug to a human or mammal body” means any adverse reaction caused by the administration of a food and / or inhalation allergen to a human or mammal body. Food allergens are generally administered orally to a human or mammal body. Inhalation allergens are typically administered by inhaling into the lungs and airways through the nose or mouth. Some drugs used to treat human and mammalian bodies may cause allergic reactions once administered systemically to such bodies. Such drugs may be administered orally, by inhalation, parenterally, or by any other route of administration to a human or mammalian body, resulting in systemic distribution of the drug within such bodies. Parenteral administration of drugs may include intramuscular, intraperitoneal, intravenous, and other routes of administration. Topical administration of drug allergens or other allergens to the skin does not cause systemic spread, so systemic administration occurs in the human or mammalian body.

[0019] Allergens that cause allergies or allergic reactions when administered to the body of a human or mammal include food allergens, drug allergens, inhaled allergens (e.g., pollen, chemicals), and all types of allergens administered to or introduced into the body of a human or mammal. Accordingly, typical allergic reactions caused by administering allergens to the body of a human or mammal, for example when the allergens are introduced into the bloodstream, include runny nose, difficulty breathing, nausea, diarrhea, nosebleeds, ear problems, wheezing, coughing, or anaphylaxis.

[0020] As used herein, “allergen” means an antigen capable of stimulating a hypersensitivity reaction in the human and / or mammalian body through an immunoglobulin E (IgE) response that typically causes the excessive release of histamine from mast cells. The term “allergen” includes—where the allergen is of biological origin and is a protein or polypeptide—and also includes fragments of such naturally occurring allergens when such fragments exhibit effects similar to those of naturally occurring allergens in relation to the release of histamine within the human or mammalian body.

[0021] As used herein, the terms “preventing” and “prevention” mean the prevention or suppression of the recurrence, onset, and development of allergies or symptoms thereof in the human and mammalian bodies resulting from the administration of the supernatant according to the present invention. In some embodiments, “preventing” and “prevention” mean the reduction of the risk of developing an allergy to a specific allergen. The term “preventing” includes measures to prevent the occurrence of an allergy, as well as to stop the progression of the allergy and, once it occurs, to reduce its consequences.

[0022] As used herein, “treatment” and “treating” mean the reduction or suppression of the progression and duration of an allergy, or the reduction or improvement of the severity of said allergy and one or more of its symptoms. “Treatment” also includes the improvement and / or reversal of symptoms of an allergy or allergic reaction. The term “treatment” means both therapeutic treatment and preventive measures. For example, people who may benefit from treatment by the compositions and methods of the present invention include people who already have allergies and people who need to prevent allergies.

[0023] According to another preferred embodiment of the present invention, at least one allergen is a biological or chemical allergen.

[0024] Biological allergens include allergens derived from biological systems such as plants, animals (e.g., insects, arachnids), or microorganisms (e.g., fungi, bacteria). In most cases, these allergens are proteins, polypeptides, or peptides.

[0025] Biological allergens are well known in the art and include allergens derived from biological systems such as plants, animals (e.g., insects, arachnids), or microorganisms (e.g., fungi, bacteria). In most cases, these allergens are proteins, polypeptides, or peptides. Biological allergens are well known in the art and are publicly available in various databases such as http: / / www.allergen.org / or http: / / www.allergome.org / .

[0026] 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.

[0027] Animal allergens are allergens containing one or more compounds found in animals, including vertebrates and invertebrates. Vertebrate allergens that may be present in a mixed allergen composition include avian allergens such as egg allergens, e.g., nGal d 1 Ovomucoid, nGal d 2 Ovalbumin, nGal d 3 Conalbumin, egg white total allergens, etc.; mammalian allergens such as milk allergens, e.g., nBos d 4 alpha-lactalbumin, nBos d 5 beta-lactoglobulin, nBos d 8 Casein, nBos d Lactoferrin, milk total allergens, etc.; and fish allergens such as, e.g., rCyp c 1, rGad c 1, cod total allergens, white fish allergens, pink fish allergens, etc. Invertebrate allergens that may be present in a mixed allergen composition include crustacean allergens such as shrimp allergens, e.g., rPen a 1 tropomyosin, shrimp complete allergen, etc.; insect allergens such as, e.g., bee sting venom allergen, wasp sting venom allergen, mosquito bite allergen, etc. Inhalable animal allergens may include cat or dog hair and dander, cockroaches, calyxes, and house dust mite excrement.

[0028] Plant allergens are allergens containing one or more compounds found in plants. Plant allergens of interest include the following: wheat allergens, e.g., rTri a 19 Omega-5 Gliadin, wheat allergen, gliadin wheat, rTri a 14 LTP, etc.; kiwi allergens, e.g., rAct d 8 PR-10, kiwi allergen, etc.; celery allergens, e.g., rApi g 1.01 PR-10, rPhl p 12, celery allergen, Bromelain CCD MUXF3, etc.; soybean allergens, e.g., rGly m 4 10 PR-10, soybean allergen, nGly m 5 Beta-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's CCD MUXF3, etc.; oat allergens, e.g., oat component allergens, oat complete allergens, etc.; sesame allergens, e.g., sesame seed component allergens, sesame reference complete allergens, etc. 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.

[0029] Allergens include allergens isolated from natural sources or recombinant or chemically produced allergens.

[0030] According to another preferred embodiment of the present invention, the PBMC cell culture comprises monocytes, T cells, B cells and / or NK cells.

[0031] According to a further preferred embodiment of the present invention, the PBMC cells are cultured in a cell culture medium selected from the group consisting of cell growth medium, preferably CellGro medium, more preferably Cellgro GMP DC medium, RPMI, DMEM, X-vivo and Ultraculture.

[0032] The PBMCs of the above PBMC cell culture are exposed to ionizing radiation before or during culture. In addition to these stress-inducing conditions, the PBMCs may be subjected to additional stress. Accordingly, according to a preferred embodiment of the present invention, the PBMCs undergo one or more additional stress-inducing conditions before or during culture.

[0033] As used herein, the term “under stress-inducing conditions” refers to culture conditions that induce stressed cells. Conditions that cause stress to cells include heat, chemicals, radiation, hypoxia, osmotic pressure, etc.

[0034] The additional stress on the cells of the present invention further increases the expression and secretion of substances beneficial for treating inflammatory skin conditions, particularly skin conditions associated with ischemia.

[0035] According to a preferred embodiment of the present invention, the stress-inducing conditions include hypoxia, ozone, heat (e.g., 2°C or higher, preferably 5°C or higher, more preferably 10°C or higher, higher than the optimal culture temperature of PBMC, i.e., 37°C), radiation (e.g., UV radiation, gamma radiation), chemicals, osmotic pressure (i.e., an osmotic pressure that is at least 10% higher than the osmotic pressure that regularly occurs in body fluids, particularly blood), or a combination thereof.

[0036] Accordingly, according to a further preferred embodiment of the present invention, the stress-inducing condition is selected from the group consisting of UV radiation, hypoxia, ozone, heat, osmotic pressure, and pH shift.

[0037] According to another preferred embodiment of the present invention, the PMC is exposed to ionizing radiation, preferably gamma rays, at a dosage of at least 10 Gy, preferably at least 20 Gy, more preferably at least 40 Gy, and more preferably at least 50 Gy.

[0038] According to a preferred embodiment of the present invention, the PBMC was cultured for at least 4 hours, preferably at least 6 hours, and more preferably at least 12 hours before isolating the supernatant.

[0039] According to a preferred embodiment of the present invention, the composition of the present invention is administered before, during, and / or after the onset of an allergic reaction and / or exposure to at least one allergen.

[0040] The composition of the present invention may be administered at different stages of an allergic reaction or even before the reaction occurs. In a particularly preferred embodiment of the present invention, the composition may be administered before a human or mammalian body is exposed to an allergen. Surprisingly, it has been found that the composition of the present invention can prevent the absorption of an allergen or a fragment thereof by antigen-presenting cells. Therefore, if such absorption can be prevented, the allergen or a fragment thereof will not be presented to the immune system of a human or mammal.

[0041] According to a preferred embodiment of the present invention, the PBMC cell culture is 1 x 10 5 Up to 1x10 8 PBMCs / ml, preferably 1x10 6 Up to 1x10 7 PBMCs / ml, more preferably 2x10 6Up to 5x10 6 It contains PBMCs / ml.

[0042] It has been found that the composition of the supernatant obtained by culturing PBMC exhibits advantageous characteristics when a certain amount of PBMC is cultured per ml of cell culture medium.

[0043] According to another preferred embodiment of the present invention, 0.1 to 5 ml supernatant / kg body weight, preferably 0.3 to 3 ml / kg body weight, more preferably 0.5 to 2 ml / kg body weight, more preferably 0.8 to 1.2 ml / kg body weight is administered to a human or mammalian body.

[0044] The composition of the present invention comprises an amount of supernatant sufficient to treat or prevent allergies and allergic reactions. As indicated above, the volume of supernatant administered per kg of body weight refers directly to the supernatant. If the said volume is too large to be administered to the body of a human or mammal, this volume may be reduced, for example, by freeze-drying. Accordingly, the volume of the composition of the present invention administered may be lower than that indicated for the supernatant. Those skilled in the art know the volume that can be administered using a specific route of administration.

[0045] According to a preferred embodiment of the present invention, the composition is administered by inhalation, topically, orally, sublingually, orally, subcutaneously, or intravenously.

[0046] The composition of the present invention may include pharmaceutically acceptable excipients such as diluents, stabilizers, carriers, etc. Each component is included according to the dosage of the formulation according to the present invention. A method for manufacturing the same is well known to those skilled in the art.

[0047] To increase the shelf life of the composition according to the present invention, the supernatant or even the entire composition may be freeze-dried. Methods for freeze-drying such formulations are well known to those skilled in the art.

[0048] Before use, lyophilized formulations may come into contact with water or aqueous solutions containing buffers, stabilizers, salts, etc.

[0049] According to another preferred embodiment of the present invention, the mammal is a horse, dog, cat, or camel.

[0050] The composition of the present invention may be used to treat any type of mammal. However, the aforementioned mammal is most preferred.

[0051] Another aspect of the present invention relates to a method for treating or preventing an allergy or allergic reaction caused by the absorption of at least one allergen into a human or mammalian body, comprising the step of administering a composition as defined above.

[0052] A further aspect of the present invention relates to a method for determining the suitability of a composition defined above for use in the treatment or prevention of an allergic reaction or allergy induced by the administration of at least one food and / or inhaled allergen or the systemic administration of at least one drug to a human or mammalian body, comprising the following steps:

[0053] a) a step of bringing at least two or more skin areas of a mammal into contact with an allergen,

[0054] b) a step of administering the composition to at least one of the skin areas as defined above, wherein the at least one of the skin areas is not treated with the composition, and

[0055] c) a step of comparing a skin area in contact with the allergen with a skin area in contact with the allergen and the composition,

[0056] d) a step of identifying differences between the above regions, and

[0057] e) A step of determining whether the above composition is suitable for treating or preventing the above disorder or disease.

[0058] A scratch assay may be used to test whether the composition of the present invention can be used for the treatment or prevention of allergies or allergic reactions caused by the administration of at least one food and / or inhaled allergen or the systemic administration of at least one drug to a human or mammalian body. If the composition of the present invention can reduce the allergic reaction at said scratch site compared to an untreated scratch site or a scratch site treated with a negative control composition, the composition of the present invention may be administered to a human or mammalian body in need thereof. Brief explanation of the drawing

[0059] Figure 1 shows that MNCaposec prevents the release of compound 48 / 80 and IgE / anti-IgE-induced mediators by primary human mast cells. Mast cell degranulation is evaluated by (A) compound 48 / 80 and (B) beta-hexaminidase released upon IgE / anti-IgE stimulation of primary human mast cells. An asterisk indicates p<.05 for Aposec compared to a medium control (CellGro). Mast cell medium refers to the medium routinely used to culture mast cells (DMEM alone). Figure 2 shows that PBMC secretome Aposec alleviates ear edema in DNFB-induced hypersensitivity. Ear thickness is evaluated by micrometer-assisted measurements 24 hours after the DNFB retest. p<0.05 Aposec versus medium control. Specific details for implementing the invention

[0060] Example 1: Degranulation of damaged mast cells in Aposec

[0061] Materials and Methods

[0062] Aposec manufacturing

[0063] The secretome (“Aposec”) of PBMC was generated as described in Wagner T et al. (Sci Rep. 2018;8(1):18016). Briefly, PBMC was obtained by Ficoll-Paque PLUS (GE Healthcare, USA)-assisted density gradient centrifugation, and 2.5 x 10⁶ 7 The concentration was adjusted to cells / mL. Subsequently, cells were exposed to 60 Gy Cesium 137 γ-irradiation (IBL 437C, Isotopen Diagnostik CIS GmbH, Germany) and cultured in phenol red-free CellGenix GMP DC medium (CellGenix GmbH, Germany) for 24 ± 2 hours. Cells and cell debris were removed by centrifugation, and the supernatant was passed through a 0.2 μm filter. Virus removal was performed using Theraflex methylene blue technology (MacoPharma, France) and γ-irradiation of the lyophilized powder (25,000 Gy, Gammatro 1500, Mediscan, Austria) as previously described (Haider T et al. Exp Neurol. 2015;267:230-42). The sterile lyophilized powder was routinely cryopreserved at -80°C.

[0064] Isolation and in vitro isolation of primary human mast cells ( in vitro ) maintain

[0065] The skin and subcutaneous adipose tissue used for mast cell isolation were obtained from patients who had undergone abdominoplasty. The subcutaneous tissue and reticular dermis were removed, and the remaining tissue was cut into small pieces and subjected to enzymatic digestion (Bacillus polymyxa, 2.4 U / mL dispase II from Roche, Switzerland) overnight at 4°C. After removing the epidermis, the dermal tissue was digested with collagenase I (Gibco, Thermo Fisher Scientific, USA) at 37°C for 2 hours. CD117 + Mast cells were enhanced by magnetic cell sorting technology (MACS System, Miltenyi Biotec, Germany) as indicated by the manufacturer. To increase the purity of the isolated cells, CD117 in the first isolation run + The isolation procedure was repeated one or more times using cells. CD117 + Mast cells were cultured in DMEM supplemented with 10% (vol / vol) heat-inactivated fetal calf serum (both Gibco), 1% (vol / vol) penicillin / streptomycin (Biochrom, Germany), and 100 ng / mL recombinant human stem cell factor (PeproTech, USA).

[0066] Compound 48 / 80-induced degranulation in primary human mast cells

[0067] Primary human mast cells were seeded at a density of 50,000 to 100,000 per well in 50 μL DMEM flat-bottom 96-well plates without colored pH indicator supplemented with serum and SCF as described above. Cells were pretreated overnight with 50 μL Aposec or 50 μL medium control (CellGenix). The next day, cells were carefully washed with 100 μL HEPES (N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid; Thermo Fisher Scientific), and degranulation was induced by adding 100 μL HEPES containing 50 μg / mL Compound 48 / 80 (Sigma Aldrich, USA). For the unstimulated control, HEPES was added. Cells were incubated at 37°C for 1 hour with normal ambient CO2. 50 μL of the supernatant was separated and preserved, and the cells were lysed in 100 μL of 0.1% Triton X-100 (Sigma Aldrich).

[0068] IgE / anti-IgE-induced degranulation of primary human mast cells

[0069] Primary human mast cells were seeded at a density of 50,000 to 100,000 per well in 50 μL DMEM flat-bottom 96-well plates without colored pH indicator supplemented with serum and SCF as described above. Cells were pre-treated with 50 μL Aposec, 50 μL medium control (CellGenix), or 50 μL 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 HEPES (N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid; Thermo Fisher Scientific), and degranulation was induced by adding 100 μL HEPES containing 5 μg / mL anti-IgE antibody (Jackson ImmunoResearch Laboratories, Inc., USA). For the unstimulated control, HEPES was added. Cells were cultured at 37°C for 1 hour with normal ambient CO2. 50 μL of the supernatant was separated and preserved, and the cells were lysed in 100 μL of 0.1% Triton X-100 (Sigma Aldrich).

[0070] Reagents for beta-hexosaminidase assay

[0071] 50 μL of substrate solution was added to 50 μL of supernatant and 50 μL of Triton-lyzed cells, respectively, and the samples were incubated at 37°C with ambient CO2 for 90 minutes. 75 μL of stop buffer was added, and the optical density at 405 nm was determined using the LUMIstar OPTIMA Reader (BMG LABTECH, Ortenberg, Germany), a plate-reading photometer, with FlUOstar OPTIMA software (version 1.20-0, BMG LABTECH).

[0072] Data and statistical analysis

[0073] The percentage of released beta-hexosaminidase is calculated using the following formula:

[0074]

[0075] Data are expressed as the arithmetic means of technical replication and the standard error of the mean. Statistical analysis was performed by a one-tailed t-test comparing the two groups, Aposec versus the medium control group, which were statistically significant at p<.05.

[0076] result

[0077] Aposec abolishes forced mast cell degranulation.

[0078] To determine whether Aposec could prevent mast cell degranulation, mast cells were pre-treated with Aposec or a medium control, and the release of beta-hexosaminidase from primary human mast cells was evaluated after stimulation with compound 48 / 80 and IgE / anti-IgE. Aposec significantly prevented both compound 48 / 80- and IgE / anti-IgE-induced mediator releases compared to the medium control (β-hexaminidase released at 27.9±3.6% and 27.3±1.1% with Aposec after stimulation with compound 48 / 80 and IgE / anti-IgE, respectively, versus mediators released at 35.7±3.4% and 36.7±3.1% in the control medium, both p < .05 aposec vs. control) (Fig. 1). In contrast, the control medium showed no effect on mast cell degranulation compared to the mast cell medium [36.7±2.3 and 36.9±1.9 released mediators with the mast cell medium after compound 48 / 80 and IgE / anti-IgE stimulation, respectively. p>.05 mast cell medium versus control medium in (A) and (B)].

[0079] conclusion

[0080] These data demonstrate that Aposec effectively prevents mediator release by primary human mast cells when stimulated with compounds 48 / 80 and IgE / anti-IgE. Enzyme release was reduced by more than 20% after chemical stimulation, whereas beta-hexosaminidase release after IgE / anti-IgE treatment was reduced by more than 25% in Aposec compared to the medium control. Taken together, these data suggest the use of Aposec for treating mast cell degranulation-mediated allergic reactions.

[0081] Example 2: In vivo ( in vivo Relief of allergic hypersensitivity symptoms by Aposec application

[0082] background

[0083] Over the past few decades, extensive research on the pathology of allergic hypersensitivity has contributed to a better understanding of immunological responses. However, the complex and multifaceted etiology of the disease represents a major obstacle to the development of effective new therapeutic interventions, and clinical treatment options remain limited to date. Since the potent anti-inflammatory effects of Aposec have been previously described, we investigated the potential of Aposec to alleviate symptoms associated with allergic hypersensitivity.

[0084] Materials and Methods

[0085] Mouse model

[0086] 1-Fluoro-2,4-dinitrobenzene (DNFB, Sigma-Aldrich) acted as an allergen inducing 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. One ear was treated with Aposec daily, and the opposite ear received vehicle medium for 6 consecutive days starting from day 0.

[0087] Micrometric measurements

[0088] 24 hours after the DNFB re-test, the ear thickness was evaluated using an electronic digital micrometer (0-25 mm, Marathon Management Inc, USA) to measure the thickness of the outer 2 / 3 of the ear. The measurement was performed four times.

[0089] Statistical analysis

[0090] Data were statistically evaluated using GraphPad Prism 6 software (GraphPad Software Inc.). A one-sided, paired t-test was performed to compare Aposec versus control media. A p-value less than 0.05 was considered statistically significant. Data are expressed as the arithmetic mean of biological replication and the standard error of the mean.

[0091] result

[0092] Aposec relieves allergen-induced tissue swelling .

[0093] Murine DNFB-induced hypersensitivity was used as a model to study the anti-inflammatory effects of Aposec in in vivo allergic reactions. The degree of ear dilation, reflecting the severity of the immune response, was found to be significantly reduced by Aposec compared to the medium control 24 hours after DNFB re-exposure (bechle-treated ear thickness 553.9±12.7 μm vs. Aposec 472.4±47.3 μm, p<.05; Aposec vs. medium control. Naive ear: 355±12.7 μm thickness) (Fig. 2).

[0094] conclusion

[0095] These data indicate that the application of Aposec effectively prevented tissue swelling following allergen re-exposure. These findings suggest that Aposec is a promising candidate for treating symptoms associated with allergic reactions.

Claims

Claim 1 A composition comprising the supernatant of a peripheral blood mononuclear cell (PBMC) cell culture for use in the treatment or prevention of an allergy or allergic reaction caused by the administration of at least one food and / or inhaled allergen or the systemic administration of at least one drug to a human or mammalian body, wherein the PBMC is exposed to ionizing radiation before or during culture. Claim 2 A composition according to claim 1, characterized in that at least one allergen is a biological or chemical allergen. Claim 3 A composition according to paragraph 2, characterized in that the biological allergen is selected from the group consisting of animal allergens, plant allergens, mold allergens, or bacterial allergens. Claim 4 A composition according to any one of claims 1 to 3, characterized in that the PBMC cell culture comprises monocytes, T cells, B cells and / or NK cells. Claim 5 A composition according to any one of claims 1 to 3, wherein the PBMC is cultured in an RPMI or DMEM cell culture medium. Claim 6 A composition according to any one of claims 1 to 3, wherein the PBMC is applied to one or more additional stress-inducing conditions before or during culture. Claim 7 A composition according to claim 6, characterized in that the stress-inducing condition is selected from the group consisting of UV radiation, hypoxia, ozone, heat, osmotic pressure, and pH shift. Claim 8 A composition according to any one of claims 1 to 3, characterized in that the PBMC is exposed to ionizing radiation at a dosage of at least 10 Gy. Claim 9 A composition according to any one of claims 1 to 3, characterized in that the PBMC is cultured for at least 4 hours before isolating the supernatant thereof. Claim 10 A composition according to any one of claims 1 to 3, characterized in that the composition is administered before, during, and / or after the onset of an allergic reaction and / or exposure to at least one allergen. Claim 11 In any one of claims 1 to 3, the PBMC cell culture is 1 x 10 5 Up to 1x10 8 A composition characterized by containing PBMCs / ml. Claim 12 A composition according to any one of claims 1 to 3, characterized in that 0.1 to 5 ml of supernatant / kg body weight is administered to a human or mammalian body. Claim 13 A composition according to any one of claims 1 to 3, characterized in that the composition is administered by inhalation, topically, orally, sublingually, orally, subcutaneously, or intravenously. Claim 14 A composition characterized in that, in any one of claims 1 to 3, the mammal is a horse, dog, cat, or camel. Claim 15 delete Claim 16 delete

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