3-Azabicyclo[3.2.1]octanecarboxylic acid and derivatives thereof for use in the treatment of inflammation

Abstract

The present invention describes 3-azabicyclo[3.2.1]octanoic acids of general formula (I), their salts and esters, for use as activators of ADAR1 in the treatment of diseases associated with infectious or non-infectious, acute or chronic inflammation, characterized by cytokine storm and / or uncontrolled immune response. [Formula 1] JPEG2023534091000009.jpg30170

Description

[Technical Field]

[0001] The present invention relates to the field of 3-azabicyclo[3.2.1]octanecarboxylic acid compounds and derivatives thereof for use in the treatment of infectious or non-infectious acute or chronic inflammation characterized by cytokine storm and / or uncontrolled immune response. [Background technology]

[0002] Inflammation is the immune system's response to harmful stimuli, such as pathogens (viruses, bacteria, fungi), toxic chemical and biological substances, cell necrosis (myocardial infarction, tissue wounds), and radiation. It represents a defense mechanism that acts by eliminating harmful stimuli and simultaneously promoting the healing process. Normally, during an inflammatory response, cellular and molecular events are tightly regulated to minimize injury. This mitigation process contributes to the restoration of tissue homeostasis and the rapid resolution of inflammation, which is defined as acute inflammation. However, if regulation of the inflammatory process is impaired, inflammation can become uncontrollable and chronic, leading to several severe inflammatory diseases or extremely dangerous syndromes, such as the so-called "cytokine storm" (cytokine storm syndrome or CSS) (Behrens and Koretzky, Arthritis & Rheumatology 2017). Regardless of etiology, a cascade of biochemical signals necessary for the elimination of harmful stimuli and the healing of damaged tissues is activated during inflammation. In particular, leukocytes that produce inflammatory cytokines are recruited from the systemic circulation to the site of injury. In general, the inflammatory response consists of a series of coordinated events involving both resident tissue cells and cells recruited from the blood. Although the types of events triggered during inflammation depend on the nature of the harmful stimulus and the type of tissue / organ involved, they all share the following common steps and mechanisms: 1) recognition of the harmful stimulus by cell surface receptors; 2) activation of inflammatory pathways; 3) release of inflammatory markers; 4) recruitment of inflammatory cells; and 5) resolution of the inflammatory process. This last step is fundamentally important because it prevents a prolonged and uncontrolled response that could result in further damage beyond that triggered by the initial pathogenic stimulus and prevents the progression from acute to chronic inflammation. Naturally, chronic inflammation can occur whenever the initial harmful stimulus is not removed. While acute or chronic inflammation is usually localized, in some cases it can become systemic and uncontrollable, leading to CSS (Gilroy and De Maeyer, Seminars in Immunology 2015). CSS is a syndrome characterized by a clinical paradigm of systemic inflammation accompanied by fever, cytopenias, coagulopathy, multiple organ failure, and hyperferritinemia, which, if untreated, can lead to death.This condition is caused by the abnormal production of cytokines and other inflammatory molecules resulting from uncontrolled immune system activation. CSS triggers can have several origins: rheumatic, neoplastic, and infectious. Among these, the most well-known form of CSS is sepsis, a condition caused by widespread infection, a hyperinflammatory syndrome characterized by hypercytokinemia, and multiple organ failure, often associated with secondary hemophagocytic syndrome (sHLH). In adults, sHLH is most often triggered by viral infection, occurring in 3.7–4.3% of sepsis cases. Key features of sHLH include persistent fever (>38.5°C), cytopenias, and hyperferritinemia, and pulmonary involvement, including acute respiratory distress syndrome (ARDS), occurs in approximately 50% of patients. However, sepsis and CSS are generally not the direct effects of a pathogen (or another type of initial insult) but rather the result of an uncontrolled immune response to that pathogen. A hallmark of CSS is an uncontrolled and dysfunctional immune response, involving the continuous activation and proliferation of both lymphocytes and macrophages, which secrete massive amounts of cytokines, resulting in a cytokine storm. Many clinical features of CSS are explained by the action of proinflammatory cytokines, such as interferons (IFNs), tumor necrosis factors (TNFs), and interleukins (ILs), such as IL-1, IL-6, and IL-18. These proinflammatory cytokines are known to be elevated in most patients with CSS. In this inflammatory context, ADAR1 plays a crucial role by regulating specific proteins involved in inflammatory activation and the release of proinflammatory cytokines. For example, during viral infections or in the presence of chemical and physical stress (UV radiation and oxidative stress) or other pathogens, PKR (protein kinase R) and RIG-I (retinoic acid-inducible gene I) are activated. In their active forms, these proteins can induce the expression of type 1 interferon (IFN-I) and other proinflammatory cytokines.Although IFN-1 is known for its antiviral activity, excessive production can lead to CSS. For this reason, several enzymes exist that can regulate its expression to maintain tissue homeostasis. One of these is ADAR1 (adenosine deaminase acting on RNA 1), a double-stranded RNA-specific adenosine deaminase that can bind to and modify viral RNA and microRNA (miRNA) (Song C. et al. Genes 2016). During infection, ADAR1 binds to viral RNA, preventing its recognition by PKR and RIG-I sensors, so that genes involved in IFN production can no longer be activated. Furthermore, ADAR1 can modify the nucleotide sequence of the viral genome through its adenosine deaminase activity, preventing its replication. Finally, a further anti-inflammatory property of ADAR1 lies in its ability to reduce the expression levels of microRNAs that target proteins with anti-inflammatory functions (e.g., miR-101 and miR-30a). Indeed, one consequence of reduced miR-101 levels is increased levels of MKP-1, a protein capable of silencing p38 MAPK, thereby preventing the production of inflammatory mediators involved in CSS. The proinflammatory activity of p38 MAPK has been widely demonstrated in several pathologies, including viral ones, due to uncontrolled cytokine release. Although it plays a central role in inflammatory responses, p38 MAPK is only one of many proteins involved in the activation of the proinflammatory cytokine cascade. It should be noted that selective p38 MAPK inhibitors cannot activate ADAR1, and as a result, such inhibitors can only partially counteract the complex mechanisms that characterize CSS, especially when CSS results from infection.

[0003] Furthermore, during viral infection, ADAR1 activity is to modify the structure of viral RNA by inhibiting its synthesis. Finally, activation of ADAR1 allows the inactivation of cellular sensors such as PKR and RIG-I, preventing excessive production of INF and thus the uncontrolled release of proinflammatory cytokines.

[0004] Activation of ADAR1 is known to be one of the innate immune mechanisms effective in neutralizing RNA viruses through editing of their genomes (Chung et al., H, Cell 2018).

[0005] Despite the existence of various medications for the treatment of acute and chronic inflammation, there is currently an unmet medical need for the treatment of diseases associated with CSS. Treatment for these diseases primarily consists of immunosuppression, complemented by control of the underlying disease, along with the use of antibiotics or antivirals for patients with infections (Behrens and Koretzky, Arthritis & Rheumatology 2017). Like most inflammatory diseases, CSS can be treated with corticosteroids or, more recently, therapies aimed at blocking specific cytokines, such as anti-IL-1, anti-IFN, and anti-IL-6 therapy. However, currently available anti-inflammatory treatments have several limitations, as they aim to control only specific cytokines or, as in the case of corticosteroids, can lead to resistance to the treatment itself. Nevertheless, because current treatments do not provide nutritional support to tissues and organs damaged by uncontrolled inflammation, systemic damage often persists even if inflammation itself can be limited. These injuries involve not only vital organs such as the lungs (especially in cases of respiratory tract infections), but also the kidneys, liver, and heart (the latter of which can lead to heart failure due to massive apoptosis), and vascular endothelium, which must be properly repaired. None of the current therapies are able to limit the multi-organ injury induced by CSS because they lack trophic and anti-apoptotic activity.

[0006] US2019 / 0359692 and WO2019 / 122909 describe p38 MAPK inhibitors for use in treating influenza with severe respiratory tract complications.

[0007] Zhou Shangxun et al. (Mediators of Inflammation, 2020; DOI: 10.1155 / 2020 / 9607535) demonstrate that ADAR1 reduces inflammation in a mouse model of sepsis.

[0008] WO2004000324, also by the same applicant, describes that derivatives of 3-azabicyclo[3.2.1]octane are useful as agonists of human neurotrophins for the treatment of diseases in which neurotrophin function, particularly NGF function, is deficient, such as: neurodegenerative disorders of the central nervous system involving neuronal apoptosis, such as Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), Huntington's disease, neuropathy, and neuronal damage caused by hypoxia, ischemia, or trauma; acquired immunodeficiency diseases associated with reduced NGF bioavailability, such as age-related immunodeficiency; diseases in which stimulation of neovascularization is dominant, such as myocardial infarction, stroke, or peripheral vascular disease; and certain ocular diseases, such as keratitis of various etiologies, glaucoma, or retinal inflammatory conditions. WO2004000324 describes (1S,4R,5R,7S)-3,4-dibenzyl-2-oxo-6,8- as a particularly preferred compound. Dioxa methyl 3-azabicyclo[3.2.1]octane-7-carboxylate (MT2) is described.

[0009] WO2013140348, again by the same applicant, describes certain carboxylic acid derivatives of 3-azabicyclo[3.2.1]octane and their medical uses, in particular for the treatment of all pathologies associated with ischemia-reperfusion following an ischemic state caused by a reduction or obstruction of blood flow followed by the restoration of oxygen / nutrient supply to tissues, or for medical procedures involving ischemia-reperfusion. WO2013140348 specifically describes the acid (1S,4R,5R,7S)-3,4-dibenzyl-2-oxo-6,8-dioxane-3-azabicyclo[3.2.1]octane-7-carboxylic acid (MT6) and its pharmaceutically acceptable salts, as well as the acid (1S,4R,5R,7S)-3,4-dibenzyl-2-oxo-6,8-dioxa-3-azabicyclo[3.2.1]octane-7-carboxylic acid lysine salt (MT8).

[0010] [ka]

[0011] The applicant has also demonstrated through appropriate non-clinical and clinical studies that MT6 acid in the form of lysine salt (referred to as MT8), sodium, potassium, or any other pharmaceutically acceptable salt, dissolved in phosphate buffer, saline buffer, or any other pharmaceutically acceptable buffer, in the absence or presence of preservatives and excipients, can be used to treat diseases in which there is a deficiency in the function of neurotrophins, particularly the function of NGF and BDNF.

[0012] On December 16, 2014, MT8 received orphan drug designation from the European Medicines Agency (EMA) for the treatment of neurotrophic keratitis 8EU / 3 / 14 / 1400.

[0013] Although there are many drugs used to reduce the damage caused by acute and chronic inflammation, there are still unmet medical needs for the treatment of these diseases. It is therefore an object of the present invention to provide at least alternative compounds for use in the treatment of acute or chronic inflammation in which the so-called CSS cytokine storm syndrome occurs.

[0014] It is therefore a further object of the present invention to provide ADAR1-activating compounds for use in the treatment of severe infectious or non-infectious inflammatory diseases characterized by cytokine storm and / or uncontrolled immune responses. Summary of the Invention

[0015] The subject of the present invention is a compound of formula (I), including its pharmaceutically acceptable salts, for use as an activator of adenosine deaminase acting on RNA 1 (ADAR1), in the treatment of acute or chronic, infectious or non-infectious inflammatory diseases characterized by a cytokine storm and / or an uncontrolled immune response, said compound of formula (I) being:

[0016] [ka] During the ceremony R1 is aryl, C 1-8 alkylaryl; R2 is C 1-8 alkylaryl; R3 is H, -C 1-8 Alkyl, C 1-8 alkylaryl.

[0017] Through a series of in vitro experiments, it was unexpectedly discovered that the compounds covered by this patent can induce: i. Hyperactivation (homodimerization) of ADAR1 significantly reduces the expression of miR-101, resulting in a decrease in the release of proinflammatory cytokines; ii. Acting to reduce the systematic production of cytokines upstream of IL-6 in the functional cascade, thereby reducing the effects resulting from the "cytokine storm" or CSS.

[0018] These actions are associated with the following activities: iii. Trophic support to hypoxic tissues at the systemic level through the reduction of damage induced by the ischemia-reperfusion process; iv. Trophic support to tissues at a systemic level via reduction of inflammatory processes or damage induced by CSS.

[0019] Therefore, administration of pharmaceutical formulations containing the compound of formula (I), the subject of the present patent, as an activator of ADAR1, an anti-inflammatory agent, and an antiviral agent is useful for treating diseases associated with acute or chronic inflammation characterized by cytokine storm and / or uncontrolled immune response. Furthermore, administration of pharmaceutical formulations containing the compound of formula (I), the subject of the present patent, is preferably, but not exclusively, useful for treating respiratory diseases, and particularly, but not exclusively, for treating respiratory diseases induced by viral agents, such as severe acute respiratory syndrome (SARS) caused by coronavirus or other viruses, by limiting biochemical and functional damage in severely damaged pulmonary endothelium, which is often already impaired by pre-existing or concurrent pathologies, as well as in hypoxic tissues of various organs (brain, kidney, liver, etc.). Detailed Description of the Invention

[0020] In the present invention, unless otherwise specified, the terms alkyl, aryl, alkylaryl are understood as follows: C 1-8 Alkyl 1-8 ) refers to a straight or branched chain alkyl group having a single bond C-C. Examples of alkyl groups according to the present invention include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, pentyl, elongated, heptyl, and octyl. The term "aryl" refers to a group containing one or more unsaturated rings, each of which is 5 to 8-membered, preferably 5 or 6. Examples of aryl groups include, but are not limited to, phenyl, biphenyl, and naphthyl.

[0021] According to the present invention, the aryl group may be substituted with one or more groups, preferably halogen, cyano, nitro, amino, hydroxy, carboxylic acid, carbonyl, and C 1-6 Alkyl 1-6 The term "halogen" refers to fluorine, chlorine, bromine, and iodine.

[0022] In the compounds of the present invention, R1 is preferably CH2Ph. Preferably, R2 is CH2Ph. Preferably, R3 is H or CH3. Optionally, the phenyl group may be substituted with one or more groups, preferably X, CN, NO, NH, OH, COOH, (C=O)Alk 1-6 wherein X is selected from the group consisting of F, Cl, Br and I.

[0023] Among the compounds of formula (I), the following are preferred: R1 is CH2Ph; R2 is CH2Ph; R3 is H or CH3; wherein the phenyl group is optionally substituted with one or more groups, preferably X, CN, NO2, NH2, OH, COOH, (C=O)Alk 1-6 wherein X is selected from the group consisting of F, Cl, Br and I.

[0024] For the purposes of the present invention, compounds of formula (IA) and (IB) are more preferred:

[0025] [ka]

[0026] Such compounds may obviously occur in a variety of stereochemical configurations.

[0027] [Table 1]

[0028] [Table 2]

[0029] For the purposes of the present invention, the compound (1S,4R,5R,7S)-3,4-dibenzyl-2-oxo-6,8- Dioxa -3-azabicyclo[3.2.1]octane-7-carboxylate methyl (MT2), (1S, 4R, 5R, 7S)-3,4-dibenzyl-2-oxo-6,8- Dioxa Among MT6 salts and its pharmaceutically acceptable salts, the following salts are particularly relevant: potassium salt, sodium salt, lysine salt, organic and inorganic quaternary ammonium salts. Thus, a particularly preferred compound is (1S,4R,5R,7S)-3,4-dibenzyl-2-oxo-6,8-dioxa-3-azabicyclo[3.2.1]octane-7-carboxylic acid L-lysine salt (MT8).

[0030] It has been found that the compounds of formula (I) above are capable of reducing the inflammatory cytokines produced by LPS-activated human monocytes / macrophages and LPS-activated human dendritic cells (see Figure 1).

[0031] In particular, under proinflammatory conditions, the compounds of the present invention are capable of activating ADAR1, an enzyme that can reduce the expression of miR-101, a microRNA (miRNA) involved in proinflammatory responses, through its RNA-editing activity. Activation of ADAR1 by the compounds of the present invention was observed in the HEK-293 TrkA cell line (see Figure 2). Indeed, treatment with the compounds has been shown to induce a significant increase in homodimer ADAR1 / ADAR1 compared to untreated cells, promoting the formation of the active form of the protein and favoring the editing process. The activity of ADAR1 to reduce miR-101 expression was observed in cells treated with the compounds of the present invention in the presence or absence of ADAR1 knockdown (Figures 3 and 4).

[0032] If ADAR1 activity is increased at the cellular level, it can protect cells from damage caused by viral infections and inflammation, and the effect can be expected even if the infection is not necessarily infectious.

[0033] The activity of ADAR1 is actually dual: - In non-infectious inflammatory processes, ADAR1 is able to bind and edit miRNAs, which, once modified, are unable to recognize their target sequences and are therefore degraded; in particular, this occurs for miR-101, but also for miR-30a, the miRNA whose expression determines the increase in pro-inflammatory cytokines such as TNF-α and IL-6. -Furthermore, in viral infections, the activity of ADAR1 consists in its structural modification by inhibiting the synthesis of viral RNA.

[0034] Finally, activation of ADAR1 allows the inactivation of cellular sensors such as PKR and RIG-I, avoiding the overproduction of INF and thus preventing the uncontrolled release of pro-inflammatory cytokines.

[0035] In conclusion, the compounds of the present invention have potent anti-inflammatory and antiviral activity such that the following can be determined: i) a reduction in viral load and consequent infection due to increased cytoplasmic levels of ADAR1, an enzyme that damages the genome of RNA viruses; ii) a reduction in the systemic production and consequent effects of cytokines resulting from the so-called "cytokine storm" in patients via activation of ADAR1 and the resulting reduction of miR-101; iii) Reduction of damage induced by the ischemia-reperfusion process that occurs under severe inflammatory conditions through metabolic support to hypoxic tissues.

[0036] Thus, the compounds for use according to the invention as activators of ADAR1 are effective anti-inflammatory and antiviral agents and are therefore useful in the treatment of diseases associated with acute or chronic inflammation characterized by cytokine storm and / or uncontrolled immune responses.

[0037] In particular, compounds for use according to the invention as activators of ADAR1 may be useful in the treatment of infectious diseases of viral origin such as: herpes virus, Epstein-Barr virus, cytomegalovirus, adenovirus, HPV, coronavirus, enteroviruses, rotavirus, Parvovirus, Influenza A virus, Ebola virus, Members of the Marburgvirus genus Members of the Dengue virus species, Hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV) infection, Panencephalitis in measles virus infection (SSPE), Hemorrhagic fever viruses (Arenaviridae, Bunyaviridae, Filoviridae, Falviviridae, and Togaviridae), measles virus, Mumps virus, rubella virus, Parechovirus, Human T-lymphotropic virus.

[0038] The compounds for use according to the invention as activators of ADAR1 and therefore anti-inflammatory agents may also be useful in the treatment of the following infectious diseases characterized by a cytokine storm: i) Bacterial origin, such as: Aeromonas hydrophila, Brucella spp., Chlamydia spp., Clostridium spp., E. coli, Legionella spp. Mycobacteria, Salmonella, Staphylococcus aureus, Acinetobacter baumannii; Mycobacterium tuberculosis Mycoplasma pneumoniae ii) Parasitic and fungal origin, such as: Plasmodium spp., Leishmania spp., Toxoplasma gondii, Entamoeba histolytica, Babesia spp. human roundworm, helminths, Candida albicans, Histoplasma, Cryptococcus neoformans, Pneumocystis spp., Penicillium marneffei. iii) Zoonotic origin, such as: Brucella, rickettsia, Ehrlichia, Coxiella burnetii, Mycobacterium avium, Clostridium, Leptospira.

[0039] The compounds for use according to the invention, as activators of ADAR1, may also be useful in diseases of non-infectious origin causing a reduction in acute and chronic inflammatory conditions such as: sepsis, hemophagocytic lymphohistiocytosis, Adult-onset Still's disease (AOSD), chronic hepatitis, Obesity, atherosclerosis, periodontitis, Cirrhosis of the liver.

[0040] The compounds for use according to the invention, as activators of ADAR1 and therefore anti-inflammatory agents, may also be useful in the treatment of autoimmune as well as degenerative diseases such as: hemophagocytic lymphohistiocytosis, lymphoproliferative syndrome, Primary and acquired immunodeficiencies not due to NGF deficiency, Dyschromatosis hereditarily (DSH), Aicardi-Goutières syndrome (AGS), Rare genetic diseases associated with IL-1 / inflammasome disorders, IFN-mediated diseases, NF-κB / ubiquitin-mediated disorders Muckle-Wells syndrome, hyper-IgD syndrome, Pediatric granulomatous arthritis, ADA2 deficiency, sepsis, Arthritis / osteoarthritis, Juvenile idiopathic arthritis, lupus erythematosus, Kawasaki disease.

[0041] The compounds for use according to the invention are also particularly useful as activators of ADAR1 and therefore as anti-inflammatory and antiviral agents in the treatment of inflammatory diseases of the airways such as: Severe acute respiratory syndrome (SARS) induced by coronavirus or other viruses, asthma, chronic obstructive pulmonary disease (COPD), bronchiectasis, interstitial lung disease or pulmonary disease, bronchiolitis, Bronchopulmonary dysplasia (BPD) of premature infants tuberculosis, Whooping cough; acute inhalation injury due to exposure to harmful and toxic substances, Occupational respiratory tract infections such as: Legionnaires' disease, Q fever, Interstitial lung disease induced by occupational activities such as: ·Pneumoconiosis, Pulmonary diseases due to metal exposure, Extrinsic allergic alveolitis, ·Ardystil syndrome; Rare lung diseases such as: ·Pulmonary vasculitis, Idiopathic eosinophilic pneumonia, ·Alveolar proteinosis, Lymphangioleiomyomatosis (LAM), Pulmonary Langerhans cell histiocytosis, Birt-Hogg-Dubé syndrome.

[0042] The compounds for use according to the invention may be formulated in conventional pharmaceutical compositions which may include one or more pharmaceutically acceptable excipients and / or diluents.

[0043] Administration of these compositions can be carried out by any conventional route of administration, for example, parenterally in the form of injectable solutions or suspensions, orally, topically, intranasally, subcutaneously, subconjunctivally, etc.

[0044] The compositions may be in the form of tablets, capsules, solutions, dispersions, suspensions, liposomal formulations, microspheres, nanospheres, foams, creams and ointments, emulsions, microemulsions and nanoemulsions, and aerosols, and may be formulated in such a way as to provide controlled or delayed release of the active ingredient.

[0045] All of the above pharmaceutical compositions may contain at least one of the present compounds of formula (I) as an active ingredient, optionally in combination with other active ingredients or adjuvants selected according to the pathological condition to be treated.

[0046] The present invention will be better understood in light of the following examples. [Brief explanation of the drawings]

[0047] [Figure 1] The graph shows the production of IL-1β, TNF-α, and IL-6 in human monocytes and human dendritic cells stimulated with LPS in the presence or absence of MT8 compound. It is clear that the production levels of proinflammatory cytokines were significantly lower in both monocytes and dendritic cells treated with MT8 compound compared to untreated cells. [Figure 2] Effect of MT8 on activation of ADAR1 homodimer complex (active form of the protein). The gel shows induction of ADAR1 / ADAR1 homodimer complex in HEK-293 TrkA cells treated with MT8 compound. The graph shows densitometric quantification of gel bands, expressed as the ratio of band density of homodimer complex ADAR1 / ADAR1 to monomeric ADAR1. The level of ADAR1 / ADAR1 homodimer induced by MT8 compound is significantly higher than that of the control. [Figure 3]Effect of MT8 on miR-101 reduction in an in vitro model of inflammation. This graph shows the effect of MT8 on reducing intracellular miR-101 expression. This phenomenon was observed in human monocytes and dendritic cells cultured in the presence of LPS, one of the compounds with the highest pro-inflammatory activity. miR-101 levels were quantified by real-time PCR using 5s ribosomal RNA and calculated using the 2-ΔCt method. [Figure 4] Effect of MT8 on miR-101 expression levels after ADAR1 knockdown. The graph shows the effect of the MT8 compound in reducing miR-101 expression in HEK-293 TrkA cells transfected with scrambled (control) siRNA, but not in cells transfected with siRNA specific for ADAR1. miR-101 levels were quantified by real-time PCR using 5s ribosomal RNA and calculated using the 2-ΔCt method. [Example]

[0048] material Acid (1S,4R,5R,7S)-3,4-dibenzyl-2-oxo-6,8-dioxa-3-azabicyclo[3.2.1]octane-7-carboxylic acid L-lysine salt (MT8) was prepared as described in WO2013140348.

[0049] Example 1 - Effect of MT8 on IL-1β, TNF-α and IL-6 production in LPS-stimulated human monocytes and human dendritic cells.

[0050] Lipopolysaccharide (LPS) is an endotoxin that induces a strong immune response. In the presence of LPS, immune system cells such as monocytes and dendritic cells respond by producing large amounts of proinflammatory cytokines, such as IL-1β, TNFα, and IL-6. To test the effect of MT8 on modulating the production of these cytokines, human monocytes (isolated from buffy coats using anti-CD14 antibodies) and human monocyte-derived dendritic cells (MDCs) were cultured at 10 °C for 1 h. 6Cells were cultured at 1000 cells / ml in complete medium and stimulated with 50 ng / ml LPS in the presence or absence of 10 μM or 30 μM MT8. After 18 h of incubation, the supernatants from both cell lines were collected, and the production of IL-1β, TNF-α, and IL-6 was evaluated using Luminex multiplex assay technology. The results (Figure 1) showed that, under the above experimental conditions, LPS was able to induce the production of IL-1β, TNF-α, and IL-6, as expected, and that such production was reduced by treatment with MT8. Notably, MT8 was able to dose-dependently reduce the amount of IL-1β in both monocytes and dendritic cells. The reduction recorded in the latter exceeded 50% (Figure 1B), while in monocytes it was approximately 17% (Figure 1A). In both cases, this reduction was statistically significant. Similar results were obtained for TNF-α production, with a reduction of approximately 17% in monocytes (Figure 1C) and 19% in dendritic cells (Figure 1D) in the presence of MT8. The reduction observed in the presence of MT8 is statistically significant. Finally, IL-6 production is also significantly reduced in the presence of MT8, by approximately 70% in monocytes (Figure 1E) and approximately 65% ​​in dendritic cells (Figure 1F). In the latter case, the reduction is also statistically significant.

[0051] Taken together, these experiments show that in all cases, the production levels of the three cytokines were significantly lower in both monocytes and dendritic cells treated with the MT8 compound compared to untreated cells.

[0052] Example 2 - Effect of MT8 on activation of ADAR1 homodimeric complexes.

[0053] To study the effect of MT8 compounds on ADAR1 enzyme, HEK-293 TrkA cells were cultured in serum-free medium for 18 hours and then incubated for another 60 minutes in the presence or absence of 10 μM MT8. The cells were then lysed in RIPA buffer (50 mM Tris-HCl, pH 7.4; 150 mM NaCl; 2 mM EDTA; 1 mM NaF; 1 mM sodium orthovanadate, 1% NP-40), and proteins were immunoprecipitated with anti-ADAR1 antibodies and subjected to biochemical analysis by Western blot. Briefly, 500 μg of total protein was immunoprecipitated using specific anti-ADAR1 antibodies. The immunoprecipitated products were loaded onto a polyacrylamide gel and transferred to a PVDF membrane. The membrane was then incubated with specific anti-ADAR1 antibodies for signal detection. The analysis highlighted the presence of the homodimeric complex ADAR1 / ADAR1, as well as monomers of ADAR1 p150 and p110. Quantification of the ADAR1 homodimeric complex was performed by densitometry and expressed as the ratio of the density of the homodimeric ADAR1 / ADAR1 band to the density of the monomeric ADAR1 p110 band. The data obtained showed that treatment with MT8 compound induced a significant increase in ADAR1 homodimer (activity) compared to untreated cells, promoting its editing process (Figure 2).

[0054] Example 3 - Effect of MT8 on miR-101 expression in an in vitro model of inflammation.

[0055] The production of IL-1β, TNFα, and IL-6 induced by proinflammatory stimuli such as LPS is determined by the activation of specific pathways, among which the expression of miR-101 is involved.

[0056] To study the effect of MT8 on miR-101 activity in a proinflammatory environment, human monocytes isolated from buffy coats were stimulated with 1 μg / ml LPS in the presence or absence of MT8 at a final concentration of 10 μM. After 60 minutes, cells were lysed in TRIzol to extract total RNA, which was then used for quantification of miR-101 by real-time PCR. Results obtained by real-time PCR showed that treatment with the MT8 compound induced a strong decrease in miR-101 expression levels compared to monocytes treated with LPS alone. Quantification was performed using the 5S ribosomal RNA gene as a housekeeping vector, and the relative increase was calculated using the 2-ΔCt method.

[0057] The graph shows the effect of the compound MT8 on reducing intracellular miR-101 expression in human monocytes cultured in the presence of LPS, one of the compounds with the highest pro-inflammatory activity.

[0058] Thus, it was surprisingly found that exposure to MT8, one of the compounds of interest in this patent, in cell and tissue systems resulted in a decrease in miR-101 within 1 hour, which explains the rapid decrease in pro-inflammatory cytokines immediately after treatment with MT8. In summary, the obtained data show that in pro-inflammatory conditions, the compound MT8 can determine a decrease in cellular inflammatory conditions by reducing the expression of miR-101 (Figure 3).

[0059] Example 4 - Effect of MT8 on miR-101 expression levels after ADAR1 knockdown.

[0060] To study the effect of MT8 on miR-101 regulation under metabolic stress conditions, HEK-293 TrkA cells were transfected with ADAR1-specific siRNA or control siRNA (scrambled) at a final concentration of 50 nM. After 48 h, cells were incubated in serum-free medium for 18 h and then stimulated with MT8 at a concentration of 10 μM for an additional 60 min. Cells were lysed with TRIzol to extract total RNA, which was then used to assay miR-101 by real-time PCR. Using the 5S ribosomal RNA gene as a housekeeping gene, miR-101 was quantified, and the relative increase was calculated using the 2-ΔCt method. The results demonstrated that treatment with MT8 compound induced a significant decrease in miR-101 expression levels in scrambled siRNA-transfected cells compared to ADAR1-specific siRNA-transfected cells, indicating that the decrease in miR-101 levels is regulated by ADAR1 activity.

[0061] Taken together, these data indicate that, under the above experimental conditions, treatment with MT8 can reduce miR-101 levels through activation of ADAR1, thereby blocking the production of proinflammatory cytokines (Figure 4).

Claims

1. A pharmaceutical composition for use as an activator of adenosine deaminase 1 (ADAR1) acting on RNA in the treatment of acute or chronic inflammatory diseases characterized by cytokine storm and / or uncontrolled immune response, said pharmaceutical composition comprising a compound of formula (IA) or (IB) or a pharmaceutically acceptable salt thereof. 【Chemistry 1】

2. 2. The pharmaceutical composition of claim 1, wherein the compound is selected from the group consisting of methyl (1S,4R,5R,7S)-3,4-dibenzyl-2-oxo-6,8-dioxa-3-azabicyclo[3.2.1]octane-7-carboxylate (MT2), (1S,4R,5R,7S)-3,4-dibenzyl-2-oxo-6,8-dioxa-3-azabicyclo[3.2.1]octane-7-carboxylic acid (MT6), and pharmaceutically acceptable salts thereof.

3. The pharmaceutical composition according to claim 2, wherein the compound is (1S, 4R, 5R, 7S)-3,4-dibenzyl-2-oxo-6,8-dioxa-3-azabicyclo[3.2.1]octane-7-carboxylic acid L-lysine salt.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the acute or chronic inflammatory disease is caused by an infection.

5. The pharmaceutical composition of claim 4, wherein the acute or chronic inflammatory disease is caused by an infection induced by a viral agent.

6. The pharmaceutical composition according to claim 5, wherein the acute or chronic inflammatory disease caused by infection is caused by an infection induced by a viral factor selected from the group consisting of: herpes virus, Epstein-Barr virus, cytomegalovirus, adenovirus, HPV, coronavirus, enteroviruses, rotavirus, Parvovirus, Influenza A virus, Ebola virus, Members of the Marburgvirus genus Members of the Dengue virus species, Hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV) infection, Measles virus infection panencephalitis (SSPE), Hemorrhagic fever viruses (Arenaviridae, Bunyaviridae, Filoviridae, Falvivilidae, and Togaviridae families), measles virus, Mumps virus, rubella virus, Parechovirus, Human T-lymphotropic virus, and Influenza virus and parainfluenza virus.

7. The pharmaceutical composition according to claim 4, wherein the acute or chronic inflammatory disease is severe due to the following origins: i) Bacterial origin selected from the following group Aeromonas hydrophylla, genus Brusera, Chlamydia genus, Clostridium spp., E. coli, Legionella spp. Mycobacteria, Mycobacterium tuberculosis, Salmonella, Staphylococcus aureus, and Acinetobacter baumannii; ii) Origins from parasites and fungi selected from the following groups: Plasmodium genus, Leishmania spp., Toxoplasma gondii Entamoeba histolytica, Babesia genus, human roundworm, helminths, Candida albicans, Histoplasma, Cryptococcus neoformans, The genus Pneumocystis, and Penicillium marnefei. iii) Origin from zoonotic diseases selected from the following group: Brucella, rickettsia, Ehrlichia, Coxiella burnetii, Mycobacterium avium, Clostridium, and Leptospira.

8. The pharmaceutical composition according to any one of claims 1 to 3, wherein the acute or chronic inflammatory disease is severe and of autoimmune and / or degenerative origin selected from the group consisting of: hemophagocytic lymphohistiocytosis, lymphoproliferative syndrome, Primary and acquired immunodeficiency disorders not caused by NGF deficiency, Hereditary somatic pigmentary disorder (DSH), IFN-mediated diseases, NF-κB / ubiquitin-mediated diseases Muckle-Wells syndrome, hyper-IgD syndrome, Pediatric granulomatous arthritis, ADA2 deficiency, sepsis, Arthritis / osteoarthritis, Juvenile idiopathic arthritis, lupus erythematosus, Kawasaki disease.

9. The pharmaceutical composition according to any one of claims 1 to 3, wherein the acute or chronic inflammatory disease is a severe inflammatory disease of the respiratory tract selected from the group consisting of: Severe acute respiratory syndrome (SARS), induced by coronavirus or other viruses. asthma, chronic obstructive pulmonary disease (COPD), bronchiectasis, interstitial lung disease or pulmonary disease, bronchiolitis, Bronchopulmonary dysplasia (BPD) of premature infants, tuberculosis, whooping cough, Acute inhalation injury due to exposure to harmful and toxic substances, Occupational respiratory infections selected from the following group Legionnaires' disease, Q fever; Interstitial lung disease induced by occupational activities selected from the following groups: ・Pneumoconiosis, - Lung disease due to metal exposure, - Extrinsic allergic alveolitis, ・Ardystil syndrome; Rare lung diseases selected from the following group: ・Pulmonary vasculitis, - Idiopathic eosinophilic pneumonia, ・Alveolar proteinosis, Lymphangioleiomyomatosis (LAM), Pulmonary Langerhans cell histiocytosis, - Birt-Hogg-Dubé syndrome. Hemophagocytic lymphohistiocytosis.

10. A pharmaceutical composition according to any one of claims 1 to 9 in combination with at least one other active ingredient or adjuvant selected according to the condition to be treated.

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