NEW THERAPEUTIC TARGETS WITH AN ANTI-INFLAMMATORY AND ANTI-INTERFERON EFFECT

Abstract

The present invention relates to an inhibitor of the ROCK-PDK1 protein kinase complex for use thereof as an anti-inflammatory and anti-interferon agent. The present invention also relates to a pharmaceutical composition comprising an inhibitor of the ROCK-PDK1 protein kinase complex as active principle and at least one pharmaceutically acceptable excipient and/or carrier and/or a diluent and/or a pharmaceutically acceptable vehicle. The present invention also relates to the use of said one pharmaceutical composition in the prevention and/or the treatment of inflammatory diseases, viral and/or bacterial infections and autoimmune diseases.

Claims

1. An inhibitor of the ROCK-PDK1 protein kinase complex for use as an anti-inflammatory and anti-interferon agent, characterized in that said inhibitor of the ROCK-PDK1 protein kinase complex is an inhibitor of the ROCK protein kinase, and in that the inhibitor of the ROCK protein kinase inhibits the secretion of inflammatory cytokines and of the interferons by an immune cell, when it is placed in contact with said immune cell.

2. The inhibitor of the ROCK-PDK1 protein kinase complex according to claim 1, in which the inhibitor of the ROCK-PDK1 protein kinase complex is selected from the compounds of formula (VII) to (X): ##STR00044## or one of the pharmaceutically acceptable salts thereof ##STR00045## or one of the pharmaceutically acceptable salts thereof, ##STR00046## or one of the pharmaceutically acceptable salts thereof, ##STR00047## or one of the pharmaceutically acceptable salts thereof.

3. The inhibitor of the ROCK-PDK1 protein kinase complex according to claim 1, in which the inhibitor of the ROCK-PDK1 protein kinase complex is the compound ##STR00048## or a pharmaceutically acceptable salt thereof.

4. A pharmaceutical composition comprising at least one inhibitor of the ROCK-PDK1 protein kinase complex according to claim 1 as active principle and at least one pharmaceutically acceptable excipient and/or carrier and/or a diluent and/or a pharmaceutically acceptable vehicle.

5. The pharmaceutical composition according to claim 4, in which the inhibitor of the ROCK-PDK1 protein kinase complex is selected from the compounds of formula (VII) to (X): ##STR00049## or one of the pharmaceutically acceptable salts thereof ##STR00050## or one of the pharmaceutically acceptable salts thereof, ##STR00051## or one of the pharmaceutically acceptable salts thereof, ##STR00052## or one of the pharmaceutically acceptable salts thereof.

6. A method comprising administering to a patient the pharmaceutical composition according to claim 4, in which the inhibitor of the ROCK-PDK1 protein kinase complex as active principle is administered to the patient at a dose of 0.5 mg to 30 mg per kg of bodyweight.

7. A method comprising administering to a patient the pharmaceutical composition according to claim 4, in which the inhibitor of the ROCK-PDK1 protein kinase complex as active principle is administered to the patient at a rate of 100 mg to 500 mg per day.

8. A method comprising administering to a patient the pharmaceutical composition according to claim 4, characterized in that the pharmaceutical composition is administered at the rate of one, two, three, four, five, six or seven times per week.

9. The pharmaceutical composition according to claim 4, characterized in that the pharmaceutical composition also comprises at least one second active principle.

10. The pharmaceutical composition according to claim 9, characterized in that the pharmaceutical composition is adapted for a simultaneous administration or a sequential administration of the inhibitor of the ROCK-PDK1 protein kinase complex and of the second active principle.

11. The pharmaceutical composition according to claim 4, in which the pharmaceutical composition is in a form adapted for administration thereof by the oral, parenteral or topical route.

12. A method comprising administering the pharmaceutical composition of claim 4 to a patient with an inflammatory disease.

13. The method of claim 12, in which inflammatory disease is selected from: amyloid neurodegenerative diseases, Alzheimer's disease, prions, Parkinson's disease, amyotrophic lateral sclerosis; inflammatory diseases of intestine, chronic inflammatory bowel disease (CIBD), irritable bowel syndrome (IBS), Crohn's disease, ulcerative colitis, hemorrhagic rectal ulcer, lesions associated with Behçet's disease, pouchitis, ulcerative colitis, ileitis and enteritis; acute inflammatory diseases, gout, septic shock, chronic inflammatory lumbar pain, inflammatory diseases of skin or dermatitis, psoriasis, atopic dermatitis, rosacea, acne erythematosa, acne, common warts, bullous skin diseases, contact eczema, skin cancers, rubor, erythemas, telangiectasias, inflammations of skin linked to exposure to UV, photo-irritation, photo-sensitization, photo-ageing, photo-carcinogenesis, lymphatic venous insufficiencies or heavy leg syndrome; arthritis, osteoarthritis, rheumatoid arthritis, juvenile idiopathic arthritis and psoriatic arthritis; chronic obstructive pulmonary disease (COPD); coeliac disease; chronic pancreatitis; Hashimoto's thyroiditis; primary biliary cirrhosis; sclerosing cholangitis; autoimmune hepatitis; vasculitides, systemic vasculitides associated with ANCA (anti-neutrophil cytoplasmic antibodies); spondyloarthropathies; chronic atrophying polychondritis; diabetes; scleroderma and cancer.

14. A method comprising administering the pharmaceutical composition of claim 4 to a patient with a viral and/or bacterial infection.

15. The method of claim 14, in which the viral infection is selected from: infections due to influenza virus, human immunodeficiency virus (HIV), herpes virus or herpes simplex virus (HSV), coronavirus, COVID-19, encephalomyocarditis virus, arboviruses, chikungunya, o'nyong'nyong, Ross River, Sindbis, Mayaro viruses, yellow fever virus, dengue fever virus, Japanese encephalitis virus, West Nile virus, temperate Eurasian tick-borne encephalitis viruses, Kyasanur Forest disease viruses, Omsk hemorrhagic fever virus, Bunyavirales viruses, Bunyamwera virus, Rift Valley fever virus, Crimean-Congo hemorrhagic fever virus, hepatitis A, B and C virus and the influenza virus.

16. The method of claim 14, in which the bacterial infection is selected from: infections due to gram-positive bacteria, bacteria of genus Staphylococcus, Staphylococcus aureus, and bacteria of genus Enterococcus, Enterococcus faecalis; infections due to gram-negative bacteria, bacteria of genus Escherichia, Escherichia coli, the bacteria of the genus Pseudomonas, in particular Pseudomonas aeruginosa, and bacteria of genus Acinetobacter, Acinetobacter baumannii.

17. A method comprising administering the pharmaceutical composition of claim 4 to a patient with an autoimmune disease.

18. The method of claim 17, in which the autoimmune disease is selected from: disseminated lupus erythematosus, systemic lupus erythematosus, multiple sclerosis, rheumatoid polyarthritis, insulin-dependent diabetes, thrombotic thrombocytopenic purpura (TTP), graft or organ rejection, graft versus host disease, different types of sclerosis, primary Sjögren's syndrome (or Gougerot-Sjögren syndrome), autoimmune polyneuropathies, multiple sclerosis, type I diabetes, autoimmune hepatitises, ankylosing spondylitis, Reiter's syndrome, gout arthritis, chronic Hashimoto's thyroiditis (hypothyroidism), Addison's disease, autoimmune hepatitises, Basedow's disease (hyperthyroidism), autoimmune cytopenias and other hematological complications of adult and child, acute or chronic autoimmune thrombocytopenias, autoimmune hemolytic anaemias, hemolytic disease of newborn (HDN), cold agglutinin disease, thrombotic thrombocytopenic purpura and autoimmune acquired hemophilia; Goodpasture syndrome, extra-membranous nephropathies, autoimmune bullous skin disorders, refractory myasthenia gravis, mixed cryoglobulinemias, inflammatory myositis, dermatomyositis and childhood systemic autoimmune disorders including antiphospholipid syndrome, conjunctive tissue disease, different types of sclerosis, autoimmune pulmonary inflammation, Guillain-Barré syndrome, chronic inflammatory demyelinating polyradiculoneuropathy (CIDP), autoimmune thyroiditis, diabetes mellitus, myasthenia gravis, autoimmune inflammatory disease of eye, neuromyelitis optica (Devic's disease), sclerodermas, pemphigus, insulin-resistant diabetes, polymyositis, Biermer's anaemia, glomerulonephritis, Wegener's disease, Horton's disease, periarthritis nodosa and Churg Strauss syndrome, Still's disease, atrophic polychondritis, Behçet's disease, monoclonal gammopathy, Wegener's granulomatosis, lupus, psoriatic rheumatism, sarcoidosis, collagenous colitis, dermatitis herpetiformis, familial Mediterranean fever, IgA deposition glomerulonephritis, Lambert-Eaton myasthenic syndrome, ophthalmia sympathica, Fiessinger-Leroy-Reiter syndrome and uveomeningoencephalitic syndrome.

Description

FIGURES

[0215] FIG. 1: FIG. 1 shows the blocking by an inhibitor of the ROCK protein kinase of formula (VII) (also called Y-27632) of the intracellular production of TNFα on monocytes of healthy donors in response to a stimulation of the Toll-like receptor TLR7/8 receptors (Resiquimod-R848), results obtained by flow cytometry.

[0216] FIG. 2: FIG. 2 shows the blocking by an inhibitor of the PDK1 protein kinase of formula (IV) (also called BX912) of the intracellular production of TNFα on monocytes of healthy donors in response to a stimulation of the Toll-like receptor TLR7/8 receptors (Resiquimod-R848), results obtained by flow cytometry.

[0217] FIG. 3: FIG. 3 shows the blocking by an inhibitor of the PDK1 protein kinase of formula (IV) (also called BX912) of the secretion of inflammatory cytokines and interferons on peripheral blood mononuclear cells (PBMCs) of healthy donors in response to a stimulation of the Toll-like receptor TLR7/8 receptors. Results obtained by LegendPlex.

[0218] FIG. 4: FIG. 4 shows the absence of toxicity of the inhibitor of the PDK1 protein kinase of formula (IV) (also called BX912) on peripheral blood mononuclear cells (PBMCs) of healthy donors.

[0219] FIG. 5: FIG. 5 shows the blocking of the intracellular production of TNFα on monocytes of healthy donors by an inhibitor of the PDK1 protein kinase of formula (IV) (also called BX912) in response to a stimulation of the Toll-like receptor 4 receptors (lipopolysaccharides-LPS), results obtained by flow cytometry.

[0220] FIG. 6: FIG. 6 shows the blocking of the intracellular production of TNFα on monocytes of healthy donors by an inhibitor of the PDK1 protein kinase of formula (IV) (also called BX912) in response to a stimulation of the Toll-like receptor 2 receptors (lipoteichoic acid-LTA), results obtained by flow cytometry.

[0221] FIG. 7: FIG. 7 shows the blocking by an inhibitor of the PDK1 protein kinase of formula (IV) (also called BX912) of the secretion of inflammatory cytokines and interferons on peripheral blood mononuclear cells (PBMCs) of healthy donors in response to a stimulation of the Toll-like receptor TLR4 receptors. Results obtained by LegendPlex.

[0222] FIG. 8: FIG. 8 shows the absence of toxicity of the inhibitor of the PDK1 protein kinase of formula (IV) (also called BX912) on peripheral blood mononuclear cells (PBMCs) of healthy donors.

[0223] FIG. 9: FIG. 9 shows the blocking of the intracellular production of IL1β on monocytes of healthy donors by an inhibitor of the PDK1 protein kinase of formula (IV) (also called BX912) in response to an activation of the inflammasome by uric acid crystals (MSU) (“gout” model), results obtained by flow cytometry.

[0224] FIG. 10: FIG. 10 shows the blocking of the intracellular production of TNFα on monocytes of healthy donors by an inhibitor of the PDK1 protein kinase of formula (IV) (also called BX912) in response to an activation of the inflammasome by uric acid crystals (MSU) (“gout” model), results obtained by flow cytometry.

[0225] FIG. 11: FIG. 11 shows the blocking of the intracellular production of IFNα on pDCs of healthy donors by an inhibitor of the PDK1 protein kinase of formula (IV) (also called BX912) in response to an infection with HIV, results obtained by flow cytometry.

[0226] FIG. 12: FIG. 12 shows the blocking of the intracellular production of TNFα on pDCs of healthy donors by an inhibitor of the PDK1 protein kinase of formula (IV) (also called BX912) in response to an infection with HIV, results obtained by flow cytometry.

[0227] FIG. 13: FIG. 13 shows the blocking by an inhibitor of the PDK1 protein kinase of formula (IV) (also called BX912) of the secretion of inflammatory cytokines and interferons on peripheral blood mononuclear cells (PBMCs) of healthy donors in response to an infection with HIV. Results obtained by LegendPlex.

[0228] FIG. 14: FIG. 14 shows the absence of toxicity of the inhibitor of the PDK1 protein kinase of formula (IV) (also called BX912) on peripheral blood mononuclear cells (PBMCs) of healthy donors.

[0229] FIG. 15A: FIG. 15A shows the blocking by the inhibitor of formula (IV) (also called BX912) of the secretion of interferons on peripheral blood mononuclear cells (PBMCs) of healthy donors, in a preventive manner, 1 h before the stimulation by Toll-like TLR 7/8 receptors. Results obtained with the STING-37 reporter cell line.

[0230] FIG. 15B: FIG. 15B shows the blocking by the inhibitor of formula (IV) (also called BX912) of the secretion of interferons on peripheral blood mononuclear cells (PBMCs) of healthy donors, during the stimulation by Toll-like TLR 7/8 receptors. Results obtained with the STING-37 reporter cell line.

[0231] FIG. 15C: FIG. 15C shows the blocking by the inhibitor of formula (IV) (also called BX912) of the secretion of interferons on peripheral blood mononuclear cells (PBMCs) of healthy donors, in a curative manner, 1 h after the stimulation by Toll-like TLR 7/8 receptors. Results obtained with the STING-37 reporter cell line.

[0232] FIG. 16: FIG. 16 shows the blocking by the inhibitor of formula (IV) (also called BX912) of the spontaneous production of TNFα on monocytes of patients suffering from juvenile idiopathic arthritis. Results obtained by SIMOA assay (digital ELISA).

[0233] FIG. 17: FIG. 17 shows the immunosuppressive effect of the inhibitor of the ROCK protein kinase of formula (VII) (also called Y27632) on human PBMCs. Human PBMCs of healthy donors were treated with the inhibitor of the ROCK protein kinase of formula (VII) for 1 h, following the doses indicated, then stimulated with Resiquimod-R848 (activators of the Toll-like receptor TLR7/8 receptors) for 24 h. (A) The interferons in the culture supernatants of the PBMCs were assayed with the STING-37 reporter cell line. (B) The activity of the NF-kB transcription factor was measured by means of the THP1-Dual line.

[0234] FIG. 18: FIG. 18 shows that the inhibitor of the ROCK protein kinase of formula (VII) (also called Y27632) attenuates the production of TNFα, IL-1β and IL-6 by the activated monocytes of healthy donors. Purified monocytes from healthy donors were treated with the inhibitor of the ROCK protein kinase of formula (VII) for 1 h, following the doses indicated, then stimulated with Resiquimod-R848 (activators of the Toll-like receptor TLR7/8 receptors) for 6 h. The cell viability and the percentage of monocytes producing TNFα, IL-1β and IL-6 were estimated. Graphical representation of the percentage of monocytes producing TNFα, IL-1β and IL-6 (A) and graphical representation of the MFI (B) (results obtained by flow cytometry).

[0235] FIG. 19: FIG. 19 shows the inhibition of the activation of T lymphocytes by the inhibitor of the ROCK protein kinase of formula (VII) (also called Y27632). Purified T lymphocytes from healthy donors were treated with the inhibitor of the ROCK protein kinase of formula (VII) for 1 h, at 1 and 5 μM, then activated with CD3/CD28 beads for 3 days. Graphical representation of the percentage of T lymphocytes expressing CD69 (A) and graphical representation of the MFI (B) (results obtained by flow cytometry).

[0236] FIG. 20: FIG. 20 shows the inhibition of the activation of B lymphocytes by the inhibitor of the ROCK protein kinase of formula (VII) (also called Y27632). Purified B lymphocytes from healthy donors were treated with the inhibitor of the ROCK protein kinase of formula (VII) for 1 h, at 1 μM, then activated with CpG-A (1 μg/ml.sup.−1) for 24 h. Graphical representation of the percentage of T lymphocytes expressing CD69 (A) and graphical representation of the MFI (B) (results obtained by flow cytometry).

[0237] FIG. 21: FIG. 21 shows the inhibition of the spontaneous production of TNFα et IL-6 cytokines by the inhibitor of the ROCK protein kinase of formula (VII) by the PBMCs of patients suffering from juvenile idiopathic arthritis.

[0238] FIG. 22: FIG. 22 shows the immunosuppressive effect of the inhibitor of the ROCK protein kinase of formula (VII) (also called compound Y27632) of the immune and non-immune cells. (A) Human PBMCs of healthy donors were treated with the inhibitor of the ROCK protein kinase of formula (VII) for 1 h, following the doses indicated, then activated with cGAMP for 24 h. The interferons in the culture supernatants of the PBMCs were assayed with the STING-37(B) reporter cell line. (B) THP1-Dual and (C) HEK293 were treated with the inhibitor of the ROCK protein kinase of formula (VII) for 1 h, following the doses indicated, then activated with cGAMP for 24 h. The production of IFN was measured via luciferase.

[0239] FIG. 23: FIG. 23 shows the immunosuppressive effect of the inhibitor of the ROCK protein kinase of formula (VIII) (also called dimethylfasudil) in the immune and non-immune cells. (A) Human PBMCs of healthy donors were treated with the inhibitor of the ROCK protein kinase of formula (VIII) for 1 h, following the doses indicated, then activated with cGAMP for 24 h. The interferons in the culture supernatants of the PBMCs were assayed with the STING-37 reporter cell line. (B) THP1-Dual and (C) HEK293 were treated with the inhibitor of the ROCK protein kinase of formula (VIII) for 1 h, following the doses indicated, then activated with cGAMP for 24 h. The production of IFN was measured via luciferase.

EXAMPLES

Example 1: Isolation and Culturing of Blood Leukocytes

[0240] The in vitro experiments were carried out using human peripheral blood mononuclear cells (PBMCs) isolated by density gradient centrifugation by means of a medium for separating leukocytes from peripheral blood (STEMCELL Technologies). The blood of healthy donors was obtained from the “Etablissement Français du Sang” [French Blood Establishment] (convention #07/CABANEL/106; Paris, France). The studies on material from patients were approved by the research ethics committee (ID-RCβ/EUDRACT: 2014-A01017-40 and 2018-A01358-47). The human monocytes were purified by positive selection with human CD14 microbeads (Miltenyi). The plasmacytoid dendritic cells (pDC) were purified by negative selection with the “EasySep Human Plasmacytoid DC” Enrichment Kit (STEMCELL Technologies). The peripheral blood mononuclear cells (PBMCs), monocytes and plasmacytoid dendritic cells (pDC) were cultured in RPMI 1640 (Sigma) containing 10% inactivated foetal bovine serum and 1 mM glutamine (Hyclone, Logan, Utah).

Example 2: Preventive Effect of the Inhibition of the PDK1 Protein Kinase by the Inhibitor of Formula (IV) (Also Called BX912) or of the Inhibition of the ROCK Protein Kinase by the Inhibitor of Formula (VII) (Also Called Y-27632) in Response to the Stimulation of the Toll 7/8 Receptors

Cell Stimulation

[0241] The PBMCs were cultured at 2.Math.10.sup.6 cells/ml. The monocytes were cultured at 1.Math.10.sup.6 cells/ml. The cells were then incubated with the inhibitor of the PDK1 protein kinase of formula (IV) (also called BX912) or the inhibitor of the ROCK protein kinase of formula (VII) (also called Y27632) at the concentrations indicated for 1 h before stimulation. They were stimulated for 16 h (flow cytometry) or 24 h (LegendPlex) with the TLR7/8 agonist, Resiquimod—R848, at 5 μg/mi.

[0242] The cells were recovered for the flow cytometry and the supernatants were collected for the detection of the cytokines. For the intracellular labelling of TNFα and IL-1β, Brefeldin A (BFA) was added to the cells 30 minutes after the stimulation.

Flow Cytometry

[0243] The cells were rinsed in PBS then incubated with a viability dye (Zombie Aqua, Biolegend) for 30 min at ambient temperature. After washing, the cells were resuspended in PBS containing 2% SVF and 2 mM EDTA then labelled with the APC Vio770 anti-CD14 (clone REA599) antibody from Miltenyi Biotec, used at 1/100°. For the intracellular labelling of TNFα and IL-1β, the “Inside Stain” kit (Miltenyi Biotec) was used according to the manufacturer's protocol. The cells were fixed for 20 min at ambient temperature with 250 μL Inside Fix solution then labelled in 100 μL Inside Perm solution containing the PE anti-TNFα (Miltenyi Biotec) and/or APC anti-IL-1β (Miltenyi Biotec) antibody at 1/50° for 30 min at ambient temperature. Data acquisition was carried out on the Canto 11 flow cytometer using the Diva software (BD Biosciences, San Jose, Calif.). The Kaluza software was used to analyze the data.

Detection of the Cytokines

[0244] An assay of the cytokines in the supernatants of the cell cultures was carried out using the “LegendPlex Antivirus Human Panel” or “LegendPlex Human Inflammation Panel” kit (Biolegend, San Diego, USA) according to the manufacturer's instructions.

[0245] Test for toxicity of the compounds on the peripheral blood mononuclear cells (PBMCs).

[0246] After 24 h of cell culture, the cell viability was determined using the CellTiter-Glo reagent (Promega), which evaluates the amount of ATP by luminescence using the EnSpire.

[0247] The results are presented in FIGS. 1 to 4.

Results:

[0248] It can be seen that: [0249] inhibition of the ROCK protein kinase by the compound of formula (VII) (also called Y27632) inhibits, according to a dosage effect, the intracellular production of inflammatory cytokines (TNFα) by the monocytes of healthy donors in response to the stimulation of the Toll-like receptor TLR 7/8 receptors (see FIG. 1). [0250] inhibition of the PDK1 protein kinase by the compound of formula (IV) (also called BX912) inhibits, according to a dosage effect, the intracellular production of inflammatory cytokines (TNFα) by monocytes of healthy donors in response to the stimulation of the Toll-like receptor TLR 7/8 receptors (see FIG. 2). [0251] inhibition of the PDK1 protein kinase by the compound of formula (IV) (also called BX912) inhibits the secretion of the inflammatory cytokines (IL-6, IL-1β, TNFα, IP10, GM-CSF, IL-10) and the interferons (IFNI1, IFNI2/3, IFNβ, IFNg) by the peripheral blood mononuclear cells (PBMCs) of healthy donors in response to the stimulation of the Toll-like receptor TLR 7/8 receptors (see FIG. 3). [0252] the compound of formula (IV) (also called BX912) has no toxicity on the immune cells (PBMCs) of healthy donors in response to the stimulation of the TLR 7/8 receptors (see FIG. 4).

[0253] Thus, the compounds of formula (VII) (also called Y27632) and of formula (IV) (also called BX912) have a preventive effect on the production of inflammatory cytokines (TNFα) by the monocytes of healthy donors in response to the stimulation of the Toll-like receptor TLR 7/8 receptors.

[0254] In addition, the compound of formula (IV) (also called BX912) is non-toxic. It has a preventive effect on the secretion of the inflammatory cytokines (IL-6, IL-1β, TNFα, IP10, GM-CSF, IL-10) and the interferons (IFNI1, IFNI2/3, IFNβ, IFNg) by the peripheral blood mononuclear cells (PBMCs) of healthy donors in response to the stimulation of the Toll-like receptor TLR 7/8 receptors.

Example 3: Preventive Effect of the Inhibition of the PDK1 Protein Kinase by the Inhibitor of Formula (IV) (Also Called BX912) in Response to the Stimulation of the TLR2/4 Receptors

Cell Stimulation

[0255] The PBMCs were cultured at 2.Math.10.sup.6 cells/ml. The monocytes were cultured at 1.Math.10.sup.6 cells/ml. The cells were then incubated with the inhibitor of the PDK1 protein kinase of formula (IV) (also called BX912) or the inhibitor of the ROCK protein kinase of formula (VII) (also called Y27632) at the concentrations indicated for 1 h before stimulation. They were stimulated for 16 h (flow cytometry) or 24 h (LegendPlex), respectively, with the TLR4 agonist, lipopolysaccharide—LPS, at 100 ng/ml and the TLR2 agonist, lipoteichoic acid—LTA, at 1 μg/ml.

[0256] The cells were recovered for the flow cytometry and the supernatants were collected for the detection of the cytokines. For the intracellular labelling of TNFα and IL-1p, Brefeldin A (BFA) was added to the cells 30 minutes after the stimulation.

Flow Cytometry

[0257] The cells were rinsed in PBS then incubated with a viability dye (Zombie Aqua, Biolegend) for 30 min at ambient temperature. After washing, the cells were resuspended in PBS containing 2% SVF and 2 mM EDTA then labelled with the APC Vio770 anti-CD14 (clone REA599) antibody from Miltenyi Biotec, used at 1/100°. For the intracellular labelling of TNFα and IL-1β, the “Inside Stain” kit (Miltenyi Biotec) was used according to the manufacturer's protocol. The cells were fixed for 20 min at ambient temperature with 250 μL Inside Fix solution then labelled in 100 μL Inside Perm solution containing the PE anti-TNFα (Miltenyi Biotec) and/or APC anti-IL-1β (Miltenyi Biotec) antibody at 1/50° for 30 min at ambient temperature. Data acquisition was carried out on the Canto II flow cytometer using the Diva software (BD Biosciences, San Jose, Calif.). The Kaluza software was used to analyze the data.

Detection of the Cytokines

[0258] An assay of the cytokines in the supernatants of the cell cultures was carried out using the “LegendPlex Antivirus Human Panel” or “LegendPlex Human Inflammation Panel” kit (Biolegend, San Diego, USA) according to the manufacturer's instructions.

Test for Toxicity of the Compounds on PBMCs

[0259] After 24 h of cell culture, the cell viability was determined using the CellTiter-Glo reagent (Promega), which evaluates the amount of ATP by luminescence using the EnSpire.

[0260] The results are presented in FIGS. 5 to 8.

Results:

[0261] It can be seen that: [0262] inhibition of the PDK1 protein kinase by the compound of formula (IV) (also called BX912) inhibits, according to a dosage effect, the production of inflammatory cytokines (TNFα) by the monocytes of healthy donors in response to the stimulation of the TLR4 receptors (see FIG. 5). [0263] inhibition of the PDK1 protein kinase by the compound of formula (IV) (also called BX912) inhibits, according to a dosage effect, the production of inflammatory cytokines (TNFα) by the monocytes of healthy donors in response to the stimulation of the TLR2 receptors (see FIG. 6). [0264] inhibition of the PDK1 protein kinase by the compound of formula (IV) (also called BX912) inhibits the secretion of the inflammatory cytokines (IL-6, IL-1β, TNFα, GM-CSF, IL-10) and the interferons (IFNI2/3, IFNβ, IFNg) by the peripheral blood mononuclear cells (PBMCs) of healthy donors in response to the stimulation of the TLR4 receptors (see FIG. 7). [0265] the compound of formula (IV) (also called BX912) has no toxicity on the peripheral blood mononuclear cells (PBMCs) of healthy donors in response to the stimulation of the TLR4 receptors (see FIG. 8).

[0266] Thus, the compound of formula (IV) (also called BX912) has a preventive effect on the production of inflammatory cytokines (TNFα) by the monocytes and the immune cells (PBMCs) of healthy donors in response to the stimulation of the TLR 4 and TLR 2 receptors.

[0267] In addition, the compound of formula (IV) (also called BX912) is non-toxic. It has a preventive effect on the secretion of the inflammatory cytokines (IL-6, IL-1β, TNFα, GM-CSF, IL-10) and the interferons (IFNI2/3, IFNβ, IFNg) by the peripheral blood mononuclear cells (PBMCs) of healthy donors in response to the stimulation of the TLR 4 receptors.

Example 4: Preventive Effect of the Inhibition of the PDK1 Protein Kinase by the Inhibitor of Formula (IV) (Also Called BX912) in Response to the Activation of the Inflammasome by Uric Acid Crystals (Mimicking “Gout”)

Cell Stimulation

[0268] The monocytes were cultured at 1.Math.10.sup.6 cells/ml. The cells were then incubated with the inhibitor of the PDK1 protein kinase of formula (IV) (also called BX912) or the inhibitor of the ROCK protein kinase of formula (VII) (also called Y27632) at the concentrations indicated for 1 h before stimulation. They were stimulated for 16 h (flow cytometry) with uric acid crystals mimicking gout (MSU) at 50 μg/ml.

[0269] The cells were recovered for the flow cytometry. For the intracellular labelling of TNFα and IL-1β, Brefeldin A (BFA) was added to the cells 30 minutes after the stimulation.

Flow Cytometry

[0270] The cells were rinsed in PBS then incubated with a viability dye (Zombie Aqua, Biolegend) for 30 min at ambient temperature. After washing, the cells were resuspended in PBS containing 2% SVF and 2 mM EDTA then labelled with the APC Vio770 anti-CD14 (clone REA599) antibody from Miltenyi Biotec, used at 1/100°. For the intracellular labelling of TNFα and IL-1β, the “Inside Stain” kit (Miltenyi Biotec) was used according to the manufacturer's protocol. The cells were fixed for 20 min at ambient temperature with 250 μL Inside Fix solution then labelled in 100 μL Inside Perm solution containing the PE anti-TNFα (Miltenyi Biotec) and/or APC anti-IL-1β (Miltenyi Biotec) antibody at 1/50° for 30 min at ambient temperature. Data acquisition was carried out on the Canto II flow cytometer using the Diva software (BD Biosciences, San Jose, Calif.). The Kaluza software was used to analyze the data.

[0271] The results are presented in FIGS. 9 to 10.

Results:

[0272] It can be seen that: [0273] inhibition of the PDK1 protein kinase by the compound of formula (IV) (also called BX912) inhibits the intracellular production of IL1β by the monocytes of healthy donors in response to an activation of the inflammasome by uric acid crystals (“gout” model) (see FIG. 9). [0274] inhibition of the PDK1 protein kinase by the compound of formula (IV) (also called BX912) inhibits the production of TNFα by the monocytes of healthy donors in response to an activation of the inflammasome by uric acid crystals (“gout” model) (see FIG. 10).

[0275] Thus, the compound of formula (IV) (also called BX912) has a preventive effect on the intracellular production of IL1β and TNFα on monocytes of healthy donors in response to an activation of the inflammasome by uric acid crystals (“gout” model).

Example 5: Preventive Effect of the Inhibition of the PDK1 Protein Kinase by the Inhibitor of Formula (IV) (Also Called BX912) in Response to an Infection with the Human Immunodeficiency Virus (HIV)

Cell Stimulation

[0276] The PBMCs were cultured at 2.Math.10.sup.6 cells/ml. The plasmacytoid dendritic cells were cultured at 1.Math.10.sup.6 cells/ml. The cells were then incubated with the inhibitor of the PDK1 protein kinase of formula (IV) (also called BX912) or the inhibitor of the ROCK protein kinase of formula (VII) (also called Y27632) at the concentrations indicated for 1 h before stimulation. They were stimulated for 16 h (flow cytometry) or 24 h (LegendPlex) with aldrithiol-2 (AT-2)-inactivated HIV-1MN, specific to the CXCR4 coreceptor.

[0277] The cells were recovered for the flow cytometry and the supernatants were collected for the detection of the cytokines. For the intracellular labelling of TNFα and IFNα, Brefeldin A (BFA) was added to the cells 30 minutes after the stimulation.

Flow Cytometry

[0278] The cells were rinsed in PBS then incubated with a viability dye (Zombie Aqua, Biolegend) for 30 min at ambient temperature. After washing, the cells were resuspended in PBS containing 2% SVF and 2 mM EDTA then labelled with the APC Vio770 anti-CD14 (clone REA599) antibody from Miltenyi Biotec, used at 1/100°. For the intracellular labelling of TNFα and IFNα, the “Inside Stain” kit (Miltenyi Biotec) was used according to the manufacturer's protocol. The cells were fixed for 20 min at ambient temperature with 250 μL Inside Fix solution then labelled in 100 μL Inside Perm solution containing the APC anti-TNFα (Miltenyi Biotec) and/or PE anti-IFNα (Miltenyi Biotec) antibody at 1/50° for 30 min at ambient temperature. Data acquisition was carried out on the Canto II flow cytometer using the Diva software (BD Biosciences, San Jose, Calif.). The Kaluza software was used to analyze the data.

Detection of the Cytokines

[0279] An assay of the cytokines in the supernatants of the cell cultures was carried out using the “LegendPlex Antivirus Human Panel” or “Legendplex Human Inflammation Panel” kit (Biolegend, San Diego, USA) according to the manufacturer's instructions.

Test for Toxicity of the Compounds on PBMCs

[0280] After 24 h of cell culture, the cell viability was determined using the CellTiter-Glo reagent (Promega), which evaluates the amount of ATP by luminescence using the EnSpire.

[0281] The results are presented in FIGS. 11 to 14.

Results:

[0282] It can be seen that: [0283] inhibition of the PDK1 protein kinase by the compound of formula (IV) (also called BX912) inhibits the intracellular production of interferon IFNα on plasmacytoid dendritic cells (pDC) of healthy donors in response to an HIV infection (see FIG. 11). [0284] inhibition of the PDK1 protein kinase by the compound of formula (IV) (also called BX912) inhibits the production of TNFα on plasmacytoid dendritic cells (pDC) of healthy donors in response to an HIV infection (see FIG. 12). [0285] inhibition of the PDK1 protein kinase by the compound of formula (IV) (also called BX912) inhibits the secretion of the inflammatory cytokines (IL-6, IL-1β, TNFα, IP10) and the interferons (IFNα2, IFNβ, IFNI1) by the peripheral blood mononuclear cells (PBMCs) of healthy donors in response to an HIV infection (see FIG. 13). [0286] the compound of formula (IV) (also called BX912) has no toxicity on the peripheral blood mononuclear cells (PBMCs) of healthy donors infected with HIV (see FIG. 14).

[0287] Thus, the compound of formula (IV) (also called BX912) has a preventive effect on the intracellular production of interferon IFNα and TNFα on plasmacytoid dendritic cells (pDC) of healthy donors in response to an HIV infection.

[0288] In addition, the compound of formula (IV) (also called BX912) is non-toxic. It has a preventive effect on the secretion of the inflammatory cytokines (IL-6, IL-1β, TNFα, IP10) and the interferons (IFNα2, IFNβ, IFNI1) by the peripheral blood mononuclear cells (PBMCs) of healthy donors in response to an HIV infection.

Example 6: Curative Effect of the Inhibition of the PDK1 Protein Kinase by the Inhibitor of Formula (IV) (Also Called BX912) in Response to the Stimulation of the TLR7/8 Receptors

Cell Stimulation

[0289] The PBMCs were cultured at 2.Math.10.sup.6 cells/ml. The cells were then incubated with the inhibitor of the PDK1 protein kinase of formula (IV) (also called BX912) at the concentrations indicated for 1 h before stimulation, at the same time as the stimulation or 1 h after the stimulation. The PBMCs were stimulated for 24 h with the TLR7/8 agonist, Resiquimod—R848, at 5 μg/ml.

[0290] The supernatants were collected for the detection of the cytokines.

Detection of the Cytokines

[0291] The assay of the interferons secreted by the PBMCs was carried out by means of the STING-37 reporter cell line. These are human HEK293 cells transfected with a reporter gene expressing luciferase under the control of the ISRE response element. The luciferase produced was revealed by the “Bright-Glo™ Luciferase Assay System” kit from Promega.

[0292] Test for toxicity of the compounds on the peripheral blood mononuclear cells (PBMCs).

[0293] After 24 h of cell culture, the cell viability was determined using the CellTiter-Glo reagent (Promega), which evaluates the amount of ATP by luminescence using the EnSpire.

[0294] The results are presented in FIGS. 15A to 15C.

Example 7: Curative Effect of the Inhibition of the PDK1 Protein Kinase by the Inhibitor of Formula (IV) (Also Called BX912) in Patients Suffering from Juvenile Idiopathic Arthritis

Cell Culture

[0295] The PBMCs were cultured at 2.Math.10.sup.6 cells/ml. The monocytes were cultured at 1.Math.10.sup.6 cells/ml. The cells were then incubated with the inhibitor of the PDK1 protein kinase of formula (IV) (also called BX912) for 24 h. The supernatants were collected for the detection of the cytokines.

Detection of the Cytokines

[0296] The assay of the TNFα secreted by the monocytes of a patient suffering from juvenile idiopathic arthritis was carried out by SIMOA (digital ELISA).

[0297] The results are presented in FIG. 16.

Results:

[0298] It can be seen that: [0299] inhibition of the PDK1 protein kinase by the compound of formula (IV) (also called BX912) inhibits the spontaneous production of TNFα on monocytes of patients suffering from juvenile idiopathic arthritis (FIG. 16).

[0300] Inhibition of PDK1 by BX912 makes it possible to drastically reduce the spontaneous production of inflammatory cytokines by the monocytes originating from patients suffering from juvenile idiopathic arthritis. This indicates that the compound of formula (IV) (also called BX912) can be used as a curative in patients suffering from a chronic auto-inflammation (auto-inflammatory disease, autoimmune diseases, interferonopathies).

Example 8: Effect of the Inhibition of the ROCK Protein Kinase by the Inhibitor of Formula (VII) (Also Called Y27632) on the Immune Cells (PBMCs, Monocytes, T Lymphocytes and B Lymphocytes)

Isolation and Culturing of Human Leukocytes.

[0301] The in vitro experiments were carried out using human peripheral blood mononuclear cells (PBMCs) isolated by density gradient centrifugation by means of a medium for separating leukocytes from peripheral blood (STEMCELL Technologies). The blood of healthy donors was obtained from the “Etablissement Français du Sang” [French Blood Establishment] (convention #07/CABANEL/106; Paris, France). The studies on material from patients were approved by the research ethics committee (ID-RCB/EUDRACT: 2014-A01017-40 and 2018-A01358-47). The human monocytes were purified by positive selection with human CD14 microbeads (Miltenyi). The T lymphocytes were purified by negative selection, using the “Pan T Cell Isolation Kit” from Miltenyi. The B lymphocytes were purified by negative selection, using the “Pan B Cell Isolation Kit” from Miltenyi. The peripheral blood mononuclear cells (PBMCs), the monocytes and T lymphocytes and B lymphocytes were cultured in RPMI 1640 (Sigma) containing 10% inactivated foetal bovine serum and 1 mM glutamine (Hyclone, Logan, Utah).

Cell Stimulation

[0302] The PBMCs were cultured at 2.Math.10.sup.6 cells/ml. The monocytes, T lymphocytes and B lymphocytes were cultured at 1.Math.10.sup.6/ml. The cells were then incubated with the inhibitor of the ROCK protein kinase of formula (VII) (also called Y27632) at the concentrations indicated for 1 h before stimulation. The PBMCs were stimulated for 24 h with the TLR7/8 agonist, Resiquimod—R848 at 5 g/ml. The culture supernatants of the PBMCs were collected for the detection of the cytokines. The monocytes were stimulated for 6 h with the TLR7/8 agonist, Resiquimod—R848 at 1 μg/ml. The cells were recovered for the flow cytometry. For the intracellular labelling of TNFα, IL-6 and IL-1β, Brefeldin A (BFA) was added to the cells 30 minutes after the stimulation. The T lymphocytes were stimulated for 3 days with beads from the “Dynabeads human T activator CD3/CD28” kit from Thermo Fisher Scientific, following their instructions. The cells were recovered for the flow cytometry. The B lymphocytes were stimulated for 24 h with CpG-A t 1 μg/ml. The cells were recovered for the flow cytometry.

Flow Cytometry

[0303] The cells were rinsed in PBS then incubated with a viability dye (Zombie Aqua, Biolegend) for 30 min at ambient temperature. After washing, the cells were resuspended in PBS containing 2% SVF and 2 mM EDTA. For the monocytes, an intracellular labelling of TNFα, IL-6 and IL-1β, with the “Inside Stain” kit (Miltenyi Biotec) was used following the manufacturer's protocol. The monocytes were fixed for 20 min at ambient temperature with 250 μL Inside Fix solution then labelled in 100 μL Inside Perm solution containing the APC anti-TNFα (Miltenyi), PE anti-IL-1β (Miltenyi) and FITC anti-IL-6 (Miltenyi) antibody at 1/50° for 30 min at ambient temperature. Data acquisition was carried out on the Attune N×T flow cytometer from Thermo Fisher. The FlowJo software was used to analyze the data. For the T lymphocytes, the cells were suspended in PBS containing 2% SVF and 2 mM EDTA then labelled with the PE anti-CD69 and APC anti-CD3 antibody from Miltenyi Biotec, used at 1/100°. After incubation for 30 min, the cells were rinsed in PBS containing 2% SVF and 2 mM EDTA. Data acquisition was carried out on the Attune N×T flow cytometer from Thermo Fisher. The FlowJo software was used to analyze the data. For the B lymphocytes, the cells were resuspended in PBS containing 2% SVF and 2 mM EDTA then labelled with the PE anti-CD69 and PE.Vio770 anti-CD19 antibody from Miltenyi Biotec, used at 1/100°. After incubation for 30 min, the cells were rinsed in PBS containing 2% SVF and 2 mM EDTA. Data acquisition was carried out on the Attune N×T flow cytometer from Thermo Fisher. The FlowJo software was used to analyze the data.

Detection of the Cytokines

[0304] The assay of the interferons secreted by the PBMCs was carried out by means of the STING-37 reporter cell line. These are human HEK293 cells transfected with a reporter gene expressing luciferase under the control of the ISRE response element. The culture supernatants of the PBMCs were added to the STING-37 cultures at 0.4.Math.10.sup.6 cells/mi for 24 h. The luciferase produced was revealed by the “Bright-Glo™ Luciferase Assay System” kit from Promega.

[0305] The activity of the NF-κB transcription factor was measured by means of the THP1-Dual reporter cell line from Invivogen. The culture supernatants of the PBMCs were added to the THP1-Dual cultures at 0.4.Math.10.sup.6 cells/ml for 24 h. The activity of the NF-κB transcription factor was measured with QUANTI-Blue, a detection agent from SEAP, following the supplier's recommendations.

[0306] The results are presented in FIGS. 17, 18, 19 and 20.

Results:

[0307] It can be seen that: [0308] inhibition of the ROCK protein kinase by the compound of formula (VII) (also called Y27632) inhibits the production of IFN and the activation of the transcription factor of the NF-κB pathway induced by the stimulation of TLR7/8 in the human PBMCs (see FIG. 17). [0309] inhibition of the ROCK protein kinase by the compound of formula (VII) (also called Y27632) inhibits, according to a dosage effect, the production of TNFα, IL-1β and IL-6 induced par R848 without affecting the viability of the monocytes (see FIG. 18). [0310] inhibition of the ROCK protein kinase by the compound of formula (VII) (also called Y27632) inhibits, according to a dosage effect, the expression of the CD69 marker induced by the CD3/CD28 beads, according to a dosage effect, at the surface of the T lymphocytes, without affecting the viability thereof (FIG. 19). [0311] inhibition of the ROCK protein kinase by the compound of formula (VII) (also called Y27632) inhibits, according to a dosage effect, the expression of the CD69 marker induced by CpG-A, ligand of TLR9, according to a dosage effect, at the surface of the B lymphocytes, without affecting the viability thereof (FIG. 20).

[0312] Thus, the compound of formula (VII) (also called Y27632) has a preventive effect on the production of inflammatory cytokines and interferons by the immune cells. but also on the activation of the T and B lymphocytes. The compound of formula (VII) (also called Y27632) has an anti-inflammatory and anti-interferon effect on the T and B lymphocytes. The compound of formula (VII) (also called Y27632) has an immunosuppressive effect on the T and B lymphocytes.

Example 9: Curative Effect of the Inhibition of the ROCK Protein Kinase by the Inhibitor of Formula (VII) (Also Called Y27632) in Patients Suffering from Juvenile Idiopathic Arthritis

Cell Culture

[0313] The PBMCs were cultured at 2.Math.10.sup.6 cells/ml. The cells were then incubated with the inhibitor of the ROCK protein kinase of formula (VII) (also called Y27632) at 1 μM for 16 h. The cells were recovered and lysed so as to carry out a RT-qPCR.

Detection of the mRNA Encoding TNFα and IL-6

[0314] Total RNA is extracted from the cells with the E.Z.N.A. Total RNA Kit 1 kit (Omega Bio-tek) following the supplier's instructions. The RNA concentration is measured using a spectrophotometer (Nanodrop ND-1000). The total RNA is reverse-transcribed to cDNA using the Prime Script RT Master Mix kit (Takara RR036A) following the supplier's instructions.

[0315] The real-time quantitative PCR is carried out according to the following conditions: cDNA diluted to 1/10th is added to the following mixture: 3.8 μL H.sub.2O, 0.1 μL sense and antisense primers (10 μM) (Table 1), 5 μL of the mixture Absolute qPCR SYBR Green containing Thermostart DNA polymerase, MgCl.sub.2, SYBR Green and dNTPs (Eurogentec). The assays are carried out in triplicate in a 384-well plate (Thermo Scientific). Amplification of the DNA by quantitative PCR is carried out in the CFX384 appliance (Bio-Rad) with the following programme: 3 min at 95° C./5 sec at 95° C. and 30 sec à 60° C. repeated 40 cycles/2 min at 72° C. and 30 sec at 95° C. followed by a dissociation step which makes it possible to verify the specificity of the amplification product. The DNA amplification data are analyzed with the Bio-Rad CFX Manager software.

TABLE-US-00001 TABLE 1 sequence of the primers used Name Sense primer Antisense primer Homo_RPL13 AACAGCTCATGAGG TGGGTCTTGAGGAC CTACGG CTCTGT (SEQ ID No. 1) (SEQ ID No. 2) Homo_TNF CCTGCTGCACTTTG GAGGGTTTGCTACA GAGTGA ACATGGG (SEQ ID No. 3) (SEQ ID No. 4) Homo_IL-6 GACAGCCACTCACC CCTCTTTGCTGCTT TCTTCA TCACAC (SEQ ID No. 5) (SEQ ID No. 6)

[0316] The results are presented in FIG. 21.

Results:

[0317] It can be seen that: [0318] inhibition of the ROCK protein kinase by the compound of formula (VII) (also called Y27632) inhibits the transcription of the genes encoding TNFα and IL-6 by the PBMCs of patients suffering from juvenile idiopathic arthritis (FIG. 21).

[0319] Inhibition of ROCK by Y27632 makes it possible to drastically reduce the transcription of the genes encoding the inflammatory cytokines by the PBMCs originating from patients suffering from juvenile idiopathic arthritis. This indicates that the compound of formula (VII) (also called Y27632) can be used as a curative in patients suffering from a chronic auto-inflammation (auto-inflammatory disease, autoimmune diseases, interferonopathies).

Example 10: Specific Anti-Inflammatory Effect of the Inhibitor of the ROCK Protein Kinase of Formula (VII) (Also Called Compound Y27632) and of the Inhibitor of the ROCK Protein Kinase of Formula (VIII) (Also Called Dimethylfasudil) on the Immune Cells in Comparison with the Non-Immune Cells

Isolation and Culturing of Human Leukocytes.

[0320] The in vitro experiments were carried out using human peripheral blood mononuclear cells (PBMCs) isolated by density gradient centrifugation by means of a medium for separating leukocytes from peripheral blood (STEMCELL Technologies). The blood of healthy donors was obtained from the “Etablissement Français du Sang” [French Blood Establishment] (convention #07/CABANEL/106; Paris, France). The peripheral blood mononuclear cells (PBMCs) and the THP1-Dual monocytes were cultured in RPMI 1640 (Sigma) containing 10% inactivated foetal bovine serum and 1 mM glutamine (Hyclone, Logan, Utah).

Culturing the Human Embryonic Kidney Cells (HEK293 Cell Line)

[0321] The HEK293 were cultured in DMEM (Sigma) containing 10% inactivated foetal bovine serum and 1% penicillin and streptomycin.

Cell Stimulation

[0322] The PBMCs were cultured at 2.Math.10.sup.6 cells/ml. The THP1-Dual and HEK293 cell lines were cultured at 1.Math.10.sup.6 cells/ml. The cells were then incubated with the inhibitor of the ROCK protein kinase of formula (VII) (also called compound Y27632) or the inhibitor of the ROCK protein kinase of formula (VIII) (also called dimethylfasudil) at the concentrations indicated for 1 h before stimulation. The PBMCs were stimulated for 24 h with the STING agonist cGAMP at 3 μg/ml. The culture supernatants of the PBMCs were collected for the detection of the cytokines. The THP1-Dual (Invivogen) were stimulated for 24 h with the STING agonist cGAMP at 3 μg/ml. The interferon response of the THP1-Dual possessed by the ISRE-ISG-Luciferase construction was revealed by means of the Quanti-Luc detection agent (Invivogen), following the supplier's instructions. The HEK293 cells were stimulated for 24 h with the STING agonist at 3 μg/ml.

Detection of the Cytokines

[0323] The assay of the interferons secreted by the PBMCs was carried out by means of the STING-37 reporter cell line. These are human HEK293 cells transfected with a reporter gene expressing luciferase under the control of the ISRE response element. The culture supernatants of the PBMCs were added to the STING-37 cultures at 0.4.Math.10.sup.6 cells/ml for 24 h. The luciferase produced was revealed by the “Bright-Glo™ Luciferase Assay System” kit from Promega.

[0324] The experiments on the HEK293 non-immune cells were carried out directly on STING-37 (HEK293 containing the ISRE-Luciferase construction). The production of interferons was revealed with the “Bright-Glo™ Luciferase Assay System” kit from Promega.

Toxicity Test

[0325] The cell toxicity of the compounds was measured by quantifying ATP with CellTiter Glo (Promega).

Results:

[0326] The results are presented in FIG. 22 and in FIG. 23.

Relating to the Inhibitor of the ROCK Protein Kinase of Formula (VII) (Also Called Compound Y27632):

[0327] The anti-inflammatory activity of the inhibitor of the ROCK protein kinase of formula (VII) (also called compound Y27632) was tested on different cell types: peripheral blood mononuclear cells (PBMCs) isolated from the blood of healthy individuals (FIG. 22A), the THP1-Dual human monocyte cell line (FIG. 22B) and the HEK293 non-immune cell line, a human embryonic kidney cell line (FIG. 22C). These cells were stimulated by cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), an agent mimicking a viral infection by binding on the STING antiviral sensor. Activation of this pathway induces the production of type I interferon. The inhibitor of the ROCK protein kinase of formula (VII) (also called compound Y27632) blocks the production of interferons (IFN) induced by the cGAMP according to a dosage effect starting from 1 μM without affecting the viability of the PBMCs (FIG. 22A) and the THP1-Dual (FIG. 22B). On the HEK293 non-immune cells, the inhibitor of the ROCK protein kinase of formula (VII) (also called compound Y27632) blocks in a limited manner the production of interferon at 1 and 5 μM induced by the STING ligand (FIG. 22C). These results demonstrate that the inhibitor of the ROCK protein kinase of formula (VII) (also called compound Y27632) exerts an immunosuppressive effect which, in a dose-dependent manner, is specific to the immune cells.

Relating to the Inhibitor of the ROCK Protein Kinase of Formula (VIII) (Also Called Dimethylfasudil):

[0328] The anti-inflammatory activity of the inhibitor of the ROCK protein kinase of formula (VIII) (also called dimethylfasudil) was tested on different cell types: peripheral blood mononuclear cells (PBMCs) isolated from the blood of healthy individuals (FIG. 23A), the THP1-Dual human monocyte cell line (FIG. 238) and the HEK293 non-immune cell line, a human embryonic kidney cell line (FIG. 23C). These cells were stimulated by cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), an agent mimicking a viral infection by binding on the STING antiviral sensor. Activation of this pathway induces the production of type I interferon. The inhibitor of the ROCK protein kinase of formula (VIII) (also called dimethylfasudil) blocks the production of interferons (IFN) induced by the cGAMP according to a dosage effect starting from 1 μM without affecting the viability of the PBMCs (FIG. 23A) and of the THP1-Dual (FIG. 23B). On the HEK293 non-immune cells, the inhibitor of the ROCK protein kinase of formula (VIII) (also called dimethylfasudil) does not block the production of interferon at 1 μM induced by the STING ligand (FIG. 23C). These results demonstrate that the inhibitor of the ROCK protein kinase of formula (VIII) (also called dimethylfasudil) exerts an immunosuppressive effect which, in a dose-dependent manner, is specific to the immune cells.