Ex vivo human model for the evaluation of allergic or pseudo-allergic inflammatory potential

Abstract

The present invention relates to an in vitro method for determining the inflammatory potential of a substance comprising the steps of: ia) administering to a skin explant, topically or by subcutaneous injection, a composition comprising the substance; which skin explant comprises the epidermis, the dermis and the epidermal appendages as well as a thickness of at least 5 millimeters of hypodermis; ib) determining the inflammatory response within the skin explant; ic) determining the level of mast cell degranulation within the skin explant; and ii) determining the inflammatory potential of the substance.

Description

Title of the invention: Ex vivo human model intended for the evaluation of allergic or pseudo-allergic inflammatory potential Technical field

[0001] The present invention relates to the field of inflammation, whether allergic, pseudo-allergic or other in nature, and proposes, more specifically, the first human ex vzvo model to determine the inflammatory potential of a compound. Prior art

[0002] The inflammatory response is an integral part of the immune system's reaction to an attack. Now, such an inflammatory response can be induced in a subject following an injection. Beyond the discomfort caused, this response can be problematic, particularly if it is an allergic response. Therefore, it is important to be able to predict, prior to any injection, the inflammatory response likely to be induced by a substance.

[0003] In connection with allergy, the biological phenomenon leading to the development of such an allergy occurs from 2 successive phases, with an initial phase called asymptomatic “sensitization” and a second phase of symptomatic “allergic reaction”.

[0004] The first phase, known as sensitization, begins when the individual comes into contact for the first time with a compound called an allergen because it generates an allergic response. This is then recognized and considered as a foreign substance by certain cells of the immune system which are present in large quantities in the skin and mucous membranes (antigen-presenting cells, e.g. dendritic cells). These cells will present the allergen on their surface and allow the production of Immunoglobulin E (IgE) by other cells.

[0005] These IgEs will quickly pass into the blood and attach themselves to cells called mast cells which are located in particular in the skin and mucous membranes (locations where allergens are likely to penetrate). This process of IgE binding is called "sensitization", because it makes the mast cells sensitive to activation in the event of a subsequent encounter with the same antigen. This first phase is silent, that is to say that the subject in the sensitization phase is asymptomatic.

[0006] During subsequent contact between the allergen and the "sensitized" organism, the allergen will bind to the IgE present on the surface of the mast cells, causing their activation. This activation results in the degranulation of the mast cells which leads to the massive release of histamine and inflammatory mediators into the body.

[0007] The mast cell is a cell present in connective tissues, which is part of the white blood cells and is characterized by the presence in its cytoplasm of very numerous granules containing chemical mediators such as serotonin, rhistamine, tryptase or heparin. When it is in contact with an allergen and presents on its surface the specific IgE of this or in contact with infectious agents, it degranulates and releases its mediators very quickly, by an exocytosis mechanism. It thus triggers immediate inflammatory reactions, sometimes serious, such as anaphylactic shock which causes hypotension. The same activation induces, in a more delayed manner (a few hours), the synthesis of numerous cytokines (such as TNF-alpha), chemokines and lipid mediators.

[0008] During allergic manifestations, this histamine will exert its effects by binding mainly to the H1 receptors present in the nose, where histamine increases edema and obstruction, and causes itching, sneezing and mucus secretions, in the skin, where histamine causes erythema, edema and itching, and in the lungs where histamine causes bronchoconstriction.

[0009] In connection with mast cells, the pseudo-allergic potential must also be taken into account. This is a mechanism that is not mediated by IgE (unlike allergy), but by the MRGPRX2 receptor (Mas-Related G-Protein coupled Receptor member X2). This mechanism is then dose-dependent. Therefore, these pseudo-allergic reactions could be prevented, or limited, in the event of re-administration of the substance by reducing the dose or decreasing the speed of administration.

[0010] Now, it is particularly difficult to predict the inflammatory potential in humans and in particular the allergic or pseudo-allergic risk associated with a compound or composition. The only means available today consists of the use of animal models in which an injection is carried out and it is observed whether or not an inflammatory reaction ensues and, in addition, degranulation of the mast cells.

[0011] It will be easily understood that it is desirable to do without such animal models in order to determine this inflammatory potential and, in particular, the allergic or pseudo-allergic risk.

[0012] Also, there is a need to determine easily and quickly, and without using animals, the inflammatory, and in particular allergic or pseudoallergic, potential in humans of an injectable composition. Statement of the invention

[0013] The inventors have previously developed an ex vivo model of human skin allowing the subcutaneous injection of a solution to be tested without the use of an animal. They have now demonstrated that not only granulocytes were present in this human skin model, but they still remained functional with a degranulation capacity. This result was all the more unexpected since it was rather a loss of mast cell functionality that was documented, with in particular significant apoptosis of the latter during the first days (see in particular KIVINEN et al., Experimental Dermatology, vol. 12, p: 53-60, 2003).

[0014] Therefore, the inventors' discovery makes it possible, following the "subcutaneous" injection or application to the epidermis of a composition in this same ex vivo human skin model, to determine the behavior of the granulocytes present and finally to deduce the inflammatory potential of a composition, injected or applied, and in particular the existence of an allergic or pseudo-allergic reaction, but also to decide on the nature of this reaction.

[0015] As a result, it becomes possible to test the inflammatory potential in humans, in particular the allergic or pseudo-allergic potential of an injectable solution without resorting to an animal model.

[0016] Also, a first object of the invention relates to an in vitro method intended to determine the inflammatory potential of a substance comprising the steps of:

[0017] ia) administration to a skin explant, topically or by subcutaneous injection, of a composition comprising the substance; which skin explant comprises the epidermis, the dermis and the epidermal appendages as well as a thickness of at least 5 millimeters of hypodermis;

[0018] ib) determination of the inflammatory response within the skin explant;

[0019] ic) determination of the level of degranulation of mast cells within the explant of skin; and

[0020] ii) determination of the inflammatory potential of the substance, preferably its allergic or pseudo-allergic potential.

[0021] Advantageously, step ia) consists of the subcutaneous injection into the explant of a composition comprising the substance.

[0022] According to a preferred embodiment, the method further comprises a step id) of determining the level of degranulation of a mast cell culture, preferably a culture of primary human mast cells, after incubation thereof in the presence of different concentrations of the substance.

[0023] This additional step allows, in addition to the confirmation of degranulation of mast cells in the presence of the substance which makes it possible to establish an allergic or pseudo-allergic potential, to determine the median effective concentration (EC50) corresponding to the necessary concentration of substance to induce median degranulation (between the absence of degranulation of mast cells and their maximum degranulation).

[0024] According to another preferred embodiment, the method further comprises a step ie) of determining the agonist potential of the substance with respect to the MRGPRX2 receptor (Mas-Related G-Protein coupled Receptor member X2).

[0025] The method according to the invention then makes it possible to decide between an allergic or pseudo-allergic potential of the substance and, in the case of a pseudo-allergic potential, to determine the maximum concentration of substance which can be injected so as not to induce a pseudo-allergic reaction. Detailed description of the invention

[0026] By "substance" is meant a substance of any nature (i.e. protein, carbohydrate, lipid, etc.), which may be of synthetic or natural origin, and of which we wish to determine the inflammatory and potentially allergenic potential.

[0027] The term “skin explant” means a fragment of skin which comprises, in addition to the epidermis, the dermis and the epidermal appendages, a thickness of at least 5 mm of hypodermis (preferably between 5 and 15 mm of hypodermis and, more preferably, between 5 and 10 mm of hypodermis).

[0028] The epidermal appendages correspond to the hair follicles, sebaceous glands and sweat glands. The hypodermis is the layer of tissue that is located immediately beneath the dermis of the skin. The hypodermis is a loose connective tissue that is richly vascularized and also contains adipose tissue.

[0029] If this skin explant is taken from a mammal, we can opt for human or pig. Now, in view of the preferred destination of the method according to the invention, we will rather opt for a human skin explant.

[0030] In relation to the origin of the skin explant, it can come from a plastic surgery from any part of the body, including plastic surgery of the abdomen, chest, buttocks, back, or even, why not, the scalp or any other part of the body comprising skin.

[0031] The skin explant is prepared as described in international application WO 2019 / 170281.

[0032] In detail, this skin explant was included, with the exception of the epidermis, in a liquid matrix capable of solidifying such as blood plasma, a solution derived from blood plasma (e.g. a dilution of blood plasma in physiological buffer, in particular a dilution of blood plasma at least 10%, 20%, 30%, or even at least 40% (weight / total weight of the matrix)), a fibrinogen solution, a collagen solution, a gelatin solution, solutions of synthetic polymers, solutions of natural polymers (e.g. agarose (agarose or agar-agar with low / low melting points), starch, polysaccharides), and mixtures thereof. For more details in link with matrices capable of solidifying and methods for placing skin explants therein, European patent no. EP 2 882 290 Bl can be consulted.

[0033] In the case of a cylindrical skin explant, we will therefore opt for a skin explant with a diameter of between 10 mm and 50 mm, preferably between 15 mm and 40 mm.

[0034] Advantageously, the skin explant is positioned within an insert which can take multiple forms and in particular correspond to a suspended insert or an insert on stilts. Now, a suspended insert will preferably be chosen. The bottom of this insert is made of a porous membrane whose diameter is between 5 and 40 mm and more preferably between 9.5 and 30 mm. As for the porosity of this membrane, it must prevent the liquid matrix from passing through it before it solidifies. Typically, this porous membrane will have a porosity of between 0.4 and 8 μm, preferably between 0.4 μm and 1.5 μm, with the interval going from 0.8 μm to 1.2 μm as the preferred porosity interval. In terms of material, a porous membrane may thus be chosen from polyethylene terephthalate (PET), nitrocellulose and polycarbonate membranes.Finally, and as an example of such inserts, we can cite those supplied by the companies Nunc, CORNING, Becton Dickinson (BD FALCON), MILLIPORE (MILLICELL), which can take the form of inserts with polycarbonate, PET or nitrocellulose membrane, which are pre-packaged in multi-well plates for 6, 8, 12 or 24 well culture plates, and whose membrane porosity can vary from 0.4 and 8 pm.

[0035] By "topical application" is meant an application of the composition to be tested on the epidermis of the skin explant.

[0036] By "subcutaneous injection" is meant an injection which is carried out in the hypodermis of the skin explant, which also gives it the name "hypodermic" injection. This type of injection, which is well known to those skilled in the art, generally requires making a skin fold using the fingers and the subcutaneous injection is then carried out in the skin fold.

[0037] According to a preferred embodiment, step ia) consists of the subcutaneous injection of a composition comprising the substance into a skin explant.

[0038] The composition in question is a composition to be tested which is in liquid form. Advantageously, the volume of this composition is between 10 μl and 1 ml, preferably between 10 μl and 500 μl and, particularly preferably, between 10 μl and 200 μl.

[0039] The needle for injecting the composition typically has a sufficient length to reach the hypodermis. Thus, needles having a length greater than or equal to 10 mm will preferably be used. As an example of such needles, needles with a length of 12, 16, 20, 25, 30, 35, 40 or even 45 mm can be used. Advantageously, the needle has a length of between 16 and 45 mm, preferably a length ranging from 20 to 40 mm. As for the diameter of the needle to be used, it can be easily identified by those skilled in the art with regard to their general knowledge. Typically, such hypodermic needles are of the type 18G, 19G, 20G, 21G, 22G, 23G, 25G, 26G, 27G, 28G, 29G, 30G or even 31 G.

[0040] This injection step can be carried out by an experimenter, who performs a pinch so as to allow the formation of a skin fold and thus facilitate the subcutaneous injection. Now, this injection step can also be carried out by an automatic injection device. Typically, the device allows an injection at a determined depth, relative to the surface of the epidermis, so as to obtain a subcutaneous injection.

[0041] Step ib) of determining the inflammatory response within the skin explant is carried out by monitoring inflammation markers which are well known to those skilled in the art. Such inflammation markers may be present within the skin explant, the matrix in which the skin explant is included and / or in the culture medium. As inflammation markers, mention may be made, but are not limited to, cytokines or antibacterial proteins. Now, mention may also be made of proteins involved in the biosynthesis of lipids or any other molecule whose level of expression varies between an inflamed state and a non-inflamed state (see in particular SERHAN & WARD, Molecular and Cellular Basis of Inflammation, Humana Press). It is also possible to analyze the monitoring of differentially expressed markers from transcriptomic, proteomic or lipidomic studies.

[0042] As cytokines that can be used as markers of inflammation, mention may be made of interleukins and their receptors. As examples of interleukins, mention may be made of IL-1A, IL-1B, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-17A, IL-17C, IL-17F, IL-19, IL-21, IL-22, IL-23, IL-27, IL-31 and IL-33, and as examples of interleukin receptors, mention may be made of IL-10RA, IL-10RB, IL-1R1, IL-5RA (CD125) and IL-9R.

[0043] As cytokines that can be used as markers of inflammation, mention may also be made of chemokines, which are chemotactic cytokines that control the migration patterns and positioning of immune cells, but also their receptors. As examples of cytokines, mention may be made of C5, Eotaxin, MCP-4, TARC, MCP-1, MIP-3A, CCL22, CCL23, MIP-1B, RANTES, MCP-3, MCP-2, CX3CL1, IL8RA, INP10, L8RB and CXCL3. Regarding chemokine receptors, mention may be made of CCL13 (MCP-4), CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR8, CX3CR1, CXCR1 and CXCR2.

[0044] It should be noted that other cytokines (than interleukins and chemokines) are conceivable as markers of inflammation. Examples of such cytokines include MCP-1, GM-CSF, TNFSF5, MCSF, GCSF, TNFSF6, IFNA2, IFNG, TNFA, TNFB, MIF, NAMPT, TRAIL and IFNA1.

[0045] Among the antibacterial proteins, we can cite antimicrobial peptides (AMPs) also called host defense peptides, which are part of the innate immune response and have a length of less than 50 amino acids. Unlike the majority of conventional antibiotics, it appears that antimicrobial peptides frequently destabilize biological membranes, can form transmembrane channels and can also strengthen immunity by acting as immunomodulators. As antimicrobial peptides, we can cite in particular defensins (e.g. beta-defensins, cathelicidins such as LL-37). Now, there are other antibacterial proteins than antimicrobial peptides, for example S100 proteins (S100A7 (psoriasin) and S100A15 (koebnerisin)).

[0046] Typically, the markers of inflammation are chosen from molecules secreted by structural cells (e.g. keratinocytes, stromal cells), adipocytes and cells of the immune system (e.g. macrophages, dendritic cells, T lymphocytes, mast cells). Examples of such inflammatory markers include MCSF, GCSF, TNFSF6, IFNA2, IFNG, RANTES, MCP-3, MCP-2, CX3CL1, TNFA, TNFB, MIF, NAMPT, TRAIL and IFNA1TNFA, MCP1, VEGF, IP-10, MDC, MIP-1B, IL-17A, IL-17C, IL-17F, TNFB, IL-27, MCP-4, MIP-1A, IL-22, IL-1B, IL-12 / IL-23p40, GMCSF, IFNG, IL-12p70, IL-23, IL-31, EOTAXIN, IL-6, IL-4, IL-13, IL-5, IL-8, IL-15, BETA-HEXOSAMINISASE, HISTAMINE, TRYPTASE and CHYMASE.

[0047] Preferably, markers of inflammation present in the culture medium will be chosen. Examples of such markers include TNFA, MCP-1, EOTAXIN-3, VEGF, IP-10, IL-2, MIP-3A, MIP-1B, IL-17A, MIP-1A, IL-22, IL-1B, IL-12 / IL-23p40, GMCSF, IFNG, IL-12p70, IL-23EOTAXIN, IL-6, IL-8, BETA-HEXOSAMINISASE, HISTAMINE and TRYPTASE.

[0048] Step ic) of determining the level of degranulation of the mast cells can be carried out using techniques well known to those skilled in the art. Techniques that can be used for this step include ELIS A or colorimetric techniques for measuring the presence of inflammatory mediators contained in the granules of the mast cells, such as histamine or tryptase, or secreted de novo such as lipid mediators or cytokines / chemokines, or else fluorescence, immunofluorescence or fluorochrome techniques specific to the granules of the mast cells.

[0049] Advantageously, this step ic) is carried out within a maximum period of 6 hours following the administration step ia), preferably within a maximum period of 4 hours.

[0050] The inventors have indeed shown that, if the granulocytes were indeed functional, the monitoring of degranulation was optimal during this interval. It should be noted that the inventors have also shown that it was not possible to induce further degranulation of the mast cells subsequently.

[0051] Advantageously, a single step ia) of administration of a composition comprising the substance is carried out by skin explant.

[0052] According to a preferred embodiment, step ic) of determining the level of degranulation of the mast cells within the skin explant is carried out by fluorescence analysis.

[0053] Advantageously, this step ic) of determining the level of degranulation of mast cells uses avidin.

[0054] Avidin is in fact a glycoprotein that binds very specifically to the heparin contained in the granules of mast cells (THARP et al., J. Histochem. Cytochem., vol.33, p:27-32,1985). Thus, during the degranulation process, the granules, as soon as they are externalized, become directly accessible to avidin. Now, and in the case of tissue fixation, the intracellular granules are accessible to avidin as soon as tissue permeabilization is carried out. This avidin can be complexed with a fluorochrome (avidin-FITC, avidin-Alexa488, avidin-sulfurhodamine 101 or any other fluorescent molecule) or with a bioluminescent molecule. Now, it is also possible to use avidin alone (uncomplexed) and in combination with a molecule that is complementary to it.Examples of such a molecule include biotin complexed with a fluorochrome, a bioluminescent molecule, or any other identifiable molecule.

[0055] Now, the determination of the level of degranulation of mast cells may use other markers, in particular of the nucleus or of the plasma membrane, so as to facilitate the identification of granulocytes externalized from mast cells. It will also be possible to measure in the culture medium tryptase, histamine, beta-hexosaminidase, chymase or any other molecules preformed within the granules of the mast cells and released during degranulation.

[0056] Typically, the determination of the level of mast cell degranulation within the skin explant can be carried out by following the protocol described in GAUDENZIO et al. (J. Clin. Invest., vol.126, p:3981-3998, 2016)

[0057] Advantageously, step ic) of determining the level of degranulation of the mast cells will be carried out on at least one histological section made from the skin explant.

[0058] To do this, one can use the well-known methods of immunohistochemistry which use the fixation of the skin explant, the inclusion of the explant (e.g. paraffin, OCT, EPON) before its preservation, and finally the production of histological sections on the inclusion block. The details of such methods are described for example in "Immunohistochemistry: Basics and Methods" by Igor BUCHWALOW (Editions SPRINGER).

[0059] The histological sections that can be used may have a very significant thickness of up to 500 μm. Advantageously, the histological section will thus have a thickness of between 1 and 500 μm. Now, it is possible to use sections with more conventional dimensions with a thickness of between 2 and 25 μm.

[0060] As for the determination of the level of mast cell granulation within the explant itself, for each identified mast cell, it is determined whether it is associated with weak, moderate or strong degranulation, then the proportion (percentage) of mast cells associated with each of these types of degranulation (weak, moderate and strong). The level of degranulation can also be analyzed in an automated manner using image analysis software or a computer algorithm or artificial intelligence techniques known as machine learning or deep learning.

[0061] In detail, a mast cell with a low level of degranulation corresponds to a mast cell with 0 to 2 granules around it (or to a cell with a smooth outline); a mast cell with a moderate level of degranulation corresponds to a mast cell with 3 to 6 granules around it (or to a cell with an outline with a granular appearance); and a mast cell with a high level of degranulation corresponds to a mast cell with more than 6 granules around it (or to a cell with an exposed shape).

[0062] For control purposes, the same steps ia), ib) and ic) may be carried out with a negative control (e.g. PBS) and / or with a composition comprising a positive control corresponding to a substance known to induce inflammation or, more specifically, degranulation of mast cells, such as, for example, IgE / antigen complexes, IgE / anti-IgE, compound 48 / 80, substance P, various secretagogues, products derived from pathogens or therapeutic molecules known to induce reactions at the injection site (e.g. anakinra, cetrorelix or icantibant).

[0063] Step ii) of determining the inflammatory potential, and more specifically the allergic or pseudo-allergic potential of the substance, can then be simply carried out with regard to the result of steps ib) and ic).

[0064] Thus, it will be possible to determine whether the substance has an inflammatory potential with regard to an increase in inflammatory markers at the end of step ib).

[0065] It will then be possible to determine whether or not this inflammatory potential is associated with an allergic or pseudo-allergic potential at the end of step ic), this with regard to the proportions of mast cells undergoing weak, moderate, or strong degranulation at the end of this step.

[0066] Thus, a substance associated with a proportion of granulocytes presenting for more than 50% a low level of degranulation and / or for less than 10% a high level of degranulation will present a low, or even zero, allergic or pseudo-allergic potential.

[0067] The inventors have thus shown that, in the cultured explant, there is a basic level of mast cell degranulation which is comparable to that observed in vivo in animals, which was not at all obvious and which supports the interest of the present model.

[0068] Conversely, a substance associated with a proportion of granulocytes presenting a high level of degranulation for more than 50% will present a high allergic or pseudo-allergic potential.

[0069] Here again, the inventors have shown that the level of degranulation observed in the culture explant is comparable to that observed in vivo in the animal with a degranulation inducer. It should also be noted that, unlike the situation observed with the basic level of degranulation, an inhomogeneous spatial distribution of mast cells undergoing degranulation is observed. The proportion of mast cells undergoing strong degranulation is then all the higher the closer one is to the injection site. This result again confirms the relevance of the model according to the invention.

[0070] Finally, a substance associated with an intermediate proportion of granulocytes, and not exceeding either of the two previous thresholds, will present an intermediate, or even moderate, allergic or pseudo-allergic potential.

[0071] According to a preferred embodiment, the method further comprises a step id) of determining the level of degranulation of a mast cell culture after incubation thereof in the presence of different concentrations of the substance.

[0072] Step ii) of determining the inflammatory potential of the substance will then make it possible, in connection with this step id), in addition to confirming degranulation by mast cells in the presence of the substance, to determine the median effective concentration (EC50) of the substance for the induction of mast cell degranulation.

[0073] Typically, a culture of primary mast cells, preferably human, will be used.

[0074] Such a mast cell culture can be obtained by methods well known to those skilled in the art. For example, the protocol described in GAUDENZIO et al. (J. Allergy Clin. Immunol., vol.l31(5), p: 1400-7, 2013) and in GAUDENZIO et al. (previously cited, 2016) which is described in the examples. To do this, mast cells are derived in appropriate culture medium from hematopoietic progenitors (e.g. CD34+, CD133+ cells) present in the circulating blood of healthy donors (e.g. adult peripheral blood, umbilical cord blood).

[0075] The determination of the degranulation of mast cells in culture can then be done by techniques well known to those skilled in the art which have been mentioned previously. Typically, mast cell markers are assayed before and after stimulation (e.g. approximately 1 hour). As markers of preformed mast cells in the secretory granules, beta-hexosaminidase, tryptase or chymase, or histamine may be mentioned. Now it is also possible to measure the secretion of cytokines and chemokines including MCSF, GCSF, TNFSF6, IFNA2, IFNG, RANTES, MCP-3, MCP-2, CX3CL1, TNFA, TNFB, MIF, NAMPT, TRAIL and IFNA1, TNFA, MCP1, VEGF, IP-10, MDC, MIP-1B, IL-17A, IL-17C, IL-17F, TNFB, IL-27, MCP-4, MIP-1A, IL-22, IL-1B, IL-12 / IL-23p40, GMCSF, IFNG, IL-12p70, IL-23, IL-31, EOTAXIN, IL-6, IL-4, IL-13, IL-5, IL-8 and IL-15.The dosage of these granulocyte markers can be carried out by colorimetric tests or ELISA-type tests in 96 or 384-well plates. In addition, it is possible to detect, by flow cytometry, the increase in exocytosis markers on the surface of mast cells such as lamp-1, annexin 5 or by labeling with fluorescent avidin or others.

[0076] A percentage of degranulation is defined relative to a positive control which is the lysis of all the mast cells in a well using a detergent (e.g. TRITON X100 used at 1%). A molecule will be defined with a low allergic or pseudoallergic potential if the percentage of degranulation of the mast cells is between 5 and 15%, with a moderate allergic or pseudoallergic potential if the percentage of degranulation of the mast cells is between 15 and 30% and with a high allergic or pseudoallergic potential if the percentage of degranulation of the mast cells is greater than 30%.

[0077] According to another preferred embodiment, the method further comprises a step ie) of determining the agonist potential of the substance with respect to the MRGPRX2 receptor (Mas-Related G-Protein coupled Receptor member X2).

[0078] Such a step can be carried out simply by a person skilled in the art and is described in the examples. To do this, cells are transformed so as to express the human MRGPRX2 receptor (see for example Accession numbers Q96LB1, NP_001290544.1, NP_473371.1, ACG60653.1, EAW68359.1 or AAH63450.1). Activation of the MRGPRX2 receptor resulting in a decrease of the intracellular calcium ion concentration, monitoring this intracellular calcium ion concentration makes it possible to extrapolate the activity of the MRGPRX2 receptor and, in the presence of the substance, the agonist potential of the latter with respect to the MRGPRX2 receptor. This measurement of receptor activation can also be done by bioluminescence (e.g. Fluo-4). Thus, if after adding the stimulating molecule, an increase in fluorescent signal is detected, then the molecule can be considered as an agonist of the MRGPRX2 receptor.

[0079] In light of the results obtained in connection with steps ib), ic) and id), this step ie) makes it possible to know whether the degranulation of the mast cells reveals an allergic potential or a pseudo-allergic potential. Indeed, the MRGPRX2 receptor is the receptor responsible for pseudo-allergies. Therefore, these pseudo-allergic reactions could be prevented, in the event of new administration of the substance, by reducing the dose or the speed of administration or co-injection of an antagonist of the MRGPRX2 receptor.

[0080] Also, step ii) of determining the inflammatory potential of the substance then makes it possible to determine whether or not the degranulation of mast cells induced by the substance results from a pseudo-allergic reaction. Once a substance is identified as having a pseudo-allergic potential, step ii) therefore makes it possible to identify the substances which should, for example, be combined with at least one mast cell stabilizer in order to facilitate their injection.

[0081] Mast cell stabilizers are drugs used to prevent or control certain allergic disorders that are well known to those skilled in the art. These compounds block the degranulation of mast cells, stabilizing the cell and thus preventing the release of histamine and associated mediators. Examples of mast cell stabilizers include loratidine, desloratidine, sodium cromoglycate, ketotifen, olopatadine, rupatadine, mepoluzimab, omolizumab, pzmirolast, nedocromil, azelastine, [>2-agonists, quercetin, luteolin, rutin, or vitamin D.

[0082] Therefore, and in the case of a pseudo-allergic reaction, the results obtained at the end of step id) also make it possible to determine the concentration of substance to be used so as not to induce degranulation of the mast cells or weak degranulation.

[0083] The following examples are given solely by way of illustration of the subject of the present invention, of which they do not constitute a limitation in any way. Examples

[0084] 1-Preparation of skin explants

[0085] Skin explants were prepared from complete skin samples from different donors, which samples included the epidermis, dermis and hypodermis (1.5 to 2 cm). The explants (epidermis, dermis and hypodermis) were then cut using a metal punch to obtain cylinders of 11 to 20 mm in diameter in which the hypodermis thickness was adjusted to the desired value (0.5 to 1 cm). Finally, these explants were kept floating in a buffered saline solution until the “inclusion” step in the solidified matrix. This inclusion step was done with a process similar to that used for the NativeSkin™ model.Briefly, the skin explant is gently placed on an insert (Millicell™ 8-well cup) with a porous membrane (PET, porosity 1 μm) at the bottom and containing a solution derived from blood plasma treated with an anticoagulant agent with reversible properties in the presence of calcium ions (sodium citrate). This solution contains 42% blood plasma, 50% 0.9% NaCl solution, 8% 1% CaCl2 saline solution, an antifibrinolytic agent (tranexamic acid or aprotinin), and 0.7% low-melting-point molten agarose (Agarose LMP GIBCOBRL, Life Technologies) (melted in an oven at 65.5°C). The antifibrinolytic agent functions to inhibit enzymes that can degrade the plasma matrix, these enzymes being secreted by the skin explant, and thus to maintain the integrity of the explant.

[0086] 2-Mast cells and skin explant

[0087] 2.1-Identification of mast cells within the skin explant

[0088] The presence of mast cells within the explant was analyzed by avidin labeling and using an anti-tryptase antibody. Mast cells were found in identical numbers between the first and fifth day of culture. Following the injection of a pro-inflammatory compound (e.g., a vaccine formulation), an increase in the secretion of pro-inflammatory cytokines was observed in the culture medium.

[0089] 2.2-Demonstration of mast cell degranulation activity within the skin explant

[0090] Following subcutaneous injection of 100 pL of a 50 pg / mL solution of compound 48 / 80 (an MRGPRX2 agonist) or control water into the skin explant, mast cell degranulation was quantified 1h, 4h, 6h or 24h post-injection. The best signal compared to the control condition was observed between 0 and 4h post-injection. It was observed that from 6h there is spontaneous degranulation of mast cells even in the absence of stimulation, which could affect the subsequent analysis. These results therefore show that the skin explant comprises mast cells, which are maintained over time and are capable of degranulation. Consequently, the skin explant therefore allows the determination of the allergic or pseudo-allergic potential of a substance without resorting to an animal model.

[0091] 3-Determination of the inflammatory potential of a substance (example cetrotide or cetrorelix)

[0092] 3.1-Inflammatory potential

[0093] 100 pL of a CETROTIDE solution having a concentration of cetrorelix of 0.25mg / l mL (luteinizing hormone inhibitor that competes by binding to its receptor) was injected into the adipose tissue of an explant as described previously using a syringe and a 27G needle of 12 mm length. As a negative and positive control, 100 pL of a PBS solution and 100 pL of a concentrated solution of compound 48 / 80 (50 pg / mL) which is a positive control MRGPRX2 agonist was injected into the adipose tissue of an explant each and as described previously.

[0094] The culture medium or the support matrix of the explants is taken and a cytokine assay is used using ELISA-type methods or any other type of method intended to detect and quantify the presence of cytokines.

[0095] The results confirmed the inflammatory potential of cetrorelix.

[0096] 3.2-Allergic or pseudo-allergic potential of the substance

[0097] 100 pL of a CETROTIDE solution having a concentration of cetrorelix of 0.25 mg / l mL (luteinizing hormone inhibitor that competes by binding to its receptor) was injected into the adipose tissue of an explant as described previously using a syringe and a 27G needle of 12 mm length. As negative and positive control, 100 pL of a PBS solution and 100 pL of a concentrated solution of compound 48 / 80 (50 pg / mL) were injected into the adipose tissue of an explant each and as described previously.

[0098] The explants were then cultured (incubator at 37°C, 5% CO2 and water-saturated atmosphere) for 1 to 4 hours before being fixed (4% paraformaldehyde solution) and embedded in a paraffin block for histological analysis.

[0099] In detail, the skin explants are dehydrated by a first alcohol bath then by a second xylene bath. Finally, a first paraffin bath allows the water previously contained in the skin explant to be replaced by paraffin. The samples impregnated with paraffin are removed from their bath and transferred to a container whose bottom is lined with absorbent paper, in order to be brought close to the inclusion station. The samples, enclosed in histology cassettes, are immersed in liquid paraffin at 56°C to remelt the paraffin which impregnates them. For each sample, the histology cassette is opened, the sample is possibly cut in two. An inclusion mold is filled with paraffin liquid and the sample (or the 2 pieces of sample) is placed in the mold and oriented in the desired direction for cutting. The mold is simultaneously transferred to a refrigerated support in order to solidify the paraffin at the bottom of the mold and hold the sample there. The lid of the histology cassette on which the sample reference appears is placed on top, so that the paraffin passes through it (possibility of adding paraffin if necessary), then everything is placed in the cold (refrigerator, freezer, cold room, etc.) for several minutes (5 to 6), in order to solidify the paraffin into a block, thereby trapping the sample in the correct orientation with the lid of the histology cassette which will become the support for the block. Once the paraffin block is well solidified, it is removed from the mold. Any excess paraffin is scraped off with a spatula on the sides of the lid of the embedding cassette.Serial sections with thicknesses varying from 4 to 5 μm are then made along the entire length of the paraffin block containing the sample.

[0100] In order to determine the level of mast cell degranulation within the tissue under different conditions, labeling is carried out using avidin coupled with a fluorochrome which allows the detection of mast cell granules. The first step allows the fixed and paraffin-impregnated sections to be deparaffinized and rehydrated. The sections are incubated for 30 minutes at room temperature in Citrate buffer pH6, then saturated and permeabilized for 40 minutes at 37°C with a solution of goat serum and 0.1% Triton. The sections are then incubated for one hour at room temperature in a humid chamber with 5pg / mL Avidin-Sulforhodamin 101 (Avidin TEXAS RED; MERCK). Staining of cell nuclei is then performed by incubating the sections with DAPI (D9542, SIGMA) at 1 / 1000 for 3 minutes at room temperature. Mounting medium is added and a coverslip is placed on the sections.The slides are then analyzed under a fluorescence microscope to determine the level of mast cell degranulation in the different explants.

[0101] The results of these experiments are presented in Table 1.

[0102] [Tables 1] Percentage of granulocytes with Compound administered PBS compound 48 / 80 Cetrotide Low level of degranulation 67.0 36.1 12.5 level of 28.0 37.1 31.6 intermediate degranulation High level of degranulation 5.0 26.8 55.9 Allergic or pseudo-allergic potential low moderate high

[0103] The results made it possible to determine the level of degranulation of the mast cells identified in the samples and, after integration, to deduce the inflammatory potential of each injected substance. In this case, the results confirm that the inflammatory potential of cetrotide is associated with degranulation of the mast cells. Consequently, cetrotide has a high allergic or pseudo-allergic potential

[0104] 3.3-Confirmation of the allergic or pseudo-allergic potential of the substance

[0105] A primary human mast cell culture was obtained according to the protocol described in GAUDENZIO et al. (J. Allergy Clin. Immunol., vol.l31(5), p: 1400-7, 2013) and in GAUDENZIO et al. (previously cited, 2016). Briefly, PBMCs from blood samples from different donors were isolated on a Ficoll gradient. Hematopoietic progenitors expressing the CD34+ marker were then isolated from them by tri-magnetic (potentially also feasible by FACS). These cells were then cultured and then matured into mast cells by culturing them in a serum-free culture medium supplemented in particular with recombinant IL-6, IL-3 and SCF for a period of approximately 3 months.

[0106] Mast cells are considered mature and ready for use when they have cytoplasmic granules that are labeled by avidin coupled to a fluorochrome and when they are capable of degranulating in response to stimulation by IgE / antigen (or IgE / anti-IgE) complexes or by MRGPRX2 receptor agonists (e.g. compound 48 / 80 or substance P).

[0107] The determination of the degranulation of mast cells in culture was then carried out by performing a dosage of granulocyte markers (e.g. beta hexosaminidase) before and after their stimulation (approximately 1 hour) by substance P, compound 48 / 80 and an IgE / anti-IgE system. This dosage is carried out by colorimetric test in 96-well plates.

[0108] The results confirm those obtained with the skin explant, namely that cetrotide has a strong allergic or pseudo-allergic potential.

[0109] 3.4-Allergic or pseudo-allergic potential for cetrotide

[0110] HEK 293 cells were transformed to express the human MRGPRX2 receptor (Accession number Q96LB1).

[0111] Activation of the MRGPRX2 receptor resulting in a decrease in the concentration Intracellular calcium ion concentration, monitoring this intracellular calcium ion concentration allows extrapolation of the activity of the MRGPRX2 receptor. To do this, a fluorescent calcium tracer was used, namely Fluo-4.

[0112] The transformed cells were placed on a microscope slide equipped with one or more culture wells. The analysis of fluorescence per cell post-acquisition is done on conventional image processing software (e.g. IMAGEJ).

[0113] The cells labeled with Fluo-4 are then incubated in the presence of cetrotide. A fluorescence analysis is then made by a video recording made on a fluorescence microscope for a period of about 300 seconds after the start of the incubation. A 20-second sequence is carried out in order to establish the baseline before adding the molecule to be tested. Then the molecule to be tested is added for a period of 100 seconds, then 100 qM of substance P are added in order to locate the cells expressing MRGPRX2. 100 seconds later, 100 qM of ionomycin are added as a positive control for visualizing calcium flux.

[0114] The results obtained showed that, after adding cetrotide, an increase in fluorescent signal is detected in the same cells that respond to substance P and therefore are transfected to express MRGPRX2. Consequently, cetrotide can be considered an agonist of the MRGPRX2 receptor.

[0115] Finally, the results demonstrated that cetrotide has a strong pseudo-allergic potential.

Claims

Claims

1. an in vztro method intended to determine the inflammatory potential of a substance and its association or not with an allergic or pseudo-allergic potential comprising the steps of: ia) administering to a skin explant, topically or by subcutaneous injection, a composition comprising the substance; which skin explant comprises the epidermis, the dermis and the epidermal appendages as well as a thickness of at least 5 millimeters of hypodermis; ib) determining the inflammatory response within the skin explant; ic) determining the level of degranulation of mast cells within the skin explant; and ii) determining the inflammatory potential of the substance and its association or not with an allergic or pseudo-allergic potential.

2. The method according to claim 1, characterized in that step ia) consists of the subcutaneous injection of a composition comprising the substance.

3. The method according to claim 1 or 2, characterized in that step ib) of determining the inflammatory response within the skin explant is carried out by monitoring inflammation markers chosen from the group comprising MCSF, GCSF, TNFSF6, IFNA2, IFNG, RANTES, MCP-3, MCP-2, CX3CL1, TNFA, TNFB, MIF, NAMPT, TRAIL and IFNA1TNFA, MCP1, VEGF, IP-10, MDC, MIP-1B, IL-17A, IL-17C, IL-17F, TNFB, IL-27, MCP-4, MIP-1A, IL-22, IL-1B, IL-12 / IL-23p40, GMCSF, IFNG, IL-12p70, IL-23, IL-31, EOTAXIN, IL-6, IL-4, IL-13, IL-5, IL-8, IL-15, BETA-HEXOSAMINISASE, HISTAMINE, TRYPTASE and CHYMASE.

4. The method according to any one of claims 1 to 3, characterized in that step ic) of determining the level of degranulation of mast cells within the skin explant uses avidin.

5. The method according to any one of claims 1 to 4, characterized in that step ic) of determining the level of degranulation of mast cells within the skin explant is carried out within a maximum of 6 hours following step ia) of administration.

6. The method according to any one of claims 1 to 5, characterized in that step ii) of determining the inflammatory potential of the substance and more specifically its allergic or pseudo-allergic potential is carried out with regard to the proportions of mast cells undergoing weak, moderate, or strong degranulation at the end of step ia), with: * a substance associated with a proportion of granulocytes presenting for more than 50% a low level of degranulation and / or for less than 10% a high level of degranulation will present a weak, or even zero, allergic or pseudo-allergic potential; * a substance associated with a proportion of granulocytes presenting for more than 50% a high level of degranulation will present a high allergic or pseudo-allergic potential.

7. The method according to any one of claims 1 to 6, characterized in that it further comprises a step id) of determining the level of degranulation of a mast cell culture after incubation thereof in the presence of different concentrations of the substance.

8. The method according to claim 7, characterized in that step ii) of determining the inflammatory potential of the substance further makes it possible to determine the median effective concentration (EC50) of the substance for the induction of mast cell degranulation and in that the method is then further intended to determine the median effective concentration (EC50) of the substance for the induction of mast cell degranulation.

9. The method according to any one of claims 1 to 8, characterized in that it further comprises a step ie) of determining the agonist potential of the substance with respect to the MRGPRX2 receptor (Mas-Related G-Protein coupled Receptor member X2).

10. The method according to claim 9, characterized in that step ii) of determining the inflammatory potential of the substance further makes it possible to determine whether the degranulation of mast cells induced by the substance results from a pseudo-allergic reaction or not and in that the method is then further intended to determine whether the Substance-induced mast cell degranulation results from a pseudo-allergic reaction or not.