6,8-dioxa-3-azabicyclo[3.2.l]Octane Carboxylic Acids And Their Derivatives For Use In The Treatment Of Inflammations

Abstract

The present invention describes 3-aza-bicyclo[3.2.1]octane acids of general formula (I), their Salts and Esters, for use as activators of ADAR1 in the treatment of diseases related to acute or chronic inflammations, infective or not., characterized by cytokine storm and/or uncontrolled immune response.

Claims

1. A method of treating inflammatory diseases characterized by cytokine storm and/or uncontrolled immune response, said method comprising administering to a subject in need thereof a compound of formula (I) as an activator of Adenosine Deaminase Acting on RNA 1 (ADAR1), said compound of formula (I) ##STR00006## wherein R.sub.1 is selected from the group consisting of aryl, C.sub.1-8alkyl-aryl; R.sub.2 is selected from the group consisting of C.sub.1-8alkyl-aryl; R.sub.3 is selected from the group H, -C.sub.1-8 alkyl, C.sub.1-8 alkyl-aryl; and including pharmaceutically acceptable salts.

2. The method according to claim 1 wherein R.sub.1 is CH.sub.2Ph; or R.sub.2 is CH.sub.2Ph; or R.sub.3 is H or CH.sub.3; and optionally the phenyl groups can be substituted with one or more groupings, and preferably one or two groupings selected from the group consisting of X, CN, NO.sub.2, NH.sub.2, OH, COOH, (C═O) Alk.sub.1-6; where X is chosen from the group consisting of F, Cl, Br and I.

3. The method according to claim 2 wherein R.sub.1 is CH.sub.2Ph; and R.sub.2 is CH.sub.2Ph; and R.sub.3 is H or CH.sub.3; and where the phenyl groups can optionally be substituted with one or more groupings, and preferably one or two groupings selected from the group consisting of X, CN, NO.sub.2, NH.sub.2, OH, COOH, (C═O) Alk.sub.1-6; where X is chosen from the group consisting of F, Cl, Br and I.

4. The method according to claim 3 wherein said compound is of formula (IA) or (IB) ##STR00007## ##STR00008## .

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

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

7. The method according to claim 1 wherein said inflammatory diseases are acute or chronic, and origin from infection.

8. The method according to claim 7 wherein said acute or chronic inflammatory diseases origin from infection induced by a viral factor.

9. The method according to claim 8 wherein the viral factor is selected from the group consisting of: Herpes Virus, Epstein-Barr virus, Cytomegalovirus, Adenovirus, HPV, Coronavirus, Enterovirus, Rotavirus, Parvovirus, Influenza A virus, Ebolavirus, members of the genus Marburgvirus, members of the dengue virus species, hepatitis A (HAV), B (HBV), C (HCV) virus infections, Pan-encephalitis (SSPE) in measles virus, Hemorrhagic fever virus (Arenaviridae, Bunyaviridae, Filoviridae, Falviviridae, and Togaviridae), Measles virus, Mumps virus, Rubella virus, Parechovirus, Human T-lymphotropic virus, and Influenza and parainfluenza viruses.

10. The method according to claim 7 wherein the inflammatory diseases are serious: i) of bacterial origin selected from the group consisting of Aeromonas hydrophila, Brucella sp., Chlamydia sp, Clostridium sp., Escherichia coli, Legionella sp., Mycobacteria, Mycobacterium tuberculosis, Salmonella, Staphylococcus aureus, and Acinetobacter baumannii; ii) of origin from parasites and fungi selected from the group consisting of: Plasmodium sp., Leishmania sp., Toxoplasma gondii, Entamoeba histolytica, Babesia sp.,y Ascaris lumbricoides, Helminths, Candida albicans, Histoplasma, Cryptococcus neoformans, Pneumocystis sp., and Penicillium marneffei; iii) of origin from zoonoses selected from the group consisting of: Brucella, Rickettsiae, Ehrlichia, Coxiella burnetiid, Mycobacterium avium, Clostridium, and Leptospira.

11. The method according to claim 1 wherein the inflammatory diseases are severe and of autoimmune and/or degenerative origin selected from the group consisting of: Hemophagocytic lymphohistiocytosis, Lymphoproliferative syndromes, Primary and acquired immunodeficiencies not due to NGF deficiency, hereditary symmetric dyschromatosis (DSH), rare genetic diseases linked to IL-1 / inflammasome disorders, IFN-mediated disorders, NF-κB / ubiquitinin mediated disorders, Muckle - Wells syndrome, hyper-IgD syndrome, pediatric granulomatous arthritis, ADA2 deficiency, sepsis, Arthritis / Osteoarthritis, Juvenile idiopathic arthritis, Lupus erythematosus, Kawasaki disease.

12. The method according to claim 1 wherein the inflammatory diseases are severe inflammatory diseases 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, pulmonary interstitial disease or pulmonary disease, bronchiolitis, bronchopulmonary dysplasia (BPD) of the premature infant, tuberculosis, whooping cough, acute inhalation injuries due to exposure to noxious and toxic substances, occupational respiratory tract infections selected from the group consisting of Legionellosis, Q fever; interstitial lung diseases induced by professional activities selected in the group consisting of: pneumoconiosis, lung diseases from exposure to metals, extrinsic allergic alveolitis, Ardystil syndrome; rare lung diseases selected from the group consisting of: pulmonary vasculitis, idiopathic eosinophilic pneumonia, pulmonary alveolar proteinosis, lymphangioleiomyomatosis (LAM), pulmonary Langerhans cell histiocytosis, Birt-Hogg-Dubé syndrome. Hemophagocytic lymphohistiocytosis.

13. The method according to claim 1 wherein the compound of formula (I) is administered in combination with at least one other active ingredient or adjuvant, chosen according to the pathological conditions to be treated.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0165] FIG. 1 - The graphs show 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 the MT8 compound. It is evident that the production levels of proinflammatory cytokines are significantly lower in both monocytes and dendritic cells treated with the MT8 compound compared to untreated cells.

[0166] FIG. 2 - Effect of MT8 on the activation of the homodimeric complex (active form of the protein) of ADAR1. The gel shows the induction of the ADAR1/ADAR1 homodimeric complex in HEK-293 TrkA cells treated with the MT8 compound. The graph shows the quantitative determination, performed by densitometry, of the gel bands and it is expressed as the ratio between the band density of the homodimeric complex ADAR1/ADAR1 and the monomer ADAR1. The ADAR1/ADAR1 homodimer levels induced by the MT8 compound are significantly higher than in the control.

[0167] FIG. 3 - Effect of MT8 on miR-101 reduction in an in vitro model of inflammation. The graph shows the ability of MT8 to decrease the expression of intracellular miR-101. This event was observed on human monocytes and dendritic cells cultured in the presence of LPS, one of the compounds with the highest pro-inflammatory activity. The level of miR-101 was quantified by Real-time PCR using 5 s ribosomal RNA for signal normalization and relative increase calculated applying the 2.sup.-ΔCt method.

[0168] FIG. 4 - Effect of MT8 on miR-101 expression levels following ADAR1 knock-down. The graph shows the ability of the MT8 compound to decrease the expression of miR-101 in HEK-293 TrkA cells transfected with scrambled (control) siRNA but not on cells transfected with specific siRNA for ADAR1. The level of miR-101 was quantified by Real-time PCR using 5 s ribosomal RNA for signal normalization and relative increase calculated applying the 2.sup.-ΔCt method.

EXPERIMENTAL PART

Materials

[0169] Acid (1S, 4R, 5R, 7S)-3,4-dibenzyl-2-oxo-6,8-dioxa-3-azabicyclo[3.2.1]octane-7-carboxylic L-lysine Salt (MT8) was prepared as described in WO2013140348.

Experiment 1 - Effect of MT8 on the Production of IL-1β, TNF-α and IL-6 in Human Monocytes and Human Dendritic Cells Stimulated With LPS

[0170] LipoPoliSaccharide (LPS) is an endotoxin that induces a strong immune reaction. In the presence of LPS, immune system cells such as monocytes and dendritic cells react by producing high amounts of inflammatory cytokines such as IL-1β, TNFα and IL-6. To test the ability of MT8 to modulate the production of these cytokines, human monocytes (isolated from buffy coat using anti-CD14 antibody) and human monocytes-derived dendritic cells (MDCs) were cultured at 10.sup.6 cells/ml in complete medium and stimulated with 50 ng/ml of LPS, in the presence or absence of MT8 at a concentration of 10 or 30 .Math.M. After 18 hours of incubation, the supernatants of both cell types were collected and the production of IL-1β, TNF-α, and IL-6 was evaluated by Luminex multiplex assay technology. The results obtained (FIG. 1) showed that in the experimental conditions described above, LPS was able, as expected, to induce the production of IL-1β, TNF-α and IL-6 and that such production is reduced by the treatment with MT8. In particular, MT8 was able to decrease the amount of IL-1β in a dose-dependent manner in both monocytes and dendritic cells. In the latter, the reduction recorded was over 50% (FIG. 1B), while in monocytes the reduction was about 17% (FIG. 1A). In any case, this reduction is statistically significant. Similar results were obtained regarding the production of TNF-α which, in the presence of MT8, is reduced by about 17% in monocytes (FIG. 1C) and by 19% in dendritic cells (FIG. 1D). Again, the reduction observed in the presence of MT8 is statistically significant. Finally, the production of IL-6 is also significantly reduced in the presence of MT8, by about 70% in monocytes (FIG. 1E) and by about 65% in dendritic cells (FIG. 1F). Also, in the latter case the reduction is statistically significant.

[0171] In summary, the experiments show that in all cases the production levels of the three cytokines are significantly lower in both monocytes and dendritic cells treated with the MT8 compound compared to untreated cells.

Experiment 2 - Effect of MT8 on the Activation of the Homodimeric Complex of ADAR1

[0172] In order to study the effect of the MT8 compound on the ADAR1 enzyme, HEK-293 TrkA cells were cultured in serum-free medium for 18 hours and incubated with or without 10 .Math.M of MT8 for another 60 minutes. The cells were then lysed in RIPA buffer (50 mM Tris-HCI, pH 7.4; 150 mM NaCl; 2 mM EDTA; 1 mM NaF; 1 mM sodium orthovanadate, 1% NP-40) and the proteins immunoprecipitated with Anti-ADAR1 antibody and subjected to biochemical analysis by Western Blot. Briefly, 500 .Math.g of total protein was immunoprecipitated using a specific Anti-ADAR1 antibody. The immunoprecipitated product was loaded onto polyacrylamide gel and transferred onto the PVDF membrane. The membrane was then incubated with specific anti-ADAR1 antibodies for signal detection. The analysis allowed to highlight the presence of homodimeric complexes ADAR1/ADAR1 and of the monomeric forms of ADAR1 p150 and p110. The quantitative determination of the homodimer complex of ADAR1, performed by densitometry, was expressed as the ratio between the density of the band of the homodimer ADAR1/ADAR1 and that of the monomer ADAR1 p110. The data obtained showed (FIG. 2) that the administration of the MT8 compound induces a high increase in the homodimeric (active) form of ADAR1 compared to untreated cells, thus promoting the editing process.

Experiment 3 - Effect of MT8 on the Expression of miR-101 in an in Vitro Model of Inflammation

[0173] The production of IL-1β and TNFα and IL-6, triggered by pro-inflammatory stimuli such as LPS, is determined by the activation of specific pathways, among which the expression of miR-101 is involved.

[0174] In order to study the effect of MT8 on the activity of miR-101 in the pro-inflammatory setting, human monocytes isolated from buffy coat were stimulated with 1 .Math.g/ml of LPS in the presence or absence of MT8 at the final concentration of 1 0 .Math.M. After 60 minutes, the cells were lysed in TRIzol for the extraction of total RNA, which was then used for the quantification of miR-101 by Real-time PCR. The results obtained by Real-time PCR showed that the administration of the MT8 compound induced a strong decrease in the miR-101 expression level compared to monocytes treated with LPS alone. The quantitative determination was performed using the 5 s ribosomal RNA gene as housekeeping and the relative increase calculated applying the 2.sup.-ΔCt method.

[0175] The graph shows the ability of the MT8 compound to decrease the expression of miR-101 within the cell. This event was observed on human monocytes cultured in the presence of LPS, one of the compounds with the greatest pro-inflammatory activity.

[0176] It was therefore surprisingly discovered that in cellular and tissue systems the exposure to MT8, one of the compounds object of the patent, determines, within 1 hour, the decrease of miR-101, this explains the rapid decrease of pro-inflammatory cytokines immediately after treatment with MT8. In summary, the data obtained (FIG. 3) show that in pro-inflammatory conditions, the compound MT8, by decreasing the expression of miR-101, is able to determine a decrease of the cellular inflammation state.

Experiment 4 - Effect of MT8 on miR-101 Expression Levels Following ADAR1 Knock-Down

[0177] In order to study the effect of MT8 on miR-101 regulatory mechanisms under metabolic stress conditions, HEK-293 TrkA cells were transfected with ADAR1-specific siRNA or control siRNA (scrambled) at the final concentration of 50 nM. After 48 hours, the cells were incubated in serum-free medium for 18 hours and stimulated with MT8 at a concentration of 10 .Math.M for a further 60 minutes. The cells were lysed with TRIzol for the extraction of total RNA, used for the assay of miR-101 by Real Time PCR. The quantitative determination of miR-101 was performed using the 5 s ribosomal RNA gene as the housekeeping gene and the relative increase calculated applying the 2.sup.-ΔCt method. The results obtained demonstrated that administration of the MT8 compound induced a marked decrease in miR-101 expression level 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.

[0178] Taken together, these data show (FIG. 4) that in the experimental conditions described above, treatment with MT8 is capable of decreasing levels of miR-101 through the activation of ADAR1, thus allowing the production of pro-inflammatory cytokines to be blocked.