WO2025078493A1 - Local administration of ripk2 inhibitors for the curative treatment of allergic asthma - Google Patents

Abstract

OF THE INVENTION LOCAL ADMINISTRATION OF RIPK2 INHIBITORS FOR THE CURATIVE TREATMENT OF ALLERGIC ASTHMA The present invention relates to a method for use in the treatment of Asthma. Here, the inventors identified the receptor-interacting serine/threonione protein kinas 2 (RIPK2) as a novel therapeutic target to improve Asthma-related diseases. They demonstrated that a local preventive administration of the RIPK2 inhibitor reduced AHR, airway eosinophilia, mucus production, Th2 cytokines and the alarmin IL-33. Moreover, they demonstrated the early role of IL-33 in the NOD1-dependent response of the epithelium to HDM. Therefore, the inventors demonstrated that the local interference of the NOD1 signaling pathway through RIPK2 inhibition may represent a new therapeutic approach in asthma. Other pulmonary diseases could also benefit of this treatment. Thus, the present invention relates to a method for use in the curative treatment of HDM-induced asthma comprising administrating to a subject in need thereof a therapeutically effective amount of an inhibitor of RIPK2.

Description

LOCAL ADMINISTRATION OF RIPK2 INHIBITORS FOR THE CURATIVE TREATMENT OF ALLERGIC ASTHMA

FIELD OF THE INVENTION

The present invention is in the field of medicine, in particular pneumology.

BACKGROUND OF THE INVENTION

House dust mite (HDM) is the most frequent allergen trigger of asthma with different innate immune mechanisms playing critical roles in outcomes. Subjects with HDM-induced asthma suffer from airway diseases such as lung inflammations. Despite type 2 immune adaptive response being one hallmark feature of HDM-related asthma, the progression of this disease involves a variety of cell types including epithelial cells and various innate immune mechanisms (Lambrecht N.D et al., 2019, Immunity Cell Press & Jacquet A et al., 2021, Front. Allergy).

The main therapies used involve the use of antihistamines and corticosteroids. However, these treatments induce many side effects such as headache, muscle loss or depression. Specific immunotherapy is also used but it is not always effective. Therefore, it is important to find new therapeutic targets and new therapies for treating asthma; in particular, HDM-induced asthma.

The Nucleotide Oligomerization Domain 1 (NODI) is a pattern recognition receptor that recognizes unique muropeptides from bacterial peptidoglycan and contributes to the development of HDM-induced asthma (Ait Yahia S et al., 2021, J. Allergy Clin. Immuno,' Hysi P et al., 2005, Hum. Mol. Genet & Ait Yahia S et al., 2014, Am. J. Respir. Crit. Care Med). The HDM-derived microbiota is sensed by NODI and potentiates allergic airway disease severity through the downstream receptor-interacting serine/threonine protein kinase 2 (RIPK2) (Ait Yahia S et al., 2021, J. Allergy Clin. Immuno).

Studies have shown that the role of RIPK2 in the promotion of HDM-induced asthma (Miller M.H et al., 2018, J.Leukoc. Biol) and have explored the use of inhibitors as a preventive measure in the HDM-induced asthma progression (Miller M.H et al., 2020, ImmunoHorizons).

SUMMARY OF THE INVENTION The present invention is defined by the claims. In particular, the present invention relates to the local administration of RIPK2 inhibitors for the curative treatment of allergic asthma.

DETAILED DESCRIPTION OF THE INVENTION

The inventors aimed to evaluate the effectiveness of a RIPK2 inhibitor administered locally as a therapeutic approach using an experimental HDM-induced allergic asthma model in Wild-type (WT) and mutant huNODl mice, and in in vitro bronchial epithelial cells from asthma patients.

They administered the RIPK2 inhibitor intra-nasally either preventively or therapeutically in the models. Airway hyperesponsiveness (AHR), bronchoalveolar lavage composition, cytokine expression and mucus production were evaluated, as well as the ex vivo effect of the inhibitor on precision lung cut slices (PLCS). Furthermore, the inhibitor was tested on airway liquid interface (ALI) epithelial cultures from asthma patients and controls, and inflammation assessed.

The inventors discovered that, in WT mice, local preventive administration of the RIPK2 inhibitor reduced AHR, airway eosinophilia, mucus production, Th2 cytokines and the alarmin IL-33. When administered after the sensitization phase, RIPK2 inhibitor failed to reduce the above parameters, except IL-33. However, in mutant huNODl mice, therapeutic local RIPK2 inhibition mitigated all asthma features. Results of PLCS emphasized an early role of IL-33 in the NODI -dependent response of the epithelium to HDM, and a late effect of NODI signalling on IL 13 effector response. Mechanistically, RIPK2 inhibitor down-regulated a number of mediators in HDM-stimulated ALI epithelial cultures from asthma patients including TSLP and chemokines.

Therefore, the inventors demonstrated that the local interference of the NODI signaling pathway through RIPK2 inhibition may represent a new therapeutic approach in asthma.

Accordingly the first object of the present invention relates to a method for the curative treatment of an allergic asthma in a patient in need thereof comprising administering locally a therapeutically effective amount of a RIPK2 inhibitor.

As used herein, the term “patient” is interchangeable with the term “individual” or “subject”, and may refer to a subject to be treated by the methods disclosed herein. In particular, the patient suffers from an allergic asthma. In some embodiments, the patient is a mammal. Non-limiting examples of mammals include rodents (e.g., mice and rats), primates (e.g., lemurs, bushbabies, monkeys, apes, and humans), rabbits, dogs (e.g., companion dogs, service dogs, or work dogs such as police dogs, military dogs, race dogs, or show dogs), horses (such as race horses and work horses), cats (e.g., domesticated cats), livestock (such as pigs, bovines, donkeys, mules, bison, goats, camels, and sheep), and deer. In some embodiments, the mammal is a human. In some embodiments, the patient is a human infant. In some embodiments, the patient is a human child. In some embodiments, the patient is a human adult.

As used herein, the term "asthma" has its general meaning in the art and refers to diseases that present as reversible airflow obstruction or bronchial hyper-responsiveness that may or may not be associated with underlying inflammation. The method of the present invention is particularly suitable for the treatment of “allergic asthma” i.e. an asthma that is caused by at least one allergen from air or blood, which can be dust mite, pollen, perfume or viral antigen. Allergic-asthma is characterized by reversible airflow limitation and airway hyperresponsiveness. These responses are typically stimulated by normal allergen-response mechanisms mediated by Th2 CD4+ T-cell activation and subsequent Th2 cytokine production. However, airway-associated allergic responses are typically more severe with an acute onset in patients with asthma than is observed in non-asthmatic patients. In particular, IL-4, IL-5, IL-9, and IL- 13 are critically important in acute allergic-asthma responses, inducing eosinophil-, macrophage-, and lymphocyte-mediated inflammatory responses, mucus hypersecretion, and airway hyperresponsiveness.

In one embodiments, the method of the present invention is particularly suitable for curing HDM-induced asthma.

As used herein, the term “HDM-induced asthma” refers to an asthma induced by house dust mites (HDM).

In one embodiment, the method of the present invention is particularly suitable for curing asthma induced by animal antigens, such as animal hairs, skin or saliva. For instance allergens from cats, dogs, guinea pigs, rabbits, horses, mice and rats can all trigger asthma. In one embodiments, the method of the present invention is particularly suitable for curing asthma induced by dog allergens. As used herein, the term “curative treatment” also known as “therapeutic treatment”, is focused on treating existing diseases, conditions, or health problems after they have already developed. The goal of curative treatment is to alleviate symptoms, eliminate the cause of the problem, and restore the patient to a healthier state. Curative treatment is reactive, as it addresses health issues that have already manifested and are causing problems for the patient. The term is thus distinguishable from the term “preventive treatment”. Indeed, the term “preventive treatment” also known as “prophylactic treatment”, aims to prevent the development of diseases, conditions, or health problems before they occur. The goal of preventive treatment is to reduce the risk factors associated with a particular health issue and promote overall well-being. Preventive treatment is proactive, as it focuses on minimizing the likelihood of health problems arising in the first place. Thus, curative treatment is administered after a health issue has developed, while preventive treatment is administered before any health problems arise. Curative treatment targets existing diseases or conditions and aims to provide relief and cure. Preventive treatment focuses on reducing the risk of developing diseases or conditions. Curative treatment addresses the symptoms and root causes of a specific health problem. Preventive treatment emphasizes lifestyle changes and interventions to minimize risk factors. The goal of curative treatment is to restore the patient's health and eliminate the disease or condition. The goal of preventive treatment is to maintain good health and avoid the onset of diseases. In particular, the method of the present invention is particularly suitable for reducing peribronchial inflammations and/or reducing mucus overproduction, and/or reducing airway hyperresponsiveness (AHR).

In one embodiment, the RIPK2 inhibitor of the present invention is used as an alternative to one or more another specific compounds used for treating allergic asthma in subjects having or developing drug resistance.

As used herein, the term “subject developing drug resistance” refers to a subject developing a resistance to one or more compounds used for treating the disease. More particular, it refers to a subject without biological reactivity after a therapeutic treatment. It also refers to a progressive decrease of the efficacy of a therapeutic treatment.

As used herein, the term “RIPK2” has its general meaning in the state of the art and refers to the receptor interacting protein-2 (RIPK2) kinase, which is also known as CARD3, RICK, CARDIAK, or RIP2. RIPK2 belongs to receptor-interacting protein family of serine/threonine kinase. RIPK2 contains a carboxy-terminal caspase-activation-and- recruitment domain (CARD) and it is a downstream signaling molecule expressed in antigen- presenting cells such as dendritic cells and macrophages (Honjo H et al., 2021, Front. Pharmacol). Its Entrez reference number is 8767 and its Uniprot reference number is 043353. RIPK2 plays a role in many compound regulation such as the activation of NF-kB (He S et al., 2018, Nature Immunology and its dysregulation is involved in many diseases such as inflammatory bowel disease or cancers (Watanabe T et al., 2019, Int. Immunol, Jaafar R et al., 2018, Biochem. Biophys. Res. Commun & Cai X et al., 2018, Oncol. Rep). Moreover, it has been shown that RIPK2 is strongly involved in the promotion of HDM-induced asthma (Miller M.H et al., 2018, J.Leukoc. Biol) and it is an important modulator of the inflammation and the immunity (He S et al., 2018, Nature Immunology).

As used herein, the term “RIPK2 inhibitor” refers to any compound natural or not which is capable of inhibiting the activity or expression of RIPK2, in particular RIPK2 kinase activity. RIPK2 inhibitors are well known in the art. The term encompasses any RIPK2 inhibitor that is currently known in the art or that will be identified in the future, and includes any chemical entity that, upon administration to a patient, results in inhibition or down-regulation of a biological activity associated with activation of the RIPK2. The term also encompasses inhibitor of expression. In some embodiments, the RIPK2 inhibitor is selective over the other kinases. By “selective” it is meant that the inhibition of the selected compound is at least 10- fold, preferably 25 -fold, more preferably 100-fold, and still preferably 300-fold higher than the inhibition of the other kinases. The RIPK2 inhibition of the compounds may be determined using various methods well known in the art. In particular, the skilled man may use any commercially available RIPK2 kinase assay (see for example the RIPK2 assay commercially available from Promega: ADP-Glo™ Kinase Assay is a luminescent kinase assay that measures ADP formed from a kinase reaction. Typical assays are also described in WO2011123609, W02011120025, W02011120026, W02011140442, W02012021580, W02012122011, and WO2013025958. Typically, the RIPK2 inhibitor is a small organic molecule.

RIPK2 inhibitors are well known in the art and typically include those described in: Tian E et al., 2023, Eur J Med Chem You J et al., 2023, Front Pharmacol Pham A.T et al., 2023 In some embodiments, the RIPK2 inhibitor is selected from the group consisting of compounds described in the International Patent Publications: WO2011120025, W02011120026, W02011123609, W02011140442, W02012021580, W02012122011, WO2013025958, and WO2014043437.

In some embodiments, the RIPK2 inhibitor belongs to 4-aminoquinolines family. Compounds from the 4-aminoquinolines family are derivatives of quinoline with an amino group at the 4-position of the quinoline (Pussard E et al., 1994, Fundam. Clin. Pharmacol)

In some embodiments, the RIPK2 inhibitor is DCAM-253 (2-dialkylamino-9-indazolyl- purine) that is disclosed in Zhao Y et al., 2012, J Immunol.

In some embodiments, the RIPK2 inhibitor of the present invention is 2-((4- (benzo[d]thiazol-5-ylamino)-6-(tert-butylsulfonyl)quinazolin-7-yl)oxy)ethyl dihydrogen phosphate (i.e. GSK2983559).

In some embodiments, the RIPK2 inhibitor is an inhibitor of RIPK2 expression. An “inhibitor of expression” refers to a natural or synthetic compound that has a biological effect to inhibit the expression of a gene. In a preferred embodiment of the invention, said inhibitor of gene expression is a siRNA, an antisense oligonucleotide or a ribozyme. For example, antisense oligonucleotides, including anti-sense RNA molecules and anti-sense DNA molecules, would act to directly block the translation of RIPK2 mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of RIPK2, and thus activity, in a cell. For example, antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the mRNA transcript sequence encoding RIPK2 can be synthesized, e.g., by conventional phosphodiester techniques. Methods for using antisense techniques for specifically inhibiting gene expression of genes whose sequence is known are well known in the art (e.g. see U.S. Pat. Nos. 6,566,135; 6,566,131; 6,365,354; 6,410,323; 6,107,091; 6,046,321; and 5,981,732). Small inhibitory RNAs (siRNAs) can also function as inhibitors of expression for use in the present invention. RIPK2 gene expression can be reduced by contacting a subject or cell with a small double stranded RNA (dsRNA), or a vector or construct causing the production of a small double stranded RNA, such that RIPK2 gene expression is specifically inhibited (i.e. RNA interference or RNAi). Antisense oligonucleotides, siRNAs, shRNAs and ribozymes of the invention may be delivered in vivo alone or in association with a vector. In its broadest sense, a "vector" is any vehicle capable of facilitating the transfer of the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid to the cells and typically cells expressing RIPK2. Typically, the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector. In general, the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid sequences. Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rous sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus. One can readily employ other vectors not named but known to the art.

As used herein, the term “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve a curative effect. A therapeutically effective amount is also one in which any toxic or detrimental effects are outweighed by the therapeutically beneficial effects. The efficient dosages and dosage regimens depend on the disease or condition to be treated and may be determined by the persons skilled in the art. A physician having ordinary skill in the art may readily determine and prescribe the effective amount required. For example, the physician could start doses at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Preferably, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the subj ect to be treated. A medicine typically contains from about 0.01 mg to about 500 mg of the active ingredient, preferably from 1 mg to about 100 mg of the active ingredient. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.

According to the present invention, the RIPK2 inhibitor is administered locally (i.e. in the respiratory tract of the subject and in particular into the lungs). In some embodiments, the RIPK2 inhibitor is administered to the patient by nasal administration. In some embodiments, the composition is administered via the nose or mouth of the patient, e.g., intranasally or via inhalation. In some embodiments, the RIPK2 inhibitor of the present invention is pulmonary administered to the patient by an aerosol. Therefore, the RIPK2 inhibitor can be formulated in the form of a spray, aerosol, solution, emulsion, or other form well-known to one of skill in the art. In particular, a liquid preparation may be placed into an appropriate device so that it may be aerosolized for inhalation through the nasal or buccal cavity. For administration by inhalation the compositions may be delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant. In particular, the active ingredients for use according to the present invention can be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant (e.g., dichlorodifluoromethane, trichlorofluoromethane, di chlorotetrafluoroethane, carbon dioxide or other suitable gas). In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges (composed of, e.g., gelatin) for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch. As used herein, the term “aerosol” is used in its conventional sense as referring to very fine liquid or solid particles carried by a propellant gas under pressure to a site of therapeutic application. A pharmaceutical aerosol can contain a therapeutically active compound, which can be dissolved, suspended, or emulsified in a mixture of a fluid carrier and a propellant. The aerosol can be in the form of a solution, suspension, emulsion, powder, or semi-solid preparation. Aerosols are intended for administration as fine, solid particles or as liquid mists via the respiratory tract of a patient. Various types of propellants can be utilized including, but not limited to, hydrocarbons or other suitable gases. Aerosols can also be delivered with a nebulizer, which generates very fine liquid particles of substantially uniform size within a gas. A liquid containing the active compound is dispersed as droplets, which can be carried by a current of air out of the nebulizer and into the respiratory tract of the patient. Alternatively, nasal drops can be instilled in the nasal cavity by tilting the head back sufficiently and apply the drops into the nares. The drops may also be inhaled through the nose.

Typically the RIPK2 inhibitor is combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions. As used herein, the term “pharmaceutically” or “pharmaceutically acceptable” refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administrated to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluents, encapsulating material or formulation auxiliary of any type. It will be appreciated that the form and character of the pharmaceutically acceptable diluent is dictated by the amount of the RIPK2 inhibitor with which it is to be combined, the route of administration and other well-known variables. The carrier(s) must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. “Carriers” or “vehicles” include any such material known in the art such as, for example, any liquid, gel, solvent, liquid diluent, solubilizer, or the like, which is non-toxic and which does not interact with any components of the composition in a deleterious manner. Examples of nutritionally acceptable carriers include, for example, water, salt solutions, alcohol, silicone, waxes, petroleum jelly, vegetable oils, polyethylene glycols, propylene glycol, liposomes, sugars, gelatin, lactose, amylose, magnesium stearate, talc, surfactants, silicic acid, viscous paraffin, perfume oil, fatty acid monoglycerides and diglycerides, petroethral fatty acid esters, hydroxymethyl-cellulose, polyvinylpyrrolidone, and the like. Spray compositions for topical delivery to the lung by inhalation may for example be formulated as aqueous solutions or suspensions or as aerosols delivered from pressurized packs, such as a metered dose inhaler, with the use of a suitable liquefied propellant. Aerosol compositions suitable for inhalation can be either a suspension or a solution and generally contain the compositions of the present invention and a suitable propellant such as a fluorocarbon or hydrogen-containing chlorofluorocarbon or mixtures thereof, particularly hydrofluoroalkanes, e.g. dichlorodifluoromethane, trichlorofluoromethane, di chlorotetrafluoroethane, especially 1 , 1 , 1 ,2-tetrafluoroethane, 1 , 1 , 1 ,2,3 ,3 ,3- heptafluoro-n-propane or a mixture thereof. Carbon dioxide or other suitable gas may also be used as propellant. The aerosol composition may be excipient free or may optionally contain additional formulation excipients well known in the art such as surfactants, e.g., oleic acid or lecithin and cosolvents, e.g. ethanol. Pressurized formulations will generally be retained in a canister (e.g. an aluminum canister) closed with a valve (e.g. a metering valve) and fitted into an actuator provided with a mouthpiece.

In some embodiments, the RIPK2 inhibitor can be administered in conjunction with other therapeutic agents. In particular, the RIPK2 inhibitor is administered to the patient as a combined preparation for simultaneous, separate or sequential use in the treatment of allergic asthma. As used herein, the term “simultaneous use” denotes the use of a RIPK2 inhibitor and at least one therapeutic agent occurring at the same time. As used herein, the term “separate use” denotes the use of a RIPK2 inhibitor and at least one therapeutic agent not occurring at the same time. As used herein, the term “sequential use” denotes the use of a RIPK2 inhibitor and at least one therapeutic agent occurring by following an order. In particular, therapeutic agents used in combination with the RIPK2 inhibitor of the present invention comprises corticoids, P- stimulant bronchodilators, bronchodilators from anticholinergics family as Tiotropium, antileukotrienes as Montelukast, anti-immunoglobulin antibodies as Omalizumab or theophylline. Further, agents may be also anti-interleukin antibodies as Benralizumab, Mepolizumab, Dupilumab, Tezepelumab.

The invention will be further illustrated by the following figures and examples. However, the examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURES

RIPK2 inhibitor as a preventive treatment on HDM-induced asthma.

Airway resistance of mice challenged with PBS, HDM or HDM and RIPK2 inhibitor

(A). Total and eosinophil BAL cell counts in mice challenged with PBS, HDM or HDM and RIPK2 inhibitor. ELISA detection of HDM-specific IgGl in serum of mice challenged with PBS, HDM or HDM and RIPK2 inhibitor (B). Quantification of stained lung sections for mucus using PAS (C). mRNA relative expression (RE) of cytokines and chemokines assessed by quantitative real-time -PCR in lung extracts from mice challenged with PBS, HDM or HDM and RIPK2 inhibitor (D).

Data are presented as mean ± SEM of n=8 to 10 animals per group. *P < .05, **P < .01, ***P < .001 versus PBS; #P < .05, ##P < .01, ###P < .001.

Figure 2: RIPK2 inhibitor as a curative treatment on HDM-induced asthma. mRNA relative expression (RE) of cytokines and chemokines assessed by quantitative real-time-PCR in lung extracts from mice challenged with PBS, HDM or HDM and RIPK2 inhibitor.

Data are presented as mean ± SEM of n=5 to 8 animals per group. *P < .05, **P < .01, ***p < .001 versus PBS; #P < .05, ##P < .01, ###P < .001. Figure 3: RIPK2 inhibitor as a curative treatment on HDM-induced asthma, in mutant NODI humanized mice.

Airway resistance in WT and mutant hNODl mice challenged with PBS, HDM or HDM and RIPK2 inhibitor (A). Total and eosinophil BAL cell counts in WT and mutant hNODl mice challenged with PBS, HDM or HDM and RIPK2 inhibitor. ELISA detection of HDM-specific IgGl response in serum WT and mutant hNODl mice challenged with PBS, HDM or HDM and RIPK2 inhibitor (B). Quantification of stained lung sections for mucus using PAS (C). mRNA relative expression (RE) of cytokines and chemokines assessed by quantitative real- time-PCR in lung extracts from WT and mutant hNODl mice challenged with PBS, HDM or HDM and RIPK2 inhibitor (D).

Data are presented as mean ± SEM of n=5 to 10 animals per group. *P < .05, **P < .01, ***p < .001 versus PBS; #P < .05, ##P < .01, ###P < .001.

Figure 4: RIPK2 inhibitor as a curative treatment on IL 13 and IL 33, in HDM stimulated PCLS obtained from mutant NODI humanized mice.

ELISA detection of IL33 in supernatants of PCLS obtained from naive WT and mutant NODI humanized mice ex vivo stimulated with PBS, HDM, the RIPK2 inhibitor alone or HDM and the RIPK2 inhibitor (A). ELISA detection of IL 13 in supernatants of PCLS obtained from WT and mutant NODI humanized mice after completion of the HDM induced asthma protocol and ex vivo stimulation with PBS, HDM, the RIPK2 inhibitor alone or HDM and the RIPK2 inhibitor (B). Data are presented as mean ± SEM of n=3 to 6 PCLS per group. *P < .05, **P < .01, ***p < .001 versus the control group; ^#P < .05, ^##P < .01, ^###P < .001.

Figure 5: RIPK2 inhibitor as a curative treatment on neutrophils and lymphocytes recruitment, in HDM stimulated BAL obtained from mutant NODI humanized mice.

BAL lymphocyte and neutrophil recruitment and total IgE in the therapeutic protocol in co-housed WT and hNODl mice. Neutrophil and lymphocyte BAL cell counts in WT and mutant hNODl mice challenged with PBS, HDM or HDM and RIPK2 inhibitor. ELISA detection of total IgE response in serum of WT and mutant hNODl mice challenged with PBS, HDM or HDM and RIPK2 inhibitor. Data are presented as mean ± SEM of n=5 to 10 animals per group. *P < .05, **P < .01, ***p < .001 versus PBS; ^#P < .05.

EXAMPLES Material & Methods

Mice.

WT female C57BL/6J mice (6 weeks of age) were purchased from Charles River. The C57BL/6J hnodl+/card4-/- (hNodl) transgenic mice harbouring a specific Nodi mutation associated with asthma (Hysi P et al., 2005, Hum Mol Genet) were generated as described previously (Zarantonelli M.L et al., 2013, Cell Host Microbe). All animals were housed under specific pathogen-free (SPF) conditions, in ventilated cages with absorbent bedding material, maintained on a 12-hour daylight cycle and with free access to commercial pelleted food and water ad libitum. All animal experiments were approved by the regional ethical committee and authorized by the ministry of research and innovation (APAFIS#7874-2016070417344442 v3), and the animals' care was in accordance with institutional guidelines.

House dust mite extract

The HDM extract used was a Dermatophagoides farinae extract kindly provided by Stallergenes/Greer (lot 9702026). An endotoxin level of 11.6 ng (= 10ng LPS) for 5IR of HDM extract was determined using the Pierce LAL Chromogenic Endotoxin Quantification Kit (Thermo Fischer Scientific).

RIPK2 inhibitor

The RIP2K inhibitor (GSK2983559) was kindly provided by Glaxo Smith Kline and resynthesized by AGV. It was previously administered orally in a trinitrobenzene sulfonic acid (TNBS) induced colitis rat model, which showed an efficacy on the histological score of the colitis starting at a dose of 7.5mg/kg (Hoiale P.A et al., 2019, J Med Chem). However, after entering a human phase I clinical trial, the recruitment was terminated in 2020 due to “non clinical toxicology findings and reduced safety margins” (GlaxoSmithKline, 2019, ClinicalTrial.gov). Therefore, local administration has been tested for reducing doses and toxicity. In vivo dose experiments were performed using preventive RIPK2 inhibitor doses of O.lmg/kg and Img/kg per intranasal instillation (i.n.) administration. Results showed similar results on AHR and the type 2 mediator profile of the lung (data not shown), therefore the lowest dose of 0. Img/kg has been chosen.

House Dust Mite-induced allergic airway inflammation and RIPK2 inhibitor interventions.

Mice were sensitized by intranasal instillation (i.n.). Starting on day seven after sensitization, mice were challenged i.n. daily for five consecutive days. Forty-eight hours after the last challenge, mice were anaesthetized, assessed for airway hyperresponsiveness and sacrificed. BAL fluids and serum were recovered. Lung samples were collected for RNA isolation, protein extraction, and histology.

In the prophylactic intervention, both the sensitization and the challenges consisted of two i.n. separated by 30 minutes. The HDM group received, a first i.n. of 25pL of PBS followed 30 minutes after by a second i.n. of 25 pL of 5IR HDM extract (i.e. 15 pg protein/ 25 pL of PBS). The group treated with the inhibitor received a first 25pL i.n. of the correspondent concentration of the RIPK2 inhibitor (O. lmg/kg), and 30 minutes after, 25 pL of HDM extract 5IR. Control mice received two i.n of 25pL of PBS (Figure 1A). Since the RIPK2 inhibitor stock vehicle solution contains DMSO, an equivalent final DMSO concentration was used in the first i.n PBS administration in the control group.

In the therapeutic intervention model, the sensitization consisted of one i.n. of 25pL of 5IR HDM extract for both the HDM and inhibitor groups. The control PBS mice received 25 pL of PBS. The challenges consisted of two i.n. separated by 30 minutes. The RIPK2 inhibitor group received a first 25pL i.n. of O. lmg/Kg inhibitor, and 30 minutes later, 25 pL i.n. of 5IR HDM extract. The HDM group received, a first i.n. of 25pL of PBS (With the equivalent addition of DMSO) followed by a second i.n. of 5IR HDM extract (i.e. 15 pg protein/ 25 pL of PBS). Control PBS mice received two i.n of 25pL of PBS (Figure 2A). An additional group of control mice consisted in the administration of the RIPK2 inhibitor together with PBS sensitization and challenges or with PBS challenges only. The data showed results similar to the PBS control group (Fig.5), thus, only the full PBS group was kept as the control group.

Airway hyperresponsiveness measurement

Mice were anaesthetized with 0.5 mg/kg medetomidine (Domitor; Pfizer) and 75 mg/kg ketamine (Imalgene 1000; Merial). Once an adequate plane of anaesthesia was achieved, mice were cannulated intratracheally with an 18-gauge catheter and connected to the Flexivent (SCIREQ) mechanical ventilator. Mice were exposed to nebulized PBS followed by increasing concentrations of methacholine (0-100 mg/mL) (Sigma- Aldrich) using an ultrasonic nebulizer (Aeron eb). Basal lung mechanics and airway resistance to increasing doses of methacholine were measured. The analysis was conducted using the FlexiWare 7 Software (Scireq).

BAL analysis

The BAL was performed using 1ml of ice-cold PBS. The BAL fluid obtained was centrifuged at 135 g for 5 min at 4°C. The BAL supernatant was recovered and stored for further analysis. Pelleted cells were then resuspended in PBS and counted. Samples of these resuspended BAL cells were spun onto slides (Shandon cytospin 4; Thermo Fisher Scientific) and stained using May-Grunwald Giemsa (Diapath) in order to perform a differential cell count.

Serum collection and analysis

Blood was drawn from the abdominal vein. Serum was collected by centrifugation at 5000 g for 5 min and stored at -20°C. Levels of total IgE and HDM-specific IgGl were measured in collected sera by ELISA. For total IgE measurements, 96-well plates (Corning Incorporated) were coated overnight with 2 pg/mL of purified rat anti -mouse IgE (clone R35- 72; BD Biosciences Pharmingen). After a blocking step and the incubation of the serum samples, a biotinylated anti -mouse IgE antibody 2pg/mL (clone R35-118; BD Biosciences Pharmingen) was used. The binding of specific antibodies was detected by the addition of a streptavidin-horseradish peroxidase (Zymed, Invitrogen) followed by the addition of TMB substrate solution (Interchim). The IgE concentration was expressed as ng/mL after comparison with murine IgE standard (0.5mg/mL, BD Biosciences Pharmingen).

For HDM-specific IgGl, microplates were coated with 10 IR/mL of HDM extract. After blocking and incubation with sera dilutions, binding of specific antibodies was detected by addition of horseradish peroxidase labelled goat anti mouse-IgGl (Southern Biotechnology, Birmingham) followed by addition of TMB substrate solution. ODs were measured on an ELISA plate reader (FLUOSTAR, Omega) at 450 nm.

Pulmonary histology

The left lobe of the lung from each mouse was fixed in Antigenfix (Diapath) and embedded in paraffin (Histowax, HistoLab) according to the manufacturer’s indications. Lung sections of 5pm were stained with a standard Hematoxylin-Eosin stain and Periodic acid-Schiff (PAS) staining kit (Diapath) to evaluate the peribronchial inflammation and mucopolysaccharide staining for mucus respectively.

Lung RNA extraction

Lung lobes were preserved using RNAlater (ThermoFisher). For lung tissue RNA extraction, the preserved tissue was homogenized with the tissue homogenizing CK28 ceramic beads (Bertin) and RLT RNeasy lysis buffer (Qiagen) using Precellys evolution homogenizer (Bertin). RNA isolation was performed with the RNeasy mini kit (Qiagen) following the manufacturer’s instructions. Quantitative Real-Time PCR

Extracted RNA was reverse-transcribed using the High Capacity cDNA reverse transcription kit (Applied Biosystems) according to the manufacturer’s instructions. The resulting cDNA was amplified using the ABsolute Blue QPCR SYBR Green Mix kit (Thermo Scientific) and detected on a StepOnePlus Real-Time PCR System (Applied Biosystems). On completion of the PCR amplification, a DNA melting curve analysis was carried out to confirm the presence of a single amplicon. Actb was used as an internal reference gene in order to normalize the transcript levels. For some cytokines, the resulting cDNA was amplified using the IDT PrimeTime Assay master mix. Real Time-PCR was performed with PrimeTime Assay primers (IDT, Leuven, Belgium). They were detected on a QuantStudio 3 (Applied Biosystems, Waltham, USA). Data were analysed via the Thermo Fisher cloud. RplpO (Ribosomal Protein Lateral Stalk Subunit P0) was used as internal reference gene to normalize the transcript levels. Relative mRNA levels (2-AACt) were determined by comparing the PCR cycle thresholds (Ct) for the gene of interest and internal reference gene (ACt) and ACt values for treated and control groups (A ACt).

Preparation of Precision cut lung slice (PCLS) and ex vivo culture.

Precision cut lung slices were prepared essentially as described before (Lyons-Cohen M.R et al., 2017, J Vis Exp). Briefly, animals were euthanized with an intraperitoneal injection of pentobarbital (Euthasol; TVM lab). A 20G cannula was introduced into the trachea and the lungs were filled with a 2% low melting temperature agarose solution (Preci si onary). Lung lobes were separated and cut into 200 pm slices using the Compresstome VF-310-0Z (Preci si onary) vibrating microtome. PCLS were cultured in DMEM-F12 L-glutamine (Gibco) supplemented with Antibiotic-antimycotic (Gibco). PCLS were stimulated for 24 hours with 1 IR of HDM alone or in combination with 5 pM of the RIPK2 inhibitor. Culture supernatants were collected for cytokine quantification and PCLS were recuperated for protein and RNA extraction.

Lung and PCLS protein extracts

Lung and PCLS were homogenized using T-PER buffer (Thermo Scientific) containing protease inhibitors (Roche diagnostics) and the tissue homogenizing CK28 ceramic beads (Bertin) in a Precellys evolution homogenizer (Bertin). The lysates were centrifuged at 13000 g for 5 minutes at 4°C and supernatants were collected. Total protein concentrations were measured using the Pierce BCA protein Assay kit (Thermo Scientific).

Cytokine ELISA Measurement

Murine cytokines (IL-33, IL-13) levels in PCLS culture supernatants were assessed using commercial ELISAs according to the instructions provided by the manufacturers (R&D Systems and Invitrogen).

Air Liquid Interface (ALI) bronchial culture, stimulation and analysis

Fully differentiated bronchial epithelial ALI cultures (Mucilair) reconstituted from primary human cells were obtained from Epithelix. Two ALI cultures were obtained from healthy donors and four from asthma patients. The 24-well size Transwell cell culture inserts were maintained in MucilAir culture medium (Epithelix) at 37°C and 5% CO2. Cultures in the presence or absence of 5pM RIPK2 inhibitor in the culture medium were stimulated by nebulization of HR of HDM in 50pl of PBS or alternatively with 50pl of PBS. 24h after stimulation culture supernatants were collected and preserved at -80°C for further analysis.

Culture supernatants were analysed with the high-throughput, multiplex proximity extension assay (PEA) inflammation kit (Olink) and evaluated twice in two different runs. The results of the PEA analysis are expressed as normalized protein expression units (NPX), which are relative log2 -transformed concentrations.

Statistical analysis

Data were analysed using Prism 9.0 (GraphPad Software). For normally distributed data, the significance of differences between groups was evaluated by one-way analysis of variance (ANOVA) with Tukey’s post hoc test for multiple comparisons. For pairwise comparisons, the two-tailed Student’s t-test was used. For airway resistance, the two-way analysis of variance test was used. Non-normally distributed data were analysed using the Kruskal -Wallis H with Dunn’s post hoc test. PEA data was evaluated by multiple t test analysis for pairwise comparisons and matched two-way ANOVA with Fisher's LSD post hoc test for multiple comparisons. The number of mice per group is indicated in the figure legends. The p values below 0.05 were considered to be statistically significant.

Results Local Preventive administration of the RIPK2 inhibitor reduces HDM-induced asthma dysregulations.

The in vivo preventive effect of the specific RIPK2 inhibitor, belonging to the 4-amino- quinolines family, was studied as a local pharmacological intervention using an experimental model of HDM-induced asthma. This preventive approach consisted of 0.1 mg/Kg RIPK2 inhibitor administration via i.n. instillation during the whole duration of the HDM-induced asthma protocol.

The preventive intranasal administration of the RIPK2 inhibitor, significantly reduced the increase in AHR measured in response to methacholine (marker of bronchial irritability) exposure in the HDM exposed group (Fig. lA). Moreover, The RIPK2 inhibitor-treated mice also exhibited a significant reduction in the bronchoalveolar lavage (BAL) eosinophil count and lymphocyte recruitment compared to the HDM group (Fig. IB). In addition, the serie levels of HDM-specific type 2 related IgGl were significantly reduced (Fig. IB). The histological analysis showed a reduction of peribronchial inflammation as well as a decrease in mucus production in the preventive RIPK2 inhibitor compared to the HDM group (Fig.lC). The intranasal preventive intervention was able of averting the upregulation of several cytokines and chemokines traditionally related to adaptive type 2 immunity including IL-4, IL-5, IL- 13 and CCL17. Furthermore, the gene expression of IL-17, the structural cell alarmin IL-33 and the neutrophil -attracting CXCL1 chemokine were also significantly reduced in the inhibitor- treated groups (Fig. ID).

Therefore, these data demonstrate that the preventive inhibition of RIPK2 mitigates the cardinal features of asthma in this experimental HDM-induced model.

RIPK2 plays an important role in the early sensitization phase.

To assess the potential therapeutic efficacy of local RIPK2 inhibition using the same model, the RIPK2 inhibitor was administered only during the challenge phase of the experimental asthma protocol (data not shown). The therapeutic intervention did not result in changes in AHR (data not shown), cell recruitment in the BAL or the levels of HDM-specific IgGl (data not shown). The inhibitor did not promote changes on mucus secretion either (data not shown).

Nonetheless, it was found that while the expression levels of IL-4, IL-5, IL-13, CCL17, and IL- 17 remained unaffected, the expression of IL-33, CCL2, and CXCL1 presented significant reductions and IFN-y significant increases (Fig.2). These results suggest that while in this murine HDM-induced asthma model, signalling through RIPK2 plays an important role in the early sensitization phase, the therapeutic inhibition still results in changes in relevant asthma mediators.

Murine and human N0D1-RIPK2 signalling axis to HDM are different and the therapeutic local RIPK2 inhibition mitigates asthma features in mutant NODI humanized mice.

It has been shown that hNODl preferentially detects Tri-diaminopimelic acid (DAP) ligands whereas mNODl detects tetra DAP ligands (Magalhaes J.G et al., 2005, EMBO Rep) and that a mutation in hNodl has been described to be associated with asthma (Hysi P et al., 2005, Hum. Mol. Genet).. Moreover, it has been shown that the main NODI peptidoglycan ligand present in HDM extracts is MTriDAP which can aggravate HDM-induced asthma (Ait Yahia S et al., 2021, J. Allergy Clin. Immuno).

The efficacy of the local therapeutic RIPK2 inhibitor intervention has been studied in transgenic mice expressing the mutant human NOD 1 and deficient for murine NOD 1. The same HDM-induced asthma protocol in the mutant hNODl and WT, induces a significant increase in AHR. However, while the RIPK2 therapeutic intervention failed to modify the AHR in the WT, there was a significant reduction in the RIPK2 inhibitor-treated mutant hNODl mice (Fig.3 A).

The BAL total cell count showed that HDM-treated mutant hNOD 1 mice exhibited more cells than the WT and that this total cell increase was significantly reduced by the RIPK2 inhibitor (Fig.3B). Similarly, the eosinophil count was significantly higher in the hNODl mice and the RIPK2 inhibition reduced the BAL eosinophilia in comparison to the HDM treated mice. Moreover, RIPK2 inhibition resulted in reduced BAL neutrophil and lymphocyte numbers (Fig.5). While no significant changes where observed in the humoral response (Fig.3B) the histological analysis showed a reduction in peribronchial inflammation and mucus production (Fig.3C).

The evaluation of the expression of type 2 cytokines and chemokines in the lung of WT and hNODl mice showed that the mutant hNODl mice displayed higher expression of IL-4, IL-5, IL-13, and CCL17. Furthermore, while the RIPK2 inhibitor failed to modify the expression of the mentioned mediators in WT mice, all of them except CCL17 were reduced in the mutant hNODl mice when compared to the HDM-treated group. Moreover, the higher level of IL-33 and IL-17 in mutant hNODl mice were found to be significantly reduced (Fig.3D).

These data underline the higher sensitivity of human NODI to HDM stimulation, and the beneficial effect of RIPK2 inhibitors in a therapeutic setting, through decreased Th2-type profile and IL-33 expression. Moreover, these results demonstrate that the therapeutic local inhibition of RIPK2 in a human NODI signalling context mitigates the main asthma hallmarks of this HDM-induced model.

RIPK2 inhibition reduces cytokine production in 11 DM stimulated PCLS obtained from mutant NODI humanized mice.

To get more insight into the early versus late events occurring in this humanized model we evaluated the ex vivo effect of the RIPK2 inhibitor using precision cut lung slices (PCLS) obtained from WT and mutant NODI humanized mice.

Ex vivo HDM stimulation of PCLS obtained from both WT and mutant hNODl naive mice promoted significant increases in the production of IL-33. However, the IL-33 concentration was significantly higher in the HDM-stimulated mutant hNODl PCLS. The addition of the RIPK2 inhibitor reduced the HDM induced IL-33 production in both WT and mutant hNODl stimulated PCLS (Fig.4A). In contrast, HDM stimulation of PCLS prepared from WT or mutant hNODl mice that had been subjected to HDM-induced asthma protocol did not result in significant changes in their IL-33 production. Besides, the RIPK2 inhibitor did not change the IL-33 supernatant concentration (data not shown).

Whilst IL- 13 was not detectable in the supernatants of PCLS obtained from WT naive mice (data not shown), PCLS from both asthmatic WT and hNODl mice produced IL- 13 in detectable concentrations without the need of HDM ex vivo stimulation. Whereas the ex vivo HDM stimulation of the asthmatic WT PCLS did not result in a significant IL- 13 increment, asthmatic mutant hNODl PCLS presented significant increased IL-13 levels. The presence of the RIPK2 inhibitor resulted in significant decreases in the IL- 13 concentration of HDM stimulated WT PCLS and a trend towards decrease in mutant hNODl mice (Fig.4B).

These results underline the differences in the murine and human NODI orthologues driven responses and emphasizes the early role of IL-33 in the NODI -dependent response of the epithelium to HDM, and a late effect of NODI signalling on IL-13 effector response.

RIPK2 inhibition modulates cytokine and chemokine production in asthmatic bronchial air liquid interface cultures with and without concomitant HDM stimulation.

To further explore the mechanistic effect of RIPK2 inhibitors in a NODI human cell signalling setting we used fully differentiated bronchial epithelium air-liquid interface cultures obtained from healthy and asthmatic individuals. A 96 inflammation-related protein PEA was used to comprehensively analyse the effects of the RIPK2 inhibitor and HDM stimulation. HDM stimulation of the bronchial ALI cultures from healthy subjects induced an increased expression of several mediators including pro-inflammatory cytokines and chemokines (IL-la, IL-6, IL-18, MCP-4, CXCL5, CXCL10, CXCL11), the pro Th2 alarmin IL-33, the growth factor FGF-19, the signalling molecule 4E-BP1 and the enzyme ADA (data not shown). Conversely, RIPK2 inhibitor treatment of these cultures led to a decreased production of a group of mediators including chemokines such as CXCL6, CCL8, CCL2, CXCL5, CXCL10, and CXCL11 (data not shown).

The PEA analysis of ALI cultures obtained from asthmatic patients displayed different protein profiles when compared to the cultures from healthy individuals. At baseline, ALI from asthmatic patients exhibited higher levels of IL-6, GDNF, VEGFA, and of MMP-1 and MMP- 10 proteases than ALI from healthy individuals (data not shown). Moreover, HDM stimulation of asthmatic ALI resulted in a change in their protein production profile, further differentiating the responses between healthy and asthmatic showing augmented levels of VEGFA, MMP-1, MMP10, and of the TSLP alarmin with decreased levels of CXCL10 (data not shown).

In asthmatics at baseline, RIPK2 treatment of PBS-stimulated ALI cultures already inhibited the production of some chemokines including CCL8, CXCL6, CXCL10 and CXCL11 and increased IL-6. Treatment of HDM-stimulated asthmatic ALI with the RIPK2 inhibitor was able to modify their protein production, and as a result, showing reduced levels of TSLP, PDL1, IL-17C, CCL8, CXCL6, CXCL9, CXCL10 and CXCL11 (data not shown). Among these molecules only TSLP was significantly increased by HDM stimulation as compared with baseline levels. Inversely, HDM stimulation and the addition of the RIPK2 inhibitor resulted in augmented levels of IL-6.

Collectively, these data demonstrate that HDM-induced asthmatic disease can be modified by therapeutic interventions through RIPK2 in the context of human NODI activation.

REFERENCES

Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

Lambrecht. N.D et al., 2019, Immunity Cell Press, DOI : 10.1016/j.immuni.2019.03.018

Jacquet A et al., 2021, Front Allergy, DOI : 10.3389/falgy.202L 662378

Ait Yahia S et al., 2021, J. Allergy Clin. Immuno, DOI : 10.1016/j.jaci.2020.12.649 Hysi P et al., 2005, Hum. Mol. Genet, DOI : 10.1093/hmg/ddi087

Ait Yahia. S et al., 2014, Am. J. Respir. Grit. Care Med, DOI : 10.1164/rccm.20131018270C

Miller M.H et al., 2018, J.Leukoc. Biol, DOI : 10.1002/JLB.4HI0118-017RR

Miller M.H et al., 2020, ImmunoHorizons, DOI : 10.4049/immunohorizons.2000073.

Honjo H et al., 2021, Front. Pharmacol, DOI : 10.3389/fphar.2021.650403

He S et al., 2018, Nature Immunology, DOI : 10.1038/s41590-018-0188-x

Watanabe. T et al., 2019, Int. Immunol, DOI : 10.1093/intimm/dxz045.

Jaafar R et al., 2018, Biochem. Biophys. Res. Commun, DOI : 10.1016/j.bbrc.2018.02.034

Cai X et al., 2018, Oncol. Rep, DOI : 10.3892/or.2018.6397

Tian E et al., 2023, Eur JMedChem, DOI : 10.1016/j.ejmech.2023.115683

You J et al., 2023, Front Pharmacol, DOI : 10.3389/fphar.2023.1192970

Pham. A.T et al., 2023, Front Pharmacol, DOI : 10.3389/fphar.2023.1127722

Pussard E et al., 1994, Fundam. Clin. Pharmacol, DOI : 10.1111/j.1472-8206.1994 ,tb00774.x

Zhao Y et al., 2012, J Immunol, DOI : 10.4049/jimmunol. l88.Supp. l69.5

Zarantonelli M L et al., 2013, Cell Host Microbe, DOI : 10.1016/j.chom.2013.04.016.

Haile P.A et al., 2019, J Med Chem, DOI : 10.1021/acs.jmedchem.9b00575

GlaxoSmithKline., 2019, ClinicalTrial.gov, GSK2983559 First Time in Human Study

Lyons-Cohen M.R et al., 2017, J Vis Exp, DOI : 10.3791/55465

Magalhaes J.G et al., 2005, EMBO Rep, DOI : 10.1038/sj.embor.7400552

Claims

CLAIMS :

1. A method of the curative treatment of an allergic asthma in a patient in need thereof comprising administering locally a therapeutically effective amount of a RIPK2 inhibitor.

2. A method for use according to claim 1 wherein the allergic asthma is a HDM-induced asthma.

3. A method for use according to claims 1 or 2 wherein the RIPK2 inhibitor reduces peribronchial inflammation, mucus overproduction and/or airway hyperresponsiveness.

4. A method according to claims 1 to 3 wherein the subject having or developing drug resistance.

5. A method according to claims 1 to 4 wherein the RIPK2 inhibitor is administered by nasal or mouth administration.

6. A method according to claims 1 to 5 wherein the RIPK2 inhibitor is an inhibitor of RIPK2 expression.

7. A method according to claims 1 to 6 wherein the RIPK2 inhibitor belongs to the 4- aminoquinolines family.

8. A method according to claims 1 to 6 wherein the RIPK2 inhibitor is DCAM-253 or 2- ((4-(benzo[d]thiazol-5-ylamino)-6-(tert-butylsulfonyl)quinazolin-7-yl)oxy)ethyl dihydrogen phosphate.

9. A method according to claims 1 to 8 wherein the RIPK2 inhibitor is administered in conjunction with at least one other therapeutic agent.

10. A method according to claim 9 wherein the therapeutic agent is corticoids, P-stimulant bronchodilators, bronchodilators from anticholinergics family as Tiotropium, antileukotrienes as Montelukast, anti-immunoglobulin antibodies as Omalizumab or theophylline. Further, agents may be also anti-interleukin antibodies as Benralizumab, Mepolizumab, Dupilumab, Tezepelumab.

11. A therapeutic composition comprising a therapeutically effective amount of an RIPK2 inhibitor for the curative treatment of an allergic asthma in a patient in need thereof.

12. A therapeutic composition according to claim 11 wherein the composition is an aerosol composition.