EP4341690A1 - Ex-vivo-humanmodell zur beurteilung des allergischen oder pseudoallergischen entzündungspotenzials - Google Patents

Ex-vivo-humanmodell zur beurteilung des allergischen oder pseudoallergischen entzündungspotenzials

Info

Publication number
EP4341690A1
EP4341690A1 EP22729664.7A EP22729664A EP4341690A1 EP 4341690 A1 EP4341690 A1 EP 4341690A1 EP 22729664 A EP22729664 A EP 22729664A EP 4341690 A1 EP4341690 A1 EP 4341690A1
Authority
EP
European Patent Office
Prior art keywords
substance
allergic
degranulation
potential
pseudo
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP22729664.7A
Other languages
English (en)
French (fr)
Inventor
Emeline Pages
Pascal Descargues
Nicolas GAUDENZIO
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Genoskin SAS
Original Assignee
Genoskin SAS
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Genoskin SAS filed Critical Genoskin SAS
Publication of EP4341690A1 publication Critical patent/EP4341690A1/de
Pending legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5082—Supracellular entities, e.g. tissue, organisms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5082—Supracellular entities, e.g. tissue, organisms
    • G01N33/5088—Supracellular entities, e.g. tissue, organisms of vertebrates

Definitions

  • the present invention relates to the field of inflammation, whether of an allergic, pseudo-allergic or other nature, and more specifically provides the first ex vivo human model for determining the inflammatory potential of a compound .
  • the inflammatory response is an integral part of the reaction of the immune system in response 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, especially if it is an allergic response. Also, it is important to be able to predict, prior to any injection, the inflammatory response likely to be induced by a substance.
  • the first so-called sensitization phase begins when the individual comes into contact for the first time with a compound that is called the 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, ex. dendritic cells). These cells will present the allergen on their surface and allow the production of immunoglobulin E (IgE) by other cells.
  • IgE immunoglobulin E
  • mast cells which are found in particular at the level of the skin and the mucous membranes (locations where the allergens are likely to penetrate). This process of IgE binding is called “sensitization” because it makes mast cells susceptible to activation upon subsequent encounter with the same antigen. This first phase is silent, ie the subject in the sensitization phase is asymptomatic.
  • the mast cell is a cell present in the connective tissues, which is part of the white blood cells and is characterized by the presence in its cytoplasm of very many granules containing chemical mediators such as serotonin, histamine, tryptase or heparin.
  • chemical mediators such as serotonin, histamine, tryptase or heparin.
  • IgEs chemical mediators
  • the same activation induces, in a more delayed manner (a few hours), the synthesis of many cytokines (such as TNF-alpha), chemokines and lipid mediators.
  • 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.
  • the pseudo-allergic potential In connection with the 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 therefore dose-dependent. Consequently, these pseudo-allergic reactions could be prevented, or limited, in the event of new administration of the substance by reducing the dose or reducing the rate of administration. [00011] 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 a composition.
  • the inventors have previously developed an ex vivo model of human skin making it possible to test the subcutaneous injection of a solution that does not require the use of animals. They have now demonstrated that not only were granulocytes present in this model of human skin, but also that they remained functional with degranulation capacity. This result was all the more unexpected since it was rather a loss of functionality of the mast cells which was documented, with in particular a significant apoptosis of the latter during the first days (see in particular Kl VINEN et al., Experimental Dermatology, vol.12 , p: 53-60, 2003).
  • 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:
  • step ia) consists of the subcutaneous injection within the explant of a composition comprising the substance.
  • the method further comprises a step id) of determining the level of degranulation of a culture of mast cells, preferably of a culture of primary human mast cells, after incubation of the latter in presence of different concentrations of the substance.
  • This additional step makes it possible, in addition to confirming mast cell degranulation 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 midline degranulation (between no mast cell degranulation and maximum mast cell degranulation).
  • EC50 median effective concentration
  • 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).
  • 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 that can be injected so as not to not induce a pseudo-allergic reaction.
  • substance is meant a substance of any kind (that is to say protein, carbohydrate, lipid, etc.), which may be of synthetic or natural origin, and of which it is desired to determine inflammatory and potentially allergenic potential.
  • explant of skin denotes 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).
  • the epidermal appendages correspond to hair follicles, sebaceous glands and sweat glands.
  • the hypodermis is the layer of tissue that is located immediately below the dermis of the skin.
  • the hypodermis is a loose connective tissue that is richly vascularized and also contains fatty tissue.
  • the skin explant is prepared as described in international application WO 2019/170281. [00035] In detail, this skin explant was included, with the exception of the epidermis, in a liquid matrix capable of solidifying like blood plasma, a solution derived from blood plasma (eg a dilution of plasma blood in physiological buffer, in particular a dilution of blood plasma to at least 10%, 20%, 30%, or even to at least 40% (weight/total weight of the matrix)), a solution of fibrinogen, a solution of collagen , gelatin solution, synthetic polymer solutions, natural polymer solutions (e.g. agarose (low/low melting point agarose or agar-agar), starch, polysaccharides), and mixtures thereof.
  • a solution derived from blood plasma eg a dilution of plasma blood in physiological buffer, in particular a dilution of blood plasma to at least 10%, 20%, 30%, or even to at least 40% (weight/total weight of the matrix)
  • liquid solution comprising at least one specific compound or composition whose concentration in said liquid solution is such that, when appropriate conditions are implemented, in particular particular temperature conditions, the liquid solution takes on a consistency of the solid or gel type .
  • Said compound or a specific composition can be of animal, vegetable or synthetic origin, its nature and its concentration are determined according to the desired physico-chemical characteristics of the matrix when it is solidified, in particular the flexibility and the resistance of the matrix. .
  • said liquid matrix capable of being able to solidify is chosen from any liquid solution, preferably nutrient, capable of solidifying or gelling under specific conditions compatible with the survival and culture of skin cells which make up said atoning.
  • Such a matrix is used in an inclusion process comprising the following steps i) the flotation of a cylindrical skin explant in a liquid matrix chosen from a solution derived from blood plasma, fibrinogen or collagen solution, or a solution of agar-agar or low melting point agarose, or a mixture of these solutions, this liquid matrix being contained in said insert, this flotation leaving the epidermal surface of the explant with emerged skin, while the dermis is immersed; ii) rapid induction of solidification of said surrounding skin explant; and, iii) The in vitro or ex vivo culture, or the survival maintenance of 1 of the skin explant in the said matrix thus solidified.
  • step i) the flotation of a cylindrical skin explant is carried out in a liquid matrix comprising a first solution chosen from a solution derived from blood plasma, a fibrinogen solution or a collagen solution, and a second low melting point agar-agar or agarose solution.
  • a first solution chosen from a solution derived from blood plasma, a fibrinogen solution or a collagen solution, and a second low melting point agar-agar or agarose solution.
  • this second solution is previously heated for a time and at a temperature sufficient to be liquid and to remain liquid at around 37°C during the sufficient time to be mixed with the first solution in said insert and until said skin explant is deposited.
  • said second solution is preheated to its melting point, or slightly higher, preferably between 65°C and 70°C.
  • Agar-agar or agarose with a low melting point is preferably an agar-agar or agarose whose maximum gelation temperature is between 24° C. and 28° C., and the melting temperature is greater than 65.5°C in 1.5% solution.
  • this agarose is the agarose named LMP Agarose (“Low melting point” (GIBCOBRL, LIFE TECHNOLOGIES).
  • said second solution is an agar-agar or low-melting-point agarose solution, the concentration of agar-agar or low-melting-point agarose of which is between 1% and 5% (preferably in a physiological solution), more preferably between 2% and 5%, between 3% and 4.5%, between 3.5% and 4.5% or alternatively between 3.8% and 4.2% or between 3.9% and 4.1%, 4% being the most preferred concentration.
  • This second agar-agar or low-melting-point agarose solution at said concentration and once heated to its melting temperature, or slightly higher, can be stored in liquid form for at least 1 hour and, preferably for at least 4 hours, 10 hours or 16 hours at 37°C.
  • the final concentration of agar-agar or low-melting-point agarose is between 0.1% and 2%, preferably between 0.2% and 1.8%.
  • Such a concentration makes it possible not only to obtain a matrix which, once solidified, makes it possible to preserve the 3D structure and to maintain said skin explant in survival, but also to obtain a solid matrix but sufficiently flexible to be non-brittle and resistant to occasional shocks.
  • the solidification of this liquid matrix taking place after deposition of the skin explant, leaving the device thus obtained at a temperature between 37°C and room temperature, preferably at 20°C.
  • the final concentration of agar-agar or low-melting-point agarose melting is between 1% and 2%, preferably between 1.25% and 1.75%, preferably between 1.4% and 1.6%, 1.5% being the most preferred concentration.
  • the final concentration of agar-agar or low-melting-point agarose melting temperature is between 0.1% and 2%, preferably between 0.2% and 1.75%, 0.25% being the most preferred concentration.
  • concentration makes it possible to obtain a matrix which, once solidified, makes it possible to preserve the 3D structure and to maintain said skin explant in survival, but also to obtain a matrix which is sufficiently flexible to be non-brittle and resistant to mechanical effects applied to the explant, for example during a effect mimicking the massage of the skin for the application of a preparation such as, for example, a cream.
  • the volume of said liquid matrix is from 1/3 to 2/3 of the total volume of the insert, preferably from 2/5 to 3/5 of the total volume, Half of the total volume of the 'insert being the preferred volume.
  • said liquid matrix capable of being able to solidify contains between 1 mM and 5 mM of Ca2+, preferably between 1.5 mM and 4.5 mM of Ca2+ According to a preferred embodiment, in step a) said liquid matrix capable of being able to solidify contains between 1 mM and 2 mM of Ca2+, preferably between 1.2 mM and 1.4 mM of Ca2+. According to another preferred embodiment, in step a) said liquid matrix capable of being able to solidify contains between 2 mM and 3 mM of Ca2+, preferably between 2.5 mM and 2.8 mM of Ca2+ and more preferentially 2 .8 mM Ca2+.
  • said liquid matrix capable of being able to solidify contains between 5 and 500 mg/mL of ascorbic acid, preferably between 25 and 75 mg/mL, 50 mg/mL of ascorbic acid being the highest concentration. most preferred.
  • said liquid matrix capable of being able to solidify is a medium containing between 1 mM and 5 mM of Ca2+ and containing between 5 and 500 mg/mL of ascorbic acid.
  • said liquid matrix capable of being able to solidify contained in the insert in step a) is a liquid matrix, the first solution of the mixture of which (the second solution being the solution of agar-agar or agarose) is preferably nutritious, and also capable of being able to solidify under the action of an increase or decrease in temperature and/or by the addition of a compound or a specific composition.
  • said liquid matrix capable of being able to solidify does not contain any growth factor or animal or human serum.
  • said liquid matrix capable of being able to solidify does not cover the upper face of the epidermis before this matrix is solidified in step b).
  • step a) of said inclusion process the skin explant is then placed on a liquid matrix capable of being able to solidify, in particular as indicated above, and which liquid matrix is chosen from any liquid solution providing all the nutritious and/or necessary ingredients for its culture, in particular for maintaining the initial physiological state of the cells which constitute it.
  • This solution is capable of solidifying or gelling under specific conditions compatible with the survival and culture of the skin explant.
  • said liquid matrix capable of being able to solidify is a liquid solution derived from blood plasma treated with an anticoagulant agent with reversible properties mixed with an agar-agar or agarose solution.
  • said liquid matrix capable of being able to solidify contains blood plasma, fibrinogen, or collagen, mixed with an agar-agar or low-melting-point agarose solution.
  • said liquid matrix capable of being able to solidify is a solution derived from blood plasma containing from 25% to 60%, preferably between 35 % and 45% (v/v) of blood plasma, from 70% to 35% of a physiological solution, such as a 0.9% NaCl solution, from 5% to 12%, preferably 8%, a 1% CaC12 saline solution, an anti-fibrinolytic agent in a concentration sufficient to obtain the desired anti-fibrinolytic activity, preferably between 5% and 2%, preferably the anti-fibrinolytic agent being chosen from tranaxemic acid or aprotinin, and an agarose solution with a low melting point between 0.5% and 4%, preferably between 1% and 2%.
  • a physiological solution such as a 0.9% NaCl solution, from 5% to 12%, preferably 8%, a 1% CaC12 saline solution
  • said liquid matrix capable of being able to solidify is a liquid solution of fibrinogen and thrombin, or collagen or blood plasma, mixed to a gelatin solution, including synthetic, natural polymeric gels such as agarose gels, in particular low/low melting point agarose or agar-agar gels, starch or polysaccharide gels and for which incubation at 37°C allows its solidification.
  • synthetic, natural polymeric gels such as agarose gels, in particular low/low melting point agarose or agar-agar gels, starch or polysaccharide gels and for which incubation at 37°C allows its solidification.
  • said liquid matrix capable of being able to solidify contains a liquid solution derived from blood plasma treated with an anticoagulant agent with reversible properties, preferably by sodium citrate; and the solidification of said matrix in step b) for this solution obtainable in the presence of calcium ions, preferably also in the presence of thrombin.
  • the solidification of said matrix in step b) can be carried out for this solution by adding thrombin or by increasing the temperature, or else using factors secreted by cells supplied to the matrix, such as primary fibroblasts.
  • said liquid matrix capable of being able to solidify is solidified after a maximum of 8 hours, preferably less than 2 hours or less than one hour, a duration of less than 30 min, preferably of less than 10 min being the most preferred duration to initiate the solidification phase of the liquid matrix after depositing the skin biopsy on this liquid matrix in step a ).
  • the explanted skin is positioned within an insert which can take multiple forms and in particular correspond to a suspended insert or to an insert on piles.
  • a suspended insert The bottom of this insert consists of a porous membrane whose diameter is between 5 and 40 mm and more preferably between 9.5 and 30 mm.
  • this porous membrane will have a porosity of between 0.4 and 8 ⁇ m, preferably between 0.4 ⁇ m and 1.5 ⁇ m, with the range of 0.8 ⁇ m and 1.2 ⁇ m as the porosity range. prefer.
  • porous membrane chosen from polyethylene terephthalate (PET), nitrocellulose and polycarbonate membranes.
  • PET polyethylene terephthalate
  • nitrocellulose polycarbonate membranes
  • MILLIPORE MILLICELL
  • topical application is meant an application of the composition to be tested on the epidermis of the skin explant.
  • Subcutaneous injection means an injection which is performed in the hypodermis of the skin explant, which also gives it the name of “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 performed in the skin fold.
  • step ia) consists of the subcutaneous injection of a composition comprising the substance in a skin explant.
  • the composition in question is a composition to be tested which is in liquid form.
  • the volume of this composition is between 10 m ⁇ and 1 ml, preferably between 10 m ⁇ and 500 m ⁇ and, particularly preferably, between 10 m ⁇ and 200 m ⁇ .
  • the needle for injecting the composition typically has a sufficient length to reach the hypodermis.
  • needles having a length greater than or equal to 10 mm will preferably be used.
  • the needle has a length of between 16 and 45 mm, preferably a length ranging from 20 to 40 mm.
  • the diameter of the needle to be used it can be identified simply by those skilled in the art with regard to his general knowledge.
  • such hypodermic needles are of the 18G, 19G, 20G, 21G, 22G, 23G, 25G, 26G, 27G, 28G, 29G, 30G or even 31G type.
  • This injection step can be performed by an experimenter, who performs pinching so as to allow the formation of a skin fold and thus facilitate subcutaneous injection.
  • this injection step can also be performed by an automatic injection device.
  • the device allows injection at a determined depth, relative to the surface of the epidermis, so as to obtain a subcutaneous injection.
  • Step ib) for 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.
  • markers of inflammation may be present within the explant of skin, of the matrix in which the explant of skin is included and/or in the culture medium.
  • markers of inflammation mention may be made, being limited thereto, of cytokines, or antibacterial proteins.
  • 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 cf. in particular SERHAN & WARD, Molecular and Cellular Basis of Inflammation, Humana Press).
  • interleukins As cytokines which can be used as markers of inflammation, mention may be made of interleukins and their receptors. By way of example 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 by way of example of interleukin receptors, mention may be made of IL-10RA, IL-10RB, IL-1R1, IL-5RA (CD125) and IL-9R.
  • cytokines which can be used as markers of inflammation mention may also be made of chemokines which are chemotactic cytokines which control the migration patterns and the positioning of the immune cells, but also their receptors.
  • chemokines include C5, Eotaxin, MCP-4, TARC, MCP-1, MIP-3A, CCL22, CCL23, MPMB, RANTES, MCP-3, MCP-2, CX3CL1, IL8RA, INP10 , L8RB and CXCL3
  • CCL13 MCP-4
  • cytokines other than interleukins and chemokines
  • cytokines include MCP-1, GM-CSF, TNFSF5, MCSF, GCSF, TNFSF6, IFNA2, IFNG, TNFA, TNFB, MIF, NAMPT, TRAIL and IFNA1.
  • antimicrobial peptides 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 enhance immunity by acting as immunomodulators.
  • antimicrobial peptides mention may in particular be made of defensins (eg beta-defensins, cathelicidins such as LL-37).
  • defensins eg beta-defensins, cathelicidins such as LL-37.
  • S100A7 psoriasin
  • S100A15 koebnerisin
  • the markers of inflammation are chosen from molecules secreted by structural cells (eg keratinocytes, stromal cells), adipocytes and cells of the immune system (eg macrophages, dendritic cells, T lymphocytes, mast cells ).
  • structural cells eg keratinocytes, stromal cells
  • adipocytes e.g macrophages, dendritic cells, T lymphocytes, mast cells.
  • MCSF MCSF
  • GCSF GCSF
  • markers of inflammation present in the culture medium will be chosen.
  • markers of inflammation present in the culture medium include TNFA, MCP-1, EOTAXIN-3, VEGF, PM0, IL-2, MIP-3A, MPMB, 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.
  • Step ic) for determining the level of mast cell degranulation can be carried out using techniques well known to those skilled in the art.
  • Techniques that can be used for this step include ELISA or colorimetric techniques making it possible to measure the presence of inflammatory mediators contained in the granules of mast cells, such as histamine or tryptase, or secreted de novo such as lipid mediators or cytokines/chemokines , or even on fluorescence, immunofluorescence or fluorochromes techniques specific to mast cell granules.
  • 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.
  • a single step ia) of administration of a composition comprising the substance is carried out per explant of the skin.
  • step ic) for determining the level of mast cell degranulation within the skin explant is carried out by fluorescence analysis.
  • this step ic) for determining the level of mast cell degranulation uses avidin.
  • Avidin is in fact a glycoprotein which 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 ).
  • THARP et al. J. Histochem. Cytochem., vol.33, p: 27-32, 1985 .
  • the granules when they are externalized, become directly accessible to avidin.
  • the intracellular granules are accessible to avidin as soon as tissue permeabilization is performed.
  • 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.
  • avidin alone (not complexed) and in combination with a molecule which is complementary to it. Mention may be made, by way of example of such a molecule, of biotin complexed with a fluorochrome, with a bioluminescent molecule or with any other identifiable molecule.
  • the determination of the level of degranulation of the mast cells can use other markers, in particular of the nucleus or the plasma membrane, so as to facilitate the identification of the granulocytes externalized from the mast cells. It will also be possible to assay tryptase, histamine, beta-hexosaminidase, chymase or any other molecules preformed within the mast cell granules and released during degranulation in the culture medium. [00088] 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)
  • step ic) of determining the level of mast cell degranulation will be carried out on at least one histological section made from the skin explant.
  • immunohistochemistry methods can be used, which use the fixation of the skin explant, the inclusion of the explant (e.g. paraffin, O.C.T., EPON) before its conservation, and finally, performing histological sections on the inclusion block.
  • the detail of such methods is described for example in “Immunohistochemistry: Basics and Methods” by Igor BUCHWALOW (SPRINGER Editions).
  • the histological sections that can be used may have a very large thickness of up to 500 ⁇ m.
  • the histological section will thus have a thickness of between 1 and 500 ⁇ m. Now, it is possible to use cuts with more classic dimensions with a thickness between 2 and 25pm.
  • the level of mast cell granulation within the explant itself it is determined, for each identified mast cell, 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 automatically using a image analysis software or a computer algorithm or artificial intelligence techniques called machine-learning or deep-learning.
  • a mast cell exhibiting a low level of degranulation corresponds to a mast cell exhibiting 0 to 2 granules around it (or to a cell exhibiting a smooth outline);
  • a mast cell presenting a moderate level of degranulation corresponds to a mast cell presenting from 3 to 6 granules around it (or to a cell presenting an outline with a granular appearance);
  • a mast cell with a high level of degranulation is a mast cell with more than 6 granules around it (or a cell with an exposed shape).
  • the same steps ia), ib) and ic) may be carried out with a negative control (eg PB S) and/or with a composition comprising a positive control corresponding to a substance known to induce a inflammation or, more specifically, degranulation of mast cells such as, for example, IgE/antigen, IgE/anti-IgE complexes, compound 48/80, substance P, various secretagogues, products from pathogens or known therapeutic molecules to induce reactions at the injection site (eg anakinra, cetrorelix or icantibant).
  • a negative control eg PB S
  • a composition comprising a positive control corresponding to a substance known to induce a inflammation or, more specifically, degranulation of mast cells such as, for example, IgE/antigen, IgE/anti-IgE complexes, compound 48/80, substance P, various secretagogues, products from pathogens or known therapeutic molecules to induce reactions at the injection site (eg anakinra
  • 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).
  • 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).
  • 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 allergic or pseudo-allergic potential, or even zero.
  • the inventors have thus shown that, in the explant in culture, there is a basic level of mast cell degranulation which is comparable to that observed in vivo in animals, which was not obvious and which reinforces the interest of this model.
  • the method further comprises a step id) of determining the level of degranulation of a culture of mast cells after incubation of the latter in the presence of different concentrations of the substance.
  • Step ii) of determining the inflammatory potential of the substance will then make it possible, in connection with this step id), in addition to the confirmation of degranulation by the mast cells in the presence of the substance, to determine the median effective concentration (EC50 ) substance for induction of mast cell degranulation.
  • EC50 median effective concentration
  • a culture of primary mast cells preferably human, will be used.
  • mast cells can be obtained by methods well known to those skilled in the art.
  • the protocol described in GAUDENZIO et al. J. Allergy Clin. Immunol., vol.131(5), p: 1400-7, 2013
  • GAUDENZIO et al. previously cited, 2016
  • the mast cells are derived in the appropriate culture medium from hematopoietic progenitors (eg CD34 + , CD133 + cells) present in the circulating blood of healthy donors (eg adult peripheral blood, blood from the umbilical cord).
  • hematopoietic progenitors eg CD34 + , CD133 + cells
  • mast cell marker assay is performed before and after stimulation (eg, about 1 hour).
  • markers of preformed mast cells in the secretory granules mention may be made of labeta-hexosaminidase, tryptase or chymase, or even histamine.
  • 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, PM0, 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.
  • cytokines and chemokines including MCSF, GCSF, TNFSF6, IFNA2, IFNG, RANTES, MCP-3, MCP-2, CX3CL1, TNFA, TNFB
  • the assay of these granulocyte markers can be carried out by colorimetric tests or ELIS A type tests in 96 or 384 well plates.
  • a percentage of degranulation is defined in relation 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 detergent e.g. TRITON X100 used at 1%.
  • 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).
  • MRGPRX2 receptor Mos-Related G-Protein coupled Receptor member X2
  • 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 to express the human MRGPRX2 receptor (see for example Accession numbers Q96LB1, NP_001290544.1, NP_473371.1, ACG60653.1, EAW68359.1 or even AAH63450.1).
  • the activation of the MRGPRX2 receptor resulting in a decrease in the intracellular concentration of calcium ions makes it possible to extrapolate the activity of the MRGPRX2 receptor and, in the presence of the substance, the agonist potential of this last with respect to the MRGPRX2 receptor.
  • This measurement of receptor activation can also be made by bioluminescence (eg Fluo-4).
  • bioluminescence eg Fluo-4
  • this step ie) makes it possible to know whether the degranulation of the mast cells reveals an allergic potential or a pseudo-allergic potential.
  • the MRGPRX2 receptor is the receptor responsible for pseudo-allergy. Therefore, these pseudo-allergic reactions could be prevented, in the event of new administration of the substance, by reducing the dose or the rate of administration or co-injection of an antagonist of the MRGPRX2 receptor.
  • step ii) of determining the inflammatory potential of the substance then makes it possible to determine whether or not the degranulation of the mast cells induced by the substance results from a pseudo-allergic reaction. As soon as a substance is identified as presenting a pseudo-allergic potential, step ii) therefore makes it possible to identify the substances which it is appropriate, for example, to combine with at least one mast cell stabilizer in order to facilitate their injection.
  • mast cell stabilizers are drugs used to prevent or control certain allergic disorders which are well known to those skilled in the art. These compounds block mast cell degranulation, stabilizing the cell and thus preventing the release of histamine and related mediators.
  • mast cell stabilizers include loratidine, desloratidine, sodium cromoglycate, ketotifen, olopatadine, rupatadine, mepoluzimab, omolizumab, pzmirolast, nedocromil, azelastine , p2-agonists, quercetin, luteolin, rutin or even vitamin D.
  • Explants of skin are prepared from complete samples of skin from different donors, which samples included the epidermis, the dermis and the hypodermis (1.5 to 2 cm). The explants (epidermis, dermis and hypodermis) were then cut using a metal punch to obtain cylinders 11 to 20 mm in diameter in which the thickness of the hypodermis is adjusted to the desired value ( 0.5 to 1cm). Finally, these explants were kept floating in a buffered saline solution until the stage of “inclusion” in the solidified matrix. This inclusion step was done with a process similar to that used for the NATIVESKINTM model.
  • the explanted skin is delicately deposited on an insert (8-well MILLICELLTM cup) with a porous membrane at the bottom (in PET, porosity 1 ⁇ m) 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% CaC12 saline solution, an anti-fibrinolytic agent (tranexamic acid or aprotinin), and melted agarose with a low melting point at 0.7% (Agarose LMP GIBCOBRL, LIFE TECHNOLOGIES) (melted in an oven at 65.5° C.).
  • the function of the anti-fibrinolytic agent is to inhibit the enzymes capable of degrading the plasma matrix, these enzymes being secreted by the skin explant, and thus to maintain the integrity of the explant.
  • explants were then cultured (incubator at 37° C., 5% C02 and atmosphere saturated with water) for 1 to 4 hours before they were fixed (4% paraformaldehyde solution) and included in a block of paraffin for histological analysis.
  • the skin explants are dehydrated by a first alcohol bath then by a second xylene bath. Finally, a first paraffin bath replaces the water previously contained in the skin explant with paraffin.
  • the paraffin-impregnated samples are taken out of 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 melt the paraffin which impregnates them. For each sample, the histology cassette is opened, the sample is possibly cut in two.
  • An embedding mold is filled with liquid paraffin and the sample (or the 2 pieces of sample) is placed in the mold and oriented in the desired direction for sectioning.
  • the mold is at the same time transferred to a refrigerated support in order to solidify the paraffin at the bottom of the mold and to hold the sample there.
  • the lid of the histology cassette on which the reference of the sample appears is placed on top, so that the paraffin passes through it (possibility of adding paraffin if necessary), then the whole thing is placed in the cold (refrigerator, freezer, cold room%) several minutes (5 to 6), in order to solidify the paraffin into a block, effectively trapping the sample in the correct orientation with the lid of the histology cassette which will become the support for the block.
  • the paraffin block Once the paraffin block has solidified, it is unmolded.
  • the excess paraffin is possibly scraped using a spatula from the sides of the lid of the embedding cassette.
  • Serial sections with a thickness varying from 4 to 5 ⁇ m are then made over the entire length of the paraffin block containing the sample.
  • labeling is carried out using avidin coupled with a fluorochrome which makes it possible to detect the mast cell granules.
  • the first step deparaffinizes and rehydrates the sections fixed and impregnated with paraffin. The sections are incubated for 30 minutes at room temperature in Citrate pH6 buffer, then saturated and permeabilized for 40 minutes at 37° C. with a 0.1% solution of goat serum and Triton. The sections are then incubated for one hour at room temperature in a humid chamber with 5 mg/ml Avidin-Sulforhodamin 101 (Avidin TEXAS RED; MERCK).
  • Staining of the cell nuclei is then carried out by incubating the sections with DAPI (D9542, SIGMA) at 1/1000 for 3 minutes at ambient temperature. Mounting medium is added and a coverslip is placed on the sections. The slides are then analyzed under a fluorescence microscope in order to determine the level of mast cell degranulation in the different explants.
  • DAPI D9542, SIGMA
  • 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 substance injected.
  • the results confirm that the inflammatory potential of cetrotide is associated with mast cell degranulation. Accordingly, cetrotide has a high allergic or pseudo-allergic potential.
  • a culture of primary human mast cells was obtained according to the protocol described in GAUDENZIO et al. (J. Allergy Clin. Immunol., vol.131(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. The hematopoietic progenitors expressing the CD34+ marker were then isolated among them by tri-magnetic (potentially also feasible by FACS). These cells were then cultured, then matured into mast cells by cultivating them in a serum-free culture medium supplemented in particular with recombinant IL-6, IL-3 and SCF for a period of approximately 3 month.
  • Mast cells are considered mature and ready for use when they present cytoplasmic granules which are labeled with avidin coupled to a fluorochrome and when they are capable of degranulating in response to stimulation by IgE complexes /antigen (or IgE/anti-IgE) or by agonists of the MRGPRX2 receptor (eg compound 48/80 or substance P).
  • IgE complexes /antigen or IgE/anti-IgE
  • agonists of the MRGPRX2 receptor eg compound 48/80 or substance P.
  • HEK 293 cells were transformed to express the human MRGPRX2 receptor (Accession number Q96LB1).
  • the transformed cells were placed on a microscope slide fitted with one or more culture wells.
  • the analysis of the fluorescence by post acquisition cell is done on a classic image processing software (ex IMAGEJ).
  • 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 over a period of about 300 seconds after starting the incubation.
  • a 20-second sequence is performed to establish the base line before adding the molecule to be tested. Then the molecule to be tested is added for a period of 100 seconds, then 100 m M of substance P are added in order to locate the cells expressing MRGPRX2. 100 seconds later, 100 m M of ionomycin is added as a positive control for visualization of calcium flux.
  • cetrotide can be considered an agonist of the MRGPRX2 receptor.
  • cetrotide has a strong pseudo-allergic potential.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Immunology (AREA)
  • Hematology (AREA)
  • Cell Biology (AREA)
  • Chemical & Material Sciences (AREA)
  • Urology & Nephrology (AREA)
  • Molecular Biology (AREA)
  • Toxicology (AREA)
  • Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
EP22729664.7A 2021-05-20 2022-05-19 Ex-vivo-humanmodell zur beurteilung des allergischen oder pseudoallergischen entzündungspotenzials Pending EP4341690A1 (de)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
FR2105293A FR3123125A1 (fr) 2021-05-20 2021-05-20 Modèle humain ex vivo destiné à l’évaluation du potentiel inflammatoire de type allergique ou pseudo-allergique
PCT/EP2022/063605 WO2022243447A1 (fr) 2021-05-20 2022-05-19 Modèle humain ex vivo destiné à l'évaluation du potentiel inflammatoire de type allergique ou pseudo-allergique

Publications (1)

Publication Number Publication Date
EP4341690A1 true EP4341690A1 (de) 2024-03-27

Family

ID=77821811

Family Applications (1)

Application Number Title Priority Date Filing Date
EP22729664.7A Pending EP4341690A1 (de) 2021-05-20 2022-05-19 Ex-vivo-humanmodell zur beurteilung des allergischen oder pseudoallergischen entzündungspotenzials

Country Status (4)

Country Link
US (1) US20240280565A1 (de)
EP (1) EP4341690A1 (de)
FR (1) FR3123125A1 (de)
WO (1) WO2022243447A1 (de)

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2990106B1 (fr) 2012-05-03 2014-05-09 Genoskin Systeme permettant la maintenance en survie et le transport de biospsies de peau et ses applications
FR3078541B1 (fr) 2018-03-05 2022-07-08 Genoskin Modele d’injection sous-cutanee ex vivo
KR20210034733A (ko) * 2019-09-20 2021-03-31 주식회사 필메카 동물실험 대체를 위한 3차원 인공 조직 및 모니터링 세포의 공동배양 시스템 및 이의 제조방법

Also Published As

Publication number Publication date
WO2022243447A1 (fr) 2022-11-24
US20240280565A1 (en) 2024-08-22
FR3123125A1 (fr) 2022-11-25

Similar Documents

Publication Publication Date Title
Talbott et al. Wound healing, fibroblast heterogeneity, and fibrosis
Bertin et al. The ion channel TRPV1 regulates the activation and proinflammatory properties of CD4+ T cells
KR102176845B1 (ko) 유도된 엑소좀을 포함하는 피부 재생 및 상처 치유용 조성물
Tomura et al. Activated regulatory T cells are the major T cell type emigrating from the skin during a cutaneous immune response in mice
CN111065731B (zh) 血管类器官、产生和使用所述类器官的方法
EP2882290B1 (de) System zur lebendhaltung und zum transport von hautbiopsien und anwendungen dieses systems
KR20160131112A (ko) 간엽줄기세포 활성화제, 활성화된 간엽줄기세포 및 그 제조 방법
EP3688141B1 (de) Ex-vivo-modell einer entzündeten menschlichen haut und deren verwendung zum screening von entzündungshemmenden verbindungen
TW202108151A (zh) 前驅調節滋胚內層細胞以及它們的用途
FR3078541A1 (fr) Modele d’injection sous-cutanee ex vivo
EP4341690A1 (de) Ex-vivo-humanmodell zur beurteilung des allergischen oder pseudoallergischen entzündungspotenzials
Banakh et al. Platelet lysate can support the development of a 3D-engineered skin for clinical application
JP2010502175A (ja) Ｃｄ１４＋単球からランゲルハンス細胞及び／又は真皮／間質性樹状細胞を製造する方法
Cavallero et al. Effects of preconditioning with TNFα and IFNγ in angiogenic potential of mesenchymal stromal cell-derived extracellular vesicles
KR20060011837A (ko) 케라티노사이트에 의한 인터루킨 18 생성의 유도 현상을이용한 저해제의 스크리닝 방법, 아토피성 피부염 유사증상의 유도 방법, 및 그 이용
US20060039990A1 (en) Biological tissue regenerative agent and method for preparing same
WO2012059703A1 (fr) Procede in vitro ou ex vivo pour la conservation et/ou la maintenance en survie d'un epiderme
US20220341918A1 (en) Cell structure and method for producing same
AU2018285579B2 (en) Blood vessel organoid, methods of producing and using said organoids
DE102004061289A1 (de) Testkit mit Testzellen und Immunzellen sowie Herstellverfahren hierfür
Lee The role of skin macrophages under conditions of stress and injury: ultraviolet irradiation and wound healing
Bloom et al. Autofluorescent granules in cells of human dermis: III. Electron microscopical observations and fluorescence characteristics
Malkmus Establishment of a 3D\(in\)\(vitro\) skin culture system for the obligatory human parasite\(Onchocerca\)\(volvulus\)
Meyer Effects of interleukin-1 on glucose uptake and energy homeostasis in lymphocytes
WO2024150259A1 (en) Biometric model of fibrosis

Legal Events

Date Code Title Description
STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: UNKNOWN

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE INTERNATIONAL PUBLICATION HAS BEEN MADE

PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: REQUEST FOR EXAMINATION WAS MADE

17P Request for examination filed

Effective date: 20231107

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

DAV Request for validation of the european patent (deleted)
DAX Request for extension of the european patent (deleted)