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WO2024153585A1 - Selective caspase-8 inhibitors and uses thereof in augmenting innate immune defenses - Google Patents

Selective caspase-8 inhibitors and uses thereof in augmenting innate immune defenses Download PDF

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Publication number
WO2024153585A1
WO2024153585A1 PCT/EP2024/050797 EP2024050797W WO2024153585A1 WO 2024153585 A1 WO2024153585 A1 WO 2024153585A1 EP 2024050797 W EP2024050797 W EP 2024050797W WO 2024153585 A1 WO2024153585 A1 WO 2024153585A1
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Prior art keywords
caspase
inhibitor
selective
pan
fmk
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PCT/EP2024/050797
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French (fr)
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Giuseppe Teti
Concetta Beninati
Agata FAMA'
Germana LENTINI
Andrea CAPPELLO
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Scylla Biotech Srl
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Publication of WO2024153585A1 publication Critical patent/WO2024153585A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/07—Tetrapeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/55—Protease inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to the field of therapeutics and in particular to the use of selective caspase-8 inhibitors that are able to unleash an endogenous, cell death-independent mechanism of activation of the innate system of body defenses to treat infections, tumors and septic shock.
  • Caspase-8 is involved in several molecular mechanisms that are activated by death receptors and by other receptors of the innate immune system. Caspase-8 is recruited into activation molecular complexes by means of the adaptor FADD. Caspase-8 activation has different effects depending on the cell type, activating stimulus and interaction with different molecular partners. Its enzymatic activities require the formation of caspase-8 homodimers or heterodimers consisting of caspase 8 and the long isoform of cFLIP, while complexation with shorter cFLIP isoforms inhibits activation.
  • caspase-8 can prevent necroptosis, a form of programmed cell death, by enzymatically inactivating RIPK1 and RIPK3, which induce necroptosis through activation of the essential mediator MLKL (Orning, P. & Lien, E. Multiple roles of caspase-8 in cell death, inflammation, and innate immunity. Journal of leukocyte biology 109, 121-141, doi : 10.1002/ JLB.3MR0420-305R 2021) .
  • caspase inhibitors have been devised for treating cell death-related conditions, including tissue damage secondary to ischemia, trauma, burns and surgery, as well as neurodegenerative and metabolic diseasesc(S. Dhani et al. Cell Death and Dis. 2021, 12:949) .
  • Non-selective inhibitors such as the peptide ketone z-VAD-fmk, affect multiple caspases (pancaspase inhibitors) and are able to reduce the extent of tissue damage in animal models of ischemia and neuro-degenerative disease, by preventing apoptosis (C.J.F. Van Noorden, The history of Z-VAD-FMK, a tool for understanding the significance of caspase inhibition, Acta Histochem. 2001;103:241-51) .
  • pan-caspase inhibitor Q-VD- OPH has therapeutic effects against dermatitis caused by Staphylococcus aureus and other bacteria by reducing the size of dermonecrotic lesions but no efficacy has been shown in treating bacterial infections affecting organs other than the skin (Alphonse et al. Pan-caspase inhibition as a potential host- directed immunotherapy against MRSA and other bacterial skin infections, Sci Transl Med. 2021; 13:eabe9887) .
  • European patent n. EP 2193801 describes the use of caspase-8 inhibitors for treating non-inf ectious inflammation in various organs and tissue, with the exception of the skin, particularly for treating inflammation following tissue damage or resection and other pathological conditions such as hepatitis , inf lammatory bowel disese, vasculitis, joint inflammation, sinusitis, scleritis, parodontitis, cervicitis, uveitis, vulvovaginitis, con unctivitis, alveolitis, esophagitis, acute glomerulonephritis, nefritis, acute bronchitis, acute cholecystitis and pancreatitis.
  • tissue damage or resection and other pathological conditions such as hepatitis , inf lammatory bowel disese, vasculitis, joint inflammation, sinusitis, scleritis, parodontitis, cervicitis, uveitis
  • U.S. patent n. US6800619 describes the use of caspase inhibitors for treatment of inflammatory diseases, autoimmune diseases, destructive bone disorders, cardiac diseases, uveitis, peritonitis, inflammatory peritonitis, lupus erythematosus, diabetes, Crohn's disease, ulcerative colitis, atopic dermatitis, transplant rejection, hemorrhagic shock, congenital cardiac insufficiency, osteoarthritis, rheumatoid arthritis, psoriasis, glomerulonephritis, graft versus host disease, intestinal inflammatory disease, sepsis, septic shock, burns, post-burn organ apoptosis, stroke, brain ischemia, traumatic brain damage, neurological damage spinal cord lesions, amyotrophic lateral sclerosis, multiple sclerosis, myocardial infarction, myocardial ischemia, atherosclerosis, acute respiratory insufficiency, adult respiratory distress syndrome, pancre
  • pan-caspase inhibitors for use in the treatment of pulmonary infections, such as SARS-CoV and related diseases.
  • pulmonary infections such as SARS-CoV and related diseases.
  • the pan-caspase inhibitor is a small molecule which is a selective inhibitor of caspase 8 selected among the following compounds: Emricasan, Nivocasan, Q-VD-OPh (1135695-98-5) , P R Inhibitor (CAS number: 608512-97-6) , Q-VD- P H (CAS 1135695-98-5) , Gly-Phe B- naphthylamide (CAS number: 21438-66-4) .
  • Emricasan and Q-VD-OPh are notorious and prototypical broad pan-caspase inhibitors (https://doi.org/10.1038/s41419-021-04240-3) ;
  • Nivocasan is a pan-caspase inhibitor with pronounced activity against caspase- 1, (Wilson, C.H., Kumar, S. Caspases in metabolic disease and their therapeutic potential. Cell Death Differ 25, 1010-1024 (2016) . https://doi.org/10.1038/s41418-018-0111-x) .
  • caspase-1/11 inhibition abrogates the protective effects of caspase-8 inhibition
  • the "P R” (sic, PKR meant) inhibitor (CAS number: 608512-97-6) is a prototypical selective protein kinase R inhibitor and therefore not a selective caspase-8 inhibitor
  • Gly-Phe B-naphthylamide (CAS number: 21438-66-4) is a cathepsin C substrate used to destroy lysosomes and cannot be considered a selective caspase-8 inhibitor
  • BI-9B1210 (CAS 848782-29-6) inhibits caspase 3 and 9 in addition to caspase-8 and therefore it isn't a specific inhibitor of caspase-8.
  • the beneficial effects described in the present invention are independent from apoptosis and the effects described in the above-mentioned publication are induced by an inhibitors of caspase-8 as well as by a pan-caspase inhibitor while the beneficial effects described in the present invention are induced by selective caspase-8 inhibitors but not by pancaspase inhibitors.
  • Emricasan The international patent application, publication n. WO 2021/064180 Al describes the property of Emricasan to alleviate M2-like polarization of macrophages and suggests that such property is due to caspase-8 inhibition. It discloses Emricasan or a caspase-8 inhibitor for use in the treatment of macrophage related diseases including solid cancer, fibrotic diseases, hepatic fibrosis or systemic sclerosis, allergy and asthma, atherosclerosis and Alzheimer's disease. Emricasan, which is a prototypical pan-caspase inhibitor which is not selective. In contrast, the present invention concerns a selective inhibitor of caspase-8, which is not a pan-caspase inhibitor and does not inhibit other caspases.
  • pan-caspase inhibitors such as Emricasan
  • Emricasan pan-caspase inhibitors
  • the latter depend on activation of neutrophils
  • international patent application, publication n. WO 2021/064180 Al deals with polarization of macrophages and does not deal with infections and septic shock.
  • caspase inhibitors as therapeutic agents is severely hampered by poor target specificity and lack of understanding of caspase function, particularly in the context of infectious disease (S. Dhani et al. Cell Death and Dis. 2021, 12:949) .
  • pan-caspase inhibitors to treat different types of infections has been variously proposed, as mentioned above in the "State of the art" section, a large body of evidence indicates that wide-spectrum inhibition affecting multiple caspases (or pan-caspase inhibition) can hamper immune host defenses due to partial or complete blockade of inflammatory caspases and consequently worsen -rather than ameliorate- the outcome of infection (Man et al.
  • cytokine storm In treating severe diseases, such as infections, cancers or septic shock, there is an unmet need to: 1) augment host defenses against the pathogenic agent; 2) protect the host against the adverse effects of his own defenses, including damage caused by excessive or generalized inflammation.
  • the main drawback of currently available strategies to augment immune responses, such as administration of exogenous cytokines (e.g. interferons) is the risk of producing adverse reactions, including the so called "cytokine storm".
  • the inflammatory response induced by selective caspase-8 inhibition involves the production of selected pro- inflammatory cytokines, including IL-lp, and the recruitment of inflammatory cells, mainly polymorphonuclear leukocytes, by a mechanism that does not require cell death; • in vitro, selective caspase-8 inhibition induces transcription of IL-lp and Cxcll mRNA in peritoneal cells and in isolated polymorphonuclear neutrophils, but not in isolated macrophages;
  • caspase-1 inhibition abrogates the cytokine-inducing and host protective effects of selective caspase-8 inhibition during infection. This indicates that the activity of caspase-1 must be preserved for the protective effects of caspase-8 inhibition to take place;
  • pan-caspase inhibitors do not show significant host protective activities against infection, despite their ability to inhibit caspase-8 activity;
  • the present invention unveils a novel molecular pathway that is constitutively activated in neutrophils and other immune cells, is kept under control by caspase-8, requires RIK3 and the activation of caspase-1, does not require cell death and leads to chemokine/cytokine production.
  • the present invention provides a method whereby : 1 ) host defenses are triggered or augmented in a diseased individual ; 2 ) said individual becomes protected against the dangerous side ef fects of exaggerated inflammatory responses .
  • a selective caspase- 8 inhibitor with the proviso that such selective caspase- 8 inhibitor is not a pan-caspase inhibitor and does not inhibit other caspases for use in the treatment of infectious disease .
  • the present invention concerns a selective caspase- 8 inhibitor, with the proviso that such selective caspase- 8 inhibitor is not a pan-caspase inhibitor and does not inhibit other caspases for use as an in vivo antimicrobial agent .
  • Another obj ect of the present invention is a selective caspase- 8 inhibitor, with the proviso that such selective caspase- 8 inhibitor is not a pan-caspase inhibitor and does not inhibit other caspases for use in the prevention and/or treatment of septic shock .
  • a selective caspase-8 inhibitor with the proviso that such selective caspase- 8 inhibitor is not a pan-caspase inhibitor and does not inhibit other caspases for use in the treatment of cancer .
  • Fig. 1 shows that selective caspase-8 inhibition promotes bacterial clearance: mice were injected i.p. with 0.2 ml of PBS (dark grey triangles) , vehicle (7.5% dimethyl sulfoxide in PBS; grey squares) or the caspase-8 inhibitor z-IETD-fmk (6 mg/kg; black circles) at 4 h before i.p challenge with 4xl0 7 CFU of GBS per mouse.
  • Peritoneal lavage fluid samples were collected at the indicated times (hours) after bacterial challenge (horizontal axis) and analyzed. The vertical axis shows bacterial numbers in peritoneal lavage fluid samples. The dashed horizontal line indicates the limit of detection of the assay. Shown are cumulative data from two experiments, each involving 4 animals per group. ***, p ⁇ 0.001 as compared to vehicle-treated mice by the Mann-Whitney U test; ns, non-signif leant .
  • Fig. 2 shows that selective caspase-8 inhibition promotes neutrophil recruitment into infection sites: mice were injected i.p. with 0.2 ml of PBS (dark grey triangles) , vehicle (7.5% dimethyl sulfoxide in PBS; grey squares) or the caspase-8 inhibitor z-IETD-fmk (6 mg/kg; black circles) at 4 h before i.p. challenge with 4xl0 7 CFU of GBS per mouse.
  • Peritoneal lavage fluid samples were collected at the indicated times after bacterial challenge (horizontal axis) and analyzed. The vertical axis shows neutrophil numbers in peritoneal lavage fluid samples. Shown are cumulative data from two experiments, each involving 4 animals per group. *, P ⁇ 0.05; **, P ⁇ 0.01 as compared to vehicle-treated mice by the Mann-Whitney U test; ns, nonsignificant .
  • Fig. 3 shows that selective caspase-8 inhibition promotes the release of the IL-lp cytokine during infection: mice were injected i.p. with 0.2 ml of PBS (dark grey triangles) , vehicle (7.5% dimethyl sulfoxide in PBS; grey squares) or the caspase-8 inhibitor z-IETD-fmk (6 mg/kg; black circles) at 4 h before i.p challenge with 4xl0 7 CFU of GBS per mouse.
  • Peritoneal lavage fluid samples were collected at the indicated times after bacterial challenge (horizontal axis) and analyzed. The vertical axis shows IL-lp concentrations in peritoneal lavage fluid samples. Shown are cumulative data from two experiments, each involving 4 animals per group. **, P ⁇ 0.01 as compared to vehicle- treated mice by the Mann-Whitney U test; ns, non-signif leant .
  • Fig. 4 shows that administration of a selective caspase-8 inhibitor is, by itself, sufficient to induce the release of interleukin 1 beta (IL-lp) , an important pro-inflammatory cytokine.
  • IL-lp interleukin 1 beta
  • Fig. 5 shows that administration of a selective caspase-8 inhibitor is, by itself, sufficient to induce the recruitment of polymorphonuclear leukocytes, a central feature of acute inflammation. Shown are the numbers of neutrophils in peritoneal lavage fluid samples from mice treated with z-IETD-fmk (6 mg/kg, i.p.; circles) or vehicle (7.5% dimethyl sulfoxide in PBS; squares) for the indicated times (hours; horizontal axis) , in the absence of other stimuli; each determination was conducted on a different animal in the course of one experiment involving 5 animals per group; the dashed line indicates the limit of detection of the test. **, P ⁇ 0.01, as compared with vehicle- treated mice by the U di Mann-Whitney test; ns, non-signif leant .
  • Fig. 6 shows that administration of a selective caspase-8 inhibitor is, by itself, sufficient to induce the release of a large number of proinf lammatory cytokines, chemokines, and growth factors. Shown are dot blot cytokine array membranes incubated with peritoneal lavage fluid samples obtained from mice at 4 h after i.p. administration of z-IETD-fmk (6 mg/kg, i.p.; upper panel) or vehicle (7.5% dimethyl sulfoxide in PBS; lower panel) , in the absence of other stimuli.
  • Fig. 7 shows that neutrophils are major IL-lp-producing cells in response to selective caspase-8 inhibition: flow cytometry plot showing cells positive for intracellular IL-lp staining (APC + cells) in peritoneal lavage fluid samples obtained from mice treated with z-IETD-fmk (6 mg/kg, i.p.) in the absence of others stimuli. Peritoneal cells were collected at 4 h after administration of vehicle (left panels) or z-IETD-fmk (right panels) . Neutrophils (upper panels) and macrophages (lower panels) were identified based on expression of Ly6G (PE+) and F4/80 (V450+) , respectively. Data are from one representative experiment of three producing similar results.
  • APC + cells intracellular IL-lp staining
  • Fig. 8 shows that the inflammatory response triggered by specific caspase-8 inhibition depends on RIPK3, but not on MLKL : flow cytometry plot showing cells positive for intracellular IL-lp staining (APC + cells) in peritoneal lavage fluid samples obtained from mice treated with z-IETD-fmk (6 mg/kg, i.p.) in the absence of others stimuli. Peritoneal cells were collected at 3 h after administration of vehicle (left panels) or z-IETD-fmk (right panels) from wild type (WT) , MLKL-/- o RIPK3-/- mice. IL-lbeta APC-A, IL-lp fluorescence; SSC-A, side scatter. Data are from one representative experiment of three producing similar results .
  • Fig. 9 shows that selective caspase-8 inhibition ameliorates high dose endotoxin (LPS) shock; C57BL/6 mice were pretreated i.v. with z-IETD-fmk (6 mg/kg; circles) or vehicle (squares) at Ih before challenge with LPS (40 mg/kg) . Mice were kept under observation and humanely euthanized when showing clinical signs of irreversible shock. Rectal temperature (vertical axis) was measured every 3 h post challenge at the indicated times (hours; horizontal axis) . Shown are cumulative data from two experiments, each involving 4 animals per group*, P ⁇ 0.05; **, P ⁇ 0.01; ***, p ⁇ 0.001, vs vehicle-treated mice, as determined by the Mann-Whitney U test.
  • LPS endotoxin
  • Fig. 10 shows that selective caspase-8 inhibition ameliorates experimental pneumococcal pneumonitis: mice were treated with z- lETD-fmk (6 mg/kg, i.v; circles) or vehicle (squares) at 24, 48, 72, 96 and 120 hours after intranasal challenge with S. pneumoniae (1 x 10 8 CFU/mouse) . Shown is a survival curve obtained by cumulating data from two experiments, each involving 8 animals per group. **, P ⁇ 0.01 vs vehicle-treated mice, as determined by Kaplan-Meier survival analysis.
  • caspase- 8 inhibition an intervention producing a decrease of the enzymatic activity, of the intracellular levels , or in the overall function of caspase- 8 or of a molecule necessary for caspase- 8 function including, but not limited to , cFLIP and FADD .
  • caspase- 8 inhibitor is meant an agent capable of producing caspase- 8 inhibition .
  • selective caspase- 8 inhibition caspase- 8 inhibition in the absence of inhibition of other caspases , including inflammatory caspases .
  • selective caspase- 8 inhibitor is meant any agent or molecule producing selective caspase- 8 inhibition .
  • pan-caspase inhibitor any agent or molecule capable of inhibiting more than one caspase at the same time in a non-selective manner .
  • the selective caspase- 8 inhibitor with the proviso that such selective caspase- 8 inhibitor is not a pan-caspase inhibitor, means that the selective caspase- 8 inhibitor inhibits only caspase- 8 and not other di f ferent caspases , therefore it is not a pan-caspase .
  • tetrapeptide an organic compound made up by four linearly arranged amino acids linked by peptide bonds .
  • cancer or tumor or cancer pathology or tumoral pathology any type of cancer and tumor or cancer pathology or tumoral pathology described in "Encyclopedic Reference of Cancer” , Manfred Schwab, Editor, Springer-Verlag, ISBN : 978-3-540-30683-2 , incorporated herein by reference in its entirety .
  • pathogen or pathogenic agent or microorganism is meant any type of microbial agent described in Topley and Wilson ' s Microbiology and Microbial Infections" , 8 Volume Set , Wiley, 10th Edition” , ISBN- 10- 0470 - 68638-3 , incorporated herein by reference in its entirety .
  • extracellular pathogen is meant a pathogenic agent causing infection by prevalently growing outside of host cells , or in which its virulence is at least partially mediated by capsule constituents .
  • antibacterial or bactericidal agent any agent or molecule capable of killing bacteria or preventing or slowing or suppressing their growth or ability to reproduce in a direct or indirect manner in vi tro or in vi vo ( e . g . , by stimulating host defenses ) .
  • bacteria are meant either gram-positive or gram-negative bacteria .
  • antimicrobial agent any agent or molecule belonging to the group of germicides , antibiotics , antiseptics , antibacterials , antivirals , anti fungals , antiprotozoarians , antiparasitic agents or any agent capable of preventing, slowing or suppressing microbial growth or ability to reproduce in a direct or indirect manner ( e . g . by stimulating host defenses ) in vitro or in vivo .
  • host-directed antimicrobial therapy is meant a form of antimicrobial therapy acting indirectly on the pathogenic agent and directly on host components by interfering with host factors necessary for pathogen' s growth or persistence or by potentiating host defenses against the pathogen .
  • the present invention concerns a selective caspase- 8 inhibitor, with the proviso that said caspase- 8 inhibitor is not a pancaspase inhibitor and does not inhibit other caspases , for use in treating infections .
  • the present invention concerns a selective caspase- 8 inhibitor, with the proviso that said caspase- 8 inhibitor is not a pancaspase inhibitor and does not inhibit other caspases , for use as an antimicrobial .
  • the selective caspase- 8 inhibitor with the proviso that said caspase- 8 inhibitor is not a pan-caspase inhibitor and does not inhibit other caspases , does not inhibit caspase- 1 and/or caspase 11 and/or caspase 4 and/or caspase-5 and there fore is not a caspase- 1 , 4 , 5 or 11 inhibitor .
  • the selective caspase- 8 inhibitor is used as an in vivo antimicrobial .
  • the selective caspase- 8 inhibitor is used as an antiviral agent , for use for the treatment of viral infections .
  • the selective caspase- 8 inhibitor with the proviso that said caspase- 8 inhibitor is not a pan-caspase inhibitor and does not inhibit other caspases is used as a host-directed antimicrobial .
  • the present invention concerns a selective caspase-8 inhibitor, with the proviso that said caspase-8 inhibitor is not a pancaspase inhibitor and does not inhibit other caspases, for use in the treatment of cancer.
  • the present invention concerns a selective caspase-8 inhibitor, with the proviso that said caspase-8 inhibitor is not a pancaspase inhibitor and does not inhibit other caspases, for use in the prevention and/or treatment of septic shock.
  • the caspase-8 inhibitor pertaining to the present invention is selective for caspase-8, and therefore does not inhibit other caspases and is devoid of a generalized (or pan-caspase) effect on multiple caspases.
  • the selective caspase-8 inhibitor which is not a pan-caspase inhibitor and does not inhibit other caspases except for caspase-8, is any molecule, optionally displaying a peptide portion, that can bind to caspase-8 thereby preventing binding of this enzyme to its physiological substrates.
  • the selective caspase-8 inhibitor which is not a pan-caspase inhibitor and does not inhibit other caspases except for caspase-8, is selected from the group consisting of: small molecule, antibody, antibody-derived molecule, nucleic acid inhibitor, anti-sense mRNA, small interfering RNA, substance capable of decreasing caspase-8 production, substance capable of increasing caspase-8 degradation, substance capable of sequestering caspase-8, substance capable of preventing the correct folding of caspase-8.
  • the selective caspase-8 inhibitor which is not a pan-caspase inhibitor and does not inhibit other caspases except for caspase-8, is a small molecule with a molecular weight lower than 5.000 daltons, more preferably is a small molecule with a molecular weight comprised between 100 and 5,000 daltons.
  • the selective caspase-8 inhibitor which is not a pan-caspase inhibitor and does not inhibit other caspases except for caspase-8, is a tetrapeptide.
  • the selective caspase-8 inhibitor which is not a pan-caspase inhibitor and does not inhibit other caspases except for caspase-8, is selected in a group consisting of: z- lETD-fmk, molecules containing the IETD, DEVD, WEHD, or VDVAD peptide sequences, cFLIP short (CASH beta) , cFLIP long (CASH alpha) , caspase-8 associated RING proteins and caspase-10 associated RING proteins (CARPs) .
  • infections are caused by pathogens selected from the group consisting of viruses, bacteria, mycetes, other eucariotic pathogens o by any other pathogen described in Topley and Wilson's Microbiology and Microbial Infections", 8 Volume Set, Wiley, 10th Edition", ISBN-10-0470 -68638-3, incorporated herein by reference in its entirety.
  • infections are those described in Topley and Wilson's Microbiology and Microbial Infections", 8 Volume Set, Wiley, 10th Edition", ISBN-10-0470 -68638-3, incorporated herein by reference in its entirety.
  • infections bacterial peritonitis, bacterial pneumonia, and sepsis In a preferred form, infections bacterial peritonitis, bacterial pneumonia, and sepsis.
  • infections are caused by extracellular pathogens.
  • extracellular pathogens are selected from a group consisting of: Staphylococcus aureus, Staphylococcus epidermidis , Staphylococcus spp , Streptococcus pyogenes , Streptococcus agalactiae , Streptococcus pneumoniae , Streptococcus spp, Enterococcus spp, Clostridium perfringens , Clostridium spp .
  • Actinomyces israelii Actinomyces spp, Escherichia coli , Klebsiella pneumoniae , Pseudomonas aeruginosa, Neisseria gonorrheae , Serratia spp, Proteus spp, Campylobacter spp, Neisseria meningitidis , Neisseria spp, Moraxella spp, Haemophylus influenzae , Haemophylus spp, Acinetobacter spp, Helicobacter pylori .
  • cancer is one of those described in "Encyclopedic Reference of Cancer” , Manfred Schwab, Editor, Springer-Verlag, ISBN : 978-3-540-30683-2 , incorporated herein by reference in its entirety .
  • cancer is selected from the group consisting of : lymphoma, leukemia, carcinoma, adenocarcinoma, teratocarcinoma, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma .
  • the invention also concerns a pharmacological composition
  • a pharmacological composition comprising as an active principle at least one selective inhibitor of caspase- 8 , which is not a pan-caspase inhibitor and does not inhibit other caspases except for caspase- 8 , and pharmaceutically acceptable vehicles and or excipients .
  • the pharmaceutical composition may optionally comprise an additional active principle selected from a group consisting of an antimicrobial agent or a chemotherapeutic antineoplastic agent .
  • compositions applicable in the herein described invention can be made according to conventional methods and techniques that are common practice in the pharmaceutical industry, such as, for example, those illustrated in Remington's Pharmaceutical Science Handbook, Mack Pub. NY - last edition.
  • pharmaceutically acceptable vehicles and/or excipients are included as useful formulation adjuvants, including for example solubilizing agents, dispersing agents, suspension agents and emulsifying agents.
  • the pharmaceutical composition contains a pharmaceutically acceptable vehicles, suitable for administering the active principle.
  • Said vehicles include antibodies and other polypeptides, genes, and other delivery agents such as liposomes, microparticles and nanoparticles on condition that such agents do not induce the production of harmful antibodies and can be administered without causing undue toxicity.
  • suitable vehicles are selected from a group consisting of large slowly metabolized macromolecules including proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers and inactive virus particles .
  • pharmaceutically acceptable vehicles include, in addition, liquids, such as water, saline, glycerol and ethanol.
  • compositions may contain auxiliary agents such as wetting agents emulsifying agents, pH buffers and similar.
  • auxiliary agents such as wetting agents emulsifying agents, pH buffers and similar.
  • Such vehicles allow the formulation of pharmaceutical compositions such as tablets, pills, sugar-coated tables, capsules, liquids, gels, syrups, doughs, suspensions and similar forms suitable for ingestion .
  • the compounds and pharmaceutical compositions pertaining to the present inventions are administered in therapeutically effective doses .
  • Therapeutically effective doses will be generally determined by a doctor based on circumstances, including conditions to be treated, administration route, administered compound, drug combination, age, body weight, response to therapy, severity of symptoms and similar.
  • the therapeutic dose can be initially estimated in cell culture assays, in animal models including those involving mice, rats , guinea pigs, rabbits dogs and pigs. Animal models can be utilized to determine the appropriate dose range and administration route. Such information can then be used to determine doses and routes useful for administration to humans.
  • HED human equivalent doses
  • the therapeutically effective doses is between 3 and 12 mg/kg and more preferably is 6 mg/kg.
  • compositions can be administered by various routes, including but not limited to, oral, intravenous, intramuscular, intramedullary, intrathecal, intraventricular, transdermal, transcutaneous, subcutaneous, intraperitoneal, intranasal, enteral, topic, sublingual, intravaginal or rectal.
  • a pharmaceutical composition suitable for oral administration can take the form of loose liquid solutions or suspensions or loose powders. More commonly, compositions are presented in a unitary form to facilitate accurate dosage.
  • unitary pharmaceutical dosage form refers to physically discrete units that are useful as unit doses for human individuals or other mammals, each unit containing a predetermined amount of active material calculated as to produce the desired therapeutic effect, in association with a suitable excipient.
  • Typical unitary dosage forms include vials or pre-loaded syringes, predosed liquid compositions, or pills tablets, capsules and similar forms in case of solid compositions. The compound may be given in a single dose or in a multiple dose program.
  • mice Six- to eight-week-old C57BL/6 and GDI wild-type (WT) female mice were obtained from Charles River Laboratories. Although data presented here were obtained with female mice only, sex- related differences in responses to caspase-8 inhibition were not detected in additional experiments. Caspl/11 ⁇ , Ripk3 ⁇ , mice were all on a C57B1/6 background.
  • mice All mice were housed in individually ventilated cages under specific pathogen-free conditions in the animal facilities of the Department of Pathology of the University of Messina.
  • the GBS WT strain H36B serotype lb was used in the present example.
  • GBS, S. pneumoniae and K were used to induce pneumonitis.
  • z-IETD-fmk was given to mice i.p. at a dose of 6 mg/kg of body weight.
  • mice were challenged with S. pneumoniae (1 x 10 8 CFU/mouse) or K. pneumoniae (5 x 10 7 CFU/mouse) by the intranasal route.
  • Peritoneal lavage fluid blood and organ homogenates were obtained and analyzed for CFU numbers, cell counts and cytokine determinations by commonly used methods.
  • Peritoneal lavage fluid was obtained by injecting 2 ml of buffered saline in the peritoneal cavity and subsequently aspirating a total of 1.7- 1.9 ml of fluid. Unconcentrated PLF samples were used to measure cytokine levels. After challenge, animals were observed for the development of clinical signs.
  • M-CASS mamouse clinical assessment score for sepsis or M-CASS
  • Animal showing signs of irreversible disease underwent euthanasia.
  • Bone marrow derived neutrophils and macrophages were obtained from the femurs and tibias of 6-8-week-old female mice. Purity of neutrophil preparations was > 97%, as assessed by flow cytometry.
  • Peritoneal cells were obtained from peritoneal lavage fluid (PLF) samples by centrifugation at 400 xg for 15min. Relative proportions of various cell types in a representative sample are reported in Table S2.
  • cells were seeded in microtiter plates at a concentration of 5 x 10 5 per well in 0.2 ml of RPMI medium with 10% fetal calf serum.
  • peritoneal cells were pre-treated in vitro with recombinant IFN- p (10 pg/ml) at 2 h before the addition of z-IETD-fmk (50 pM) or vehicle. Cells were then cultured for the indicated length of time in the presence of z-IETD-fmk or vehicle.
  • Cytokine and mRNA levels were measured in culture supernatants and cell pellets, respectively, by ELISA and Real Time-PCR as described below. Samples were assayed using the Pierce LDH cytotoxicity assay kit (Thermo Fisher Scientific) or for cytokine/chemokine concentrations using the following assays (all from R&D Systems) : Proteome Profiler Mouse Cytokine Array Kit; CXCL1/KC DuoSet; CXCL2/MIP-2 DuoSet; TNF-a DuoSet; IL-ip DuoSet; IL-l" DuoSet; IL-18 DuoSet. The lower detection limits of these assays were 15.6 (IL-lp, IL-la, CXCL1 and 2) , 31.3 (TNF- a) and 46.9 pg/ml (IL-18) .
  • cytokines were measured in PLF or peritoneal cell cultures obtained from neutrophil-depleted mice. Neutrophil depletion was achieved by i.v. injection of 100 pg of rat monoclonal anti-mouse Ly-6G Ab (clone 1A8 ) or rat IgG2a control (isotype control) at 24 h before z-IETD-fmk (6 mg/kg, i.p.) or vehicle treatment for 4 h in the absence of other stimuli. Under these conditions, anti-Ly6G was sufficient to reduce neutrophil blood and peritoneal counts to ⁇ 1% at 24 h after treatment.
  • Expression of the genes encoding IL-lp, Cxcll, Cxcl2, CxcllO, IL-6, IFN-p, IL12b and TNF-a was determined by qPCR using a CFX96 Touch Real-Time PCR Detection System (Bio-Rad) as described in: Biondo, C. et al. MyD88 and TLR2, but not TLR4, are required for host defence against Cryptococcus neo f ormans .
  • mice were collected from mice at 4 h after i.p. injection with z-IETD-fmk or vehicle, washed three times with ice-cold PBS and lysed by vigorous vortexing in RIPA lysis buffer [50 mM Tris -HC1, pH 7.5, 100 mM NaCl, 1% Triton X- 100, 20% glycerol, lx protease inhibitor cocktail] . Lysates were then centrifuged at 13, 000 x g for 15 min at 4°C to eliminate cellular debris.
  • Protein concentration in each sample was determined using the Micro BCA Protein Assay Kit. Protein samples (30 pg of protein per lane) were run on precast Bolt Bis-Tris 4- 12% gels with lx MOPS buffer and transferred on PVDF (polyvinylidene difluoride) membranes. Membranes were washed in TBS-T (Tris Buffered Saline with 0.1% Tween-20) and blocked with TBS-T containing 5% bovine serum albumin (BSA) for 2 h. Membranes were subsequently incubated with primary antibodies in TBS-T containing 1% BSA at 4 °C overnight.
  • TBS-T Tris Buffered Saline with 0.1% Tween-20
  • BSA bovine serum albumin
  • the following primary antibodies were used: phospho-RIP (Serl66) (E7G6O) rabbit mAb, RIP (D94C12) XP rabbit mAb, anti-mouse IL-1 beta/IL-lF2 antibody and anti-beta actin. After incubation, membranes were washed with TBS-T and incubated with secondary antibody (anti-rabbit or anti-goat IgG HRP-linked antibodies) for 2 h at room temperature in TBS-T containing 1% BSA. Protein bands were visualized by Immobilon Forte Western HRP substrate and detected using a BioRad's ChemiDoc XRS system. Beta-actin was used as loading control.
  • peritoneal cells were collected from mice at the indicated times after various treatments, washed three times with DPBS and stained in the dark for 20 minutes with LIVE/DEAD Fixable Aqua Dead Cell Stain Kit, according to the manufacturer's instructions. Cells were then blocked with 0.5 pg Fc Block for 20 minutes at room temperature and stained for surface markers for 20 minutes in the dark with rat anti-mouse Ly-6G (clone 1A8 ) , rat anti-mouse F4/80 (Clone BM8) , anti-mouse pro-IL-1 beta (clone NJTEN3) or isotype control monoclonal antibodies.
  • Intracellular Fixation & Permeabilization Buffer Set (eBioscience ) was used, following the manufacturer's instruction. Briefly, cells were incubated for 30 min in the dark with IC Fixation Buffer and washed twice with Permeabilization Buffer IX (Perm Buffer) . Cells were then stained with the anti-mouse IL-1 beta (Pro-form) Monoclonal Antibody (clone NJTEN3) APC for 30 min in the dark at 4°C. Following two washes with Perm Buffer, cells were resuspended in PBS and 100,000 events per sample were collected on a FACS Canto II flow cytometer (BD Biosciences) . Data analysis was performed using FlowJo version 10 software.
  • BD Trucount Absolute Counting Tubes (BD Trucount Absolute Counting Tubes; BD Biosciences) . Survival data were analyzed by Kaplan-Meier survival plots. All other data were analyzed by the Mann-Whitney test using GraphPad Prism 5.0 (GraphPad Software, Inc., San Diego, CA) . Differences were considered significant when P values were less than 0.05.
  • mice were given the caspase-8 inhibitor z-IETD-fmk at a dose that was found in preliminary experiments to inhibit in vivo activation of caspase-8, but not caspase-1 (see Methods) .
  • mice were challenged i.p with a highly lethal dose of group B streptococcus (GBS) , an important agent of sepsis and meningitis that has been used over the years to model anti-bacterial innate immune responses.
  • GBS group B streptococcus
  • z-IETD-fmk The protective effect of z-IETD-fmk was not due to direct antibacterial activity since this compound did not affect in vitro GBS growth at concentrations up to 2 mg/ml.
  • IL-lp interleukin Ip
  • IETD may crossinhibit other caspases in addition to caspase-8
  • cytokine/chemokine elevations were measured as early as 1 (Cxcll and 2) , 2 (IL-lp and IL-18) and 3 h (IL-la) after treatment (for IL-lp levels, see Fig. 4) , while their levels were low or undetectable in samples from vehicle-treated mice or from mice treated with the pan-caspase inhibitor z-VAD-fmk. Cytokine production occurred concomitantly with neutrophil influx in the peritoneal cavity (Fig. 5) and in the absence of detectable cell death since no significant decrease in peritoneal cell viability occurred over 5 h after administration of z-IETD-fmk.
  • Cytokine appearance was preceded by activation of selected cytokine genes and was concomitant in timing with neutrophil recruitment into the peritoneal cavity. These effects were not due to endotoxin contamination since endotoxin levels in the z-IETD-fmk preparations employed were ⁇ 0.01 EU/ml. Further analysis of PLF supernatants using a protein array revealed that z-IETD-fmk induced the release of a range of chemokines, cytokines and growth factors, including CCL2, CCL12, Cxcll/2/10/12/13, IL-16, IL-17, G-CSF and M-CSF, as well as the complement component C5a and the metalloproteinase inhibitor TIMP-1 (Fig.6) .
  • Cxcll or IL-lp production could not be induced by z-IETD-fmk treatment in cultures of mouse macrophages isolated from a variety of sources, in macrophage cell lines or in mast cells.
  • increased transcription of IL-lp and TNF-a genes and cytokine release was detected in murine bone marrow neutrophils, but not in macrophages, in the presence of z-IETD-fmk.
  • production of CXCL8 and MIP-la was detected in human blood after addition of z-IETD-fmk.
  • Immunoreactive ILlp was detected in 1- 2% of peritoneal cells obtained from mice inoculated with vehicle (Fig. 7 and 8) , PBS or from untreated mice (data not shown) .
  • the number of IL-lp-producing cells increased concomitantly with neutrophil influx, and at 4 h neutrophils and macrophages represented 60-70 and 10-20%, respectively, of the IL-lp-producing cells (Fig. 7) .
  • neutrophils are activated both in vitro and in vivo by z-IETD-fmk and represent most of the cells producing IL-lp cells after i.p. treatment with the inhibitor.
  • mice lacking key signaling proteins involved in cytokine responses and programmed cell death we sought to formally confirm that IETD produces its effects by specifically acting on caspase-8. To this end, we used mice lacking both caspase-8 and MLKL, the essential executioner of necroptotic cell death, since the isolated absence of caspase 8 is embryonically lethal due to uncontrolled necroptosis.
  • TLRs Tolllike receptors
  • IL-lp Partially processed IL-lp was detected in peritoneal cells from wild type mice after i.p. administration of z-IETD-fmk and such processing was reduced in caspase-1/11 double KO animals. Residual IL-lp processing in these animals after z-IETD-fmk treatment was possibly related to the activities of neutrophil serine proteases. Notably, z-IETD administration was unable to reduce bacterial burden in GBS- challenged caspase-1/11 double KO mice, and co-administration of the caspase 1/11 inhibitor z-YVAD-fmk in wild type mice abrogated the beneficial effects on infection observed when z-IETD-fmk was given alone.
  • necroptosis which requires RIPK1-dependent activation of RIPK3 and MLKL, was involved in the in vivo inflammatory response triggered by caspase-8 inhibition.
  • Phosphorylation of RIPK1 was detected by western blot in lysates of peritoneal cells obtained after i.p. administration of z- lETD-fmk.
  • Pre-treatment with the RIPK1 kinase-inhibitor necrostatin-1 significantly attenuated lETD-induced pro- inflammatory changes, while these were completely abrogated by the absence of RIPK3 (Fig. 8) .
  • z-IETD-fmk treatment largely prevented hypothermia and death induced by high-dose endotoxin, despite producing moderate elevations in TNF-a and IL-p blood levels (Fig. 9) .
  • z-IETD-fmk treatment significantly protected mice in a gram positive shock model involving the i.v. administration of live GBS bacteria, by inducing moderate elevations in cytokine blood levels and reducing bacterial burden.
  • z-IETD-fmk treatment did not ameliorate lethality or increase circulating cytokine levels in mice lacking Caspl/11 or RIPK3, indicating that these molecules are required for the therapeutic effects of IETD.
  • IFN-p Exogenous administration of recombinant IFN-p moderately ameliorated GBS-induced sepsis in wild type mice, in general agreement with the notion that IFN-p promotes host defenses against GBS and other extracellular bacteria.
  • IFN-p treatment was ineffective in Caspl/11 or RIPK3 KO mice, indicating that these proteins are involved the therapeutic activities of the cytokine.
  • mice were challenged withl xlO 8 CFU of the serotype 2 D39 reference strain by the intranasal route and, after 24 h, were treated daily with z-IETD-fmk (6 mg/kg i.v.) or vehicle. Under these conditions, 62% of the mice receiving vehicle showed signs of irreversible disease and were humanely euthanized within 5 days, while only 12% of z-IETD-fmk-treated animals succumbed to infection (p ⁇ 0.05; Fig. 10) . A significantly lower bacterial burden was detected in z-IETD-fmk-treated mice compared with control animals.
  • z-IETD-fmk had similar protective activities in a model of pneumonia caused by carbapenem-resistant Klebsiella pneumoniae .
  • our data indicate that z- lETD-fmk administration produces marked protective effects in models of septic shock or invasive pneumonia caused by partially or extremely antibiotic-resistant pathogens.
  • caspase-8 suppresses a proinf lammatory program that is spontaneously activated in neutrophils, depends on RIPK3, and is sustained by tonic IFN-p production. Accordingly, it was found that exposure of bone marrow neutrophils to a caspase-8 inhibitor is sufficient to induce the production of pro-inflammatory cytokines at both the mRNA and protein level in the absence of other external stimuli .
  • chemokines and cytokines known to be potentially released by neutrophils upon activation.
  • This distinctive cytokine signature included several members of Cxcl chemokine family, CCL2, IL-17, TIMP-1, and IL-lp, while TNF- a/IL-12 responses were less pronounced.
  • neutrophils were found to represent most of the IL-lp-producing cells and to be required for cytokine elevations in lETD-induced peritoneal exudates, as shown by, respectively, immunofluorescence and in vivo neutrophil depletion experiments.
  • the caspase-8 inhibitor z-IETD-fmk was carefully titrated here in comparison with other fmk-based inhibitors, to provide maximal selectivity. Under the conditions used, the proinf lammatory changes induced by z-IETD-fmk were due to specific caspase-8 inhibition, since they were completely absent in animals lacking caspase-8. Moreover, these effects depended on the kinase activity of RIPK1 and on RIPK3, which are both substrates of caspase-8 but not of other caspases. Therefore, evidence obtained from multiple approaches indicates that the effects of z-IETD-fmk at the used doses are due to selective inhibition of caspase-8.
  • caspase-8 Although completely dependent on the presence of RIPK3, the mechanisms unleashed by selective caspase-8 inhibition are independent from MLKL, the essential executioner of necroptosis, and occur in the absence of other forms of cell death. Permanent deletion or inactivation of caspase 8 can produce detrimental effects in genetically modified mice. However, according to the present invention, temporary inhibition of caspase-8 had no detrimental effects and ameliorated the outcome of lethal infections due to augmented neutrophil-mediated bacterial clearance.
  • selective caspase-8 inhibition augments the production of pro-inflammatory cytokines while, at the same time, preventing their toxic effects. Therefore, the proinf lammatory program described according to the present invention can be harnessed against dif f icult-to-treat bacterial diseases, such as those caused by antibiotic-resistant pathogens.
  • the pancaspase inhibitor z-VAD-fmk was ineffective against bacterial infections that were effectively prevented by z-IETD-fmk under conditions resulting in selective caspase-8 inhibition.

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Abstract

Selective caspase-8 inhibitors are herein described for use in treating infections, tumors, and septic shock. Said inhibitors are not pan-caspase inhibitors and unleash an innate immune activation mechanism in a cell death-independent fashion.

Description

Selective caspase-8 inhibitors and uses thereof in augmenting innate immune defenses
******
TECHNICAL FIELD
The present invention relates to the field of therapeutics and in particular to the use of selective caspase-8 inhibitors that are able to unleash an endogenous, cell death-independent mechanism of activation of the innate system of body defenses to treat infections, tumors and septic shock.
STATE OF THE ART
Caspase-8 is involved in several molecular mechanisms that are activated by death receptors and by other receptors of the innate immune system. Caspase-8 is recruited into activation molecular complexes by means of the adaptor FADD. Caspase-8 activation has different effects depending on the cell type, activating stimulus and interaction with different molecular partners. Its enzymatic activities require the formation of caspase-8 homodimers or heterodimers consisting of caspase 8 and the long isoform of cFLIP, while complexation with shorter cFLIP isoforms inhibits activation. In complex with CFLIPL, caspase-8 can prevent necroptosis, a form of programmed cell death, by enzymatically inactivating RIPK1 and RIPK3, which induce necroptosis through activation of the essential mediator MLKL (Orning, P. & Lien, E. Multiple roles of caspase-8 in cell death, inflammation, and innate immunity. Journal of leukocyte biology 109, 121-141, doi : 10.1002/ JLB.3MR0420-305R 2021) .
Several caspase inhibitors have been devised for treating cell death-related conditions, including tissue damage secondary to ischemia, trauma, burns and surgery, as well as neurodegenerative and metabolic diseasesc(S. Dhani et al. Cell Death and Dis. 2021, 12:949) . Non-selective inhibitors, such as the peptide ketone z-VAD-fmk, affect multiple caspases (pancaspase inhibitors) and are able to reduce the extent of tissue damage in animal models of ischemia and neuro-degenerative disease, by preventing apoptosis (C.J.F. Van Noorden, The history of Z-VAD-FMK, a tool for understanding the significance of caspase inhibition, Acta Histochem. 2001;103:241-51) .
It was recently reported that the pan-caspase inhibitor Q-VD- OPH has therapeutic effects against dermatitis caused by Staphylococcus aureus and other bacteria by reducing the size of dermonecrotic lesions but no efficacy has been shown in treating bacterial infections affecting organs other than the skin (Alphonse et al. Pan-caspase inhibition as a potential host- directed immunotherapy against MRSA and other bacterial skin infections, Sci Transl Med. 2021; 13:eabe9887) .
International patent application publication number W02021/202530 describes the use of non-selective inhibitors of caspases, such as the pan-caspase inhibitor Q-VD-OPH for the treatment of viral infections such as COVID-19 and for inducing tissue regeneration after cutaneous and intestinal tissue damage .
European patent n. EP 2193801 describes the use of caspase-8 inhibitors for treating non-inf ectious inflammation in various organs and tissue, with the exception of the skin, particularly for treating inflammation following tissue damage or resection and other pathological conditions such as hepatitis , inf lammatory bowel disese, vasculitis, joint inflammation, sinusitis, scleritis, parodontitis, cervicitis, uveitis, vulvovaginitis, con unctivitis, alveolitis, esophagitis, acute glomerulonephritis, nefritis, acute bronchitis, acute cholecystitis and pancreatitis.
United States patent application publication n. US2006/0205771 describes caspase-9, caspase-3, caspase-8 or pan-caspase inibitors for cancer treatment.
U.S. patent n. US6800619 describes the use of caspase inhibitors for treatment of inflammatory diseases, autoimmune diseases, destructive bone disorders, cardiac diseases, uveitis, peritonitis, inflammatory peritonitis, lupus erythematosus, diabetes, Crohn's disease, ulcerative colitis, atopic dermatitis, transplant rejection, hemorrhagic shock, congenital cardiac insufficiency, osteoarthritis, rheumatoid arthritis, psoriasis, glomerulonephritis, graft versus host disease, intestinal inflammatory disease, sepsis, septic shock, burns, post-burn organ apoptosis, stroke, brain ischemia, traumatic brain damage, neurological damage spinal cord lesions, amyotrophic lateral sclerosis, multiple sclerosis, myocardial infarction, myocardial ischemia, atherosclerosis, acute respiratory insufficiency, adult respiratory distress syndrome, pancreatitis, kidney and liver diseases, alcohol abuse diseases, hepatitis, cancer.
China patent n. CN10469448 describes Z-IETD-EMK to increase viral titers in in vitro cultures of the Porcine Reproductive and Respiratory syndrome (PRRSV) .
United States patent application, publication n. US 2020/0000869 proposes the use of caspase inhibitors for treating infections caused by Borrelia burgdorferi, Escherichia coli, Acinetobacter baumannii, Helicobacter pyloris, Legionella pneumophilia, Mycobacteria, Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus faecalis, Streptococcus bovis, Streptococcus pneumoniae, Campylobacter, Helicobacter pyloris, Legionella pneumophilia, Mycobacteria, Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes, Streptococcus agalactiae, Clostridium perfringens, Clostridium perfringens Pasteurella multocida, Fusobacterium nucleatum, Streptobacillus moniliformis, Treponema pallidum, Treponema pertenue, Leptospira, Rickettsia and Actinomyces israelii.
International Patent application, publication n. WO03068242 describes caspase inhibitors for treatment of IL-l-mediated diseases, apoptosis-mediated diseases, inflammatory diseases, autoimmune diseases, destructive bone disorders, proliferative diseases, infectious diseases, degenerative diseases, cell death-associated diseases, diseases associated with alcohol abuse, viral diseases, uveitis, inflammatory peritonitis, osteoarthritis, pancreatitis, asthma, adult respiratory distress syndrome, glomerulonephritis, rheumatoid arthritis, systemic lupus erythematosus, scleroderma, chronic thyroiditis, Grave's diseases, autoimmune gastritis, diabetes, hemolytic autoimmune anemia, autoimmune neutropenia, thrombocytopenia, chronic hepatitis, myasthenia gravis, inflammatory bowel disease, Crohn's disease, psoriasis, atopic dermatitis, wound healing, transplant rejection, osteoporosis, leukemia, myelodysplastic syndrome, multiple myeloma-related bone diseases, melanoma, Kaposi's sarcoma, myeloma, hemorrhagic shock, sepsis, septic shock, burns, shigellosis, Alzheimer's disease, Parkinson's disease, Huntington's disease, Kennedy's disease, prion disease, cerebral ischemia, epilepsy, myocardial ischemia, acute and chronic cardiac diseases, myocardial infarction, congenital cardiac insufficiency, arteriosclerosis, coronary bypass, spinal muscle atrophy, amyotrophic lateral sclerosis, multiple sclerosis, HIV-related encephalopathy, aging, alopecia, stroke neurological damage, ulcerative colitis, traumatic brain damage, spinal cord lesions hepatitis B, hepatitis C, hepatitis G, yellow fever, dengue fever, Japanese encephalitis, various forms of hepatic disease, renal diseases, duodenal and gastric ulcers caused by H. pylori, HIV infection, tuberculosis and meningitis.
International patent application, publication n. W02022008597 describes pan-caspase inhibitors for use in the treatment of pulmonary infections, such as SARS-CoV and related diseases. Although said document clearly refers in its entirety to a pancaspase inhibitor and not to a selective caspase-8 inhibitor, inpage 11, lines 6-10, it discloses that the pan-caspase inhibitor is a small molecule which is a selective inhibitor of caspase 8 selected among the following compounds: Emricasan, Nivocasan, Q-VD-OPh (1135695-98-5) , P R Inhibitor (CAS number: 608512-97-6) , Q-VD- P H (CAS 1135695-98-5) , Gly-Phe B- naphthylamide (CAS number: 21438-66-4) . BI-9B1210 (CAS 848782- 29-6)". The above definition is a contradiction in terms, since the definition of a pan-caspase inhibitor is incompatible with that of selective caspase-8 inhibitor and the terms are mutually exclusive (S. Dhani et al. Cell Death and Dis. 2021, 12:949) ; 2) none of the cited compounds can be considered as a selective caspase-8 inhibitor as it is known to those skilled in the art. For example, Emricasan and Q-VD-OPh (CAS 1135695-98-5) are notorious and prototypical broad pan-caspase inhibitors (https://doi.org/10.1038/s41419-021-04240-3) ; Nivocasan is a pan-caspase inhibitor with pronounced activity against caspase- 1, (Wilson, C.H., Kumar, S. Caspases in metabolic disease and their therapeutic potential. Cell Death Differ 25, 1010-1024 (2018) . https://doi.org/10.1038/s41418-018-0111-x) . The present inventors demonstrated that caspase-1/11 inhibition abrogates the protective effects of caspase-8 inhibition; the "P R" (sic, PKR meant) inhibitor (CAS number: 608512-97-6) is a prototypical selective protein kinase R inhibitor and therefore not a selective caspase-8 inhibitor; Gly-Phe B-naphthylamide (CAS number: 21438-66-4) is a cathepsin C substrate used to destroy lysosomes and cannot be considered a selective caspase-8 inhibitor; BI-9B1210 (CAS 848782-29-6) inhibits caspase 3 and 9 in addition to caspase-8 and therefore it isn't a specific inhibitor of caspase-8.
International patent application, Publication no. WO 2021/202530 deals with pan-caspase inhibition and not with specific caspase- 8. In particular, selective caspase-8 inhibition is not mentioned at page 3, lines 18-23, at page 38, lines 25-33, or in the claims. The caspase-8 inhibitor z-IETD-fmk is mentioned at page 38 line 32 under the "materials and methods section" just to describe how this and similar compounds were dissolved. No description or claim is made in the said patent application about any beneficial effects or therapeutic use of Z-IETD-EMK against any disease condition.
St. Luis et al., "The equine arteritis virus induces apoptosis via caspase-8 and mitochondria-dependent caspase-9 activation", VIROLOGY, ELSEVIER, AMSTERDAM, vol. 367, no. 1, 2007 shows that the equine arteritis virus (EAV) can induce apoptosis in monkey kidney cells and that such apoptosis can be prevented by treating cells with a pan-caspase inhibitor (Z-VAD-EMK) , a caspase-8 inhibitor (Z-IETD-EMK) or a caspase-9 inhibitor ( Z-LEHD-EMK) , and concludes that caspases 8 and 9 are involved in apoptosis caused by EAV. Said scientific publication does not suggest that caspase inhibitors should be used or even considered for treatment of diseases by EAV or and other infections, cancer or indeed any disease condition. In fact, blocking apoptosis in this context would be detrimental to the host since apoptosis is unanimously considered a form of host defense against viruses and other intracellular pathogens because it eliminates the pathogens' replication niche (Orning and Lien J Leukoc Biol. 2020;l-21) ; 2) . The beneficial effects described in the present invention are independent from apoptosis and the effects described in the above-mentioned publication are induced by an inhibitors of caspase-8 as well as by a pan-caspase inhibitor while the beneficial effects described in the present invention are induced by selective caspase-8 inhibitors but not by pancaspase inhibitors.
The international patent application, publication n. WO 2021/064180 Al describes the property of Emricasan to alleviate M2-like polarization of macrophages and suggests that such property is due to caspase-8 inhibition. It discloses Emricasan or a caspase-8 inhibitor for use in the treatment of macrophage related diseases including solid cancer, fibrotic diseases, hepatic fibrosis or systemic sclerosis, allergy and asthma, atherosclerosis and Alzheimer's disease. Emricasan, which is a prototypical pan-caspase inhibitor which is not selective. In contrast, the present invention concerns a selective inhibitor of caspase-8, which is not a pan-caspase inhibitor and does not inhibit other caspases. As remarked above, pan-caspase inhibitors, such as Emricasan, are not effective in terms of the therapeutic activities described and claimed in the present invention. Indeed, the latter depend on activation of neutrophils, while international patent application, publication n. WO 2021/064180 Al deals with polarization of macrophages and does not deal with infections and septic shock.
Kucklemburg et al. "Endothelial cell apoptosis induced by bacteria-activated platelets requires caspase-8 and -9 and generation of reactive oxygen species", THROMBOSIS AND HAEMOSTASIS, SCHATTAUER GMBH, DE, vol. 99, no. 2, 2008, shows that bacteria-activated platelets can induce apoptosis in bovine endothelial cells and that such apoptosis can be prevented by treating cells with a pan-caspase inhibitor (Z-VAD-EMK) , a caspase-8 inhibitor (Z-IETD-EMK) or a caspase-9 inhibitor (Z- LEHD-EMK) . The paper concludes that caspases 8 and 9 are involved in apoptosis caused by bacteria-activated platelets. Said paper This paper reports in vitro study not indicating that caspase inhibitors should be considered for treatment of septic shock, because there is no reason to believe that blocking apoptosis of endothelial cells is beneficial in septic shock. Indeed, septic shock is a hyperinf lammatory condition while apoptosis is considered anti-inflammatory (Orning and Lien J Leukoc Biol. 2020; 1-21) . Moreover, the effects described in the publication cited above are induced not only by z-IETD-fmk, but also by pancaspase inhibitors while the beneficial effects described in the present invention are induced only by selective caspase-8 inhibitors and not by pan-caspase inhibitors.
TECHNICAL PROBLEM
The use of currently available caspase inhibitors as therapeutic agents is severely hampered by poor target specificity and lack of understanding of caspase function, particularly in the context of infectious disease (S. Dhani et al. Cell Death and Dis. 2021, 12:949) . Although the use of pan-caspase inhibitors to treat different types of infections has been variously proposed, as mentioned above in the "State of the art" section, a large body of evidence indicates that wide-spectrum inhibition affecting multiple caspases (or pan-caspase inhibition) can hamper immune host defenses due to partial or complete blockade of inflammatory caspases and consequently worsen -rather than ameliorate- the outcome of infection (Man et al. Molecular mechanisms and functions of pyroptosis, inflammatory caspases and inf lammasomes in infectious diseases, Immunol Rev. 2017; 277:61-75) . Moreover, suppressing the enzymatic activities of multiple caspases carries an inherent risk of increased toxicity .
In treating severe diseases, such as infections, cancers or septic shock, there is an unmet need to: 1) augment host defenses against the pathogenic agent; 2) protect the host against the adverse effects of his own defenses, including damage caused by excessive or generalized inflammation. The main drawback of currently available strategies to augment immune responses, such as administration of exogenous cytokines (e.g. interferons) , is the risk of producing adverse reactions, including the so called "cytokine storm".
It was discovered in the present invention that -in the context of the above-mentioned diseases- selective caspase-8 inhibition achieves an unexpected, dual effect. On the one hand, selective caspase-8 inhibition potentiates host defenses. On the other it protects against deadly damage caused by generalized inflammation, including septic shock. The herein disclosed discovery was made possible by the identification of a novel cellular and molecular mechanism whereby caspase-8 controls the production of a specific set of inflammatory cytokines, chemokines, and growth factors. In particular, it was discovered that :
• by activating cells of the innate immune system, such as neutrophils, selective caspase-8 inhibition is sufficient to induce, in vivo, by itself a full inflammatory response: the presence of other stimuli is not required;
• the inflammatory response induced by selective caspase-8 inhibition involves the production of selected pro- inflammatory cytokines, including IL-lp, and the recruitment of inflammatory cells, mainly polymorphonuclear leukocytes, by a mechanism that does not require cell death; • in vitro, selective caspase-8 inhibition induces transcription of IL-lp and Cxcll mRNA in peritoneal cells and in isolated polymorphonuclear neutrophils, but not in isolated macrophages;
• selective caspase-8 inhibition can potentiate pathogen- triggered pro-inflammatory responses, thereby increasing neutrophil infiltration and cytokine/chemokine production;
• caspase-1 inhibition abrogates the cytokine-inducing and host protective effects of selective caspase-8 inhibition during infection. This indicates that the activity of caspase-1 must be preserved for the protective effects of caspase-8 inhibition to take place;
• pan-caspase inhibitors do not show significant host protective activities against infection, despite their ability to inhibit caspase-8 activity;
• the host-protective activities of specific caspase-8 inhibition require RIPK1 phosphorylation and the presence of RIPK3, two kinases that are known to be degraded and inactivated by caspase-8, but not by other caspases;
• the host-protective activities of specific caspase-8 inhibition do not require necroptosis or other forms of cell death;
In summary, the present invention unveils a novel molecular pathway that is constitutively activated in neutrophils and other immune cells, is kept under control by caspase-8, requires RIK3 and the activation of caspase-1, does not require cell death and leads to chemokine/cytokine production.
It was found unexpectedly that this pathway can be harnessed to potentiate immune defenses against pathogenic agents such as infectious microorganisms and cancer cells. Additionally, it was found according to the present invention that selective caspase- 8 inhibition has remarkable protective activities against septic shock and prevents hypothermia and death caused by shock-inducing agents , such as high-dose bacterial endotoxin .
Therefore , the present invention provides a method whereby : 1 ) host defenses are triggered or augmented in a diseased individual ; 2 ) said individual becomes protected against the dangerous side ef fects of exaggerated inflammatory responses .
OBJECT OF THE INVENTION
The technical problem is therefore solved by providing : a selective caspase- 8 inhibitor, with the proviso that such selective caspase- 8 inhibitor is not a pan-caspase inhibitor and does not inhibit other caspases for use in the treatment of infectious disease .
The present invention concerns a selective caspase- 8 inhibitor, with the proviso that such selective caspase- 8 inhibitor is not a pan-caspase inhibitor and does not inhibit other caspases for use as an in vivo antimicrobial agent .
Another obj ect of the present invention is a selective caspase- 8 inhibitor, with the proviso that such selective caspase- 8 inhibitor is not a pan-caspase inhibitor and does not inhibit other caspases for use in the prevention and/or treatment of septic shock .
The technical problem is also solved by providing a selective caspase- 8 inhibitor, with the proviso that such selective caspase- 8 inhibitor is not a pan-caspase inhibitor and does not inhibit other caspases for use in the treatment of cancer . A selective caspase-8 inhibitor, with the proviso that such selective caspase-8 inhibitor is not a pan-caspase inhibitor, means that the selective caspase-8 inhibitor inhibits only caspase-8 and not other different caspases, therefore it is not a pan-caspase.
Further technical characteristics of the invention will be clarified by the following detailed technical description with reference to the experimental data and figures provided.
BRIEF DESCRIPTION OF THE FIGURES
Fig. 1 shows that selective caspase-8 inhibition promotes bacterial clearance: mice were injected i.p. with 0.2 ml of PBS (dark grey triangles) , vehicle (7.5% dimethyl sulfoxide in PBS; grey squares) or the caspase-8 inhibitor z-IETD-fmk (6 mg/kg; black circles) at 4 h before i.p challenge with 4xl07 CFU of GBS per mouse. Peritoneal lavage fluid samples were collected at the indicated times (hours) after bacterial challenge (horizontal axis) and analyzed. The vertical axis shows bacterial numbers in peritoneal lavage fluid samples. The dashed horizontal line indicates the limit of detection of the assay. Shown are cumulative data from two experiments, each involving 4 animals per group. ***, p <0.001 as compared to vehicle-treated mice by the Mann-Whitney U test; ns, non-signif leant .
Fig. 2 shows that selective caspase-8 inhibition promotes neutrophil recruitment into infection sites: mice were injected i.p. with 0.2 ml of PBS (dark grey triangles) , vehicle (7.5% dimethyl sulfoxide in PBS; grey squares) or the caspase-8 inhibitor z-IETD-fmk (6 mg/kg; black circles) at 4 h before i.p. challenge with 4xl07 CFU of GBS per mouse. Peritoneal lavage fluid samples were collected at the indicated times after bacterial challenge (horizontal axis) and analyzed. The vertical axis shows neutrophil numbers in peritoneal lavage fluid samples. Shown are cumulative data from two experiments, each involving 4 animals per group. *, P<0.05; **, P<0.01 as compared to vehicle-treated mice by the Mann-Whitney U test; ns, nonsignificant .
Fig. 3 shows that selective caspase-8 inhibition promotes the release of the IL-lp cytokine during infection: mice were injected i.p. with 0.2 ml of PBS (dark grey triangles) , vehicle (7.5% dimethyl sulfoxide in PBS; grey squares) or the caspase-8 inhibitor z-IETD-fmk (6 mg/kg; black circles) at 4 h before i.p challenge with 4xl07 CFU of GBS per mouse. Peritoneal lavage fluid samples were collected at the indicated times after bacterial challenge (horizontal axis) and analyzed. The vertical axis shows IL-lp concentrations in peritoneal lavage fluid samples. Shown are cumulative data from two experiments, each involving 4 animals per group. **, P<0.01 as compared to vehicle- treated mice by the Mann-Whitney U test; ns, non-signif leant .
Fig. 4 shows that administration of a selective caspase-8 inhibitor is, by itself, sufficient to induce the release of interleukin 1 beta (IL-lp) , an important pro-inflammatory cytokine. Shown is the concentration of IL-lp in peritoneal lavage fluid samples from mice treated with z-IETD-fmk (6 mg/kg, i.p.; circles) or vehicle (7.5% dimethyl sulfoxide in PBS; squares) for the indicated times (hours; horizontal axis) , in the absence of other stimuli; each determination was conducted on a different animal in the course of one experiment involving 5 animals per group; the dashed line indicates the limit of detection of the test. **, P <0.01, as compared with vehicle- treated mice by the U di Mann-Whitney test; ns, non-signif leant .
Fig. 5 shows that administration of a selective caspase-8 inhibitor is, by itself, sufficient to induce the recruitment of polymorphonuclear leukocytes, a central feature of acute inflammation. Shown are the numbers of neutrophils in peritoneal lavage fluid samples from mice treated with z-IETD-fmk (6 mg/kg, i.p.; circles) or vehicle (7.5% dimethyl sulfoxide in PBS; squares) for the indicated times (hours; horizontal axis) , in the absence of other stimuli; each determination was conducted on a different animal in the course of one experiment involving 5 animals per group; the dashed line indicates the limit of detection of the test. **, P <0.01, as compared with vehicle- treated mice by the U di Mann-Whitney test; ns, non-signif leant .
Fig. 6 shows that administration of a selective caspase-8 inhibitor is, by itself, sufficient to induce the release of a large number of proinf lammatory cytokines, chemokines, and growth factors. Shown are dot blot cytokine array membranes incubated with peritoneal lavage fluid samples obtained from mice at 4 h after i.p. administration of z-IETD-fmk (6 mg/kg, i.p.; upper panel) or vehicle (7.5% dimethyl sulfoxide in PBS; lower panel) , in the absence of other stimuli.
Fig. 7 shows that neutrophils are major IL-lp-producing cells in response to selective caspase-8 inhibition: flow cytometry plot showing cells positive for intracellular IL-lp staining (APC+ cells) in peritoneal lavage fluid samples obtained from mice treated with z-IETD-fmk (6 mg/kg, i.p.) in the absence of others stimuli. Peritoneal cells were collected at 4 h after administration of vehicle (left panels) or z-IETD-fmk (right panels) . Neutrophils (upper panels) and macrophages (lower panels) were identified based on expression of Ly6G (PE+) and F4/80 (V450+) , respectively. Data are from one representative experiment of three producing similar results.
Fig. 8 shows that the inflammatory response triggered by specific caspase-8 inhibition depends on RIPK3, but not on MLKL : flow cytometry plot showing cells positive for intracellular IL-lp staining (APC+ cells) in peritoneal lavage fluid samples obtained from mice treated with z-IETD-fmk (6 mg/kg, i.p.) in the absence of others stimuli. Peritoneal cells were collected at 3 h after administration of vehicle (left panels) or z-IETD-fmk (right panels) from wild type (WT) , MLKL-/- o RIPK3-/- mice. IL-lbeta APC-A, IL-lp fluorescence; SSC-A, side scatter. Data are from one representative experiment of three producing similar results .
Fig. 9 shows that selective caspase-8 inhibition ameliorates high dose endotoxin (LPS) shock; C57BL/6 mice were pretreated i.v. with z-IETD-fmk (6 mg/kg; circles) or vehicle (squares) at Ih before challenge with LPS (40 mg/kg) . Mice were kept under observation and humanely euthanized when showing clinical signs of irreversible shock. Rectal temperature (vertical axis) was measured every 3 h post challenge at the indicated times (hours; horizontal axis) . Shown are cumulative data from two experiments, each involving 4 animals per group*, P<0.05; **, P <0.01; ***, p <0.001, vs vehicle-treated mice, as determined by the Mann-Whitney U test.
Fig. 10 shows that selective caspase-8 inhibition ameliorates experimental pneumococcal pneumonitis: mice were treated with z- lETD-fmk (6 mg/kg, i.v; circles) or vehicle (squares) at 24, 48, 72, 96 and 120 hours after intranasal challenge with S. pneumoniae (1 x 108 CFU/mouse) . Shown is a survival curve obtained by cumulating data from two experiments, each involving 8 animals per group. **, P <0.01 vs vehicle-treated mice, as determined by Kaplan-Meier survival analysis.
DETAILED DESCRIPTION OF THE INVENTION Within the scope of the present invention, by caspase- 8 inhibition is meant an intervention producing a decrease of the enzymatic activity, of the intracellular levels , or in the overall function of caspase- 8 or of a molecule necessary for caspase- 8 function including, but not limited to , cFLIP and FADD . By caspase- 8 inhibitor is meant an agent capable of producing caspase- 8 inhibition .
Within the scope of the present invention, by selective caspase- 8 inhibition is meant caspase- 8 inhibition in the absence of inhibition of other caspases , including inflammatory caspases . By selective caspase- 8 inhibitor is meant any agent or molecule producing selective caspase- 8 inhibition .
Within the scope of the present invention, by pan-caspase inhibitor is meant any agent or molecule capable of inhibiting more than one caspase at the same time in a non-selective manner .
Within the scope of the present invention, the selective caspase- 8 inhibitor, with the proviso that such selective caspase- 8 inhibitor is not a pan-caspase inhibitor, means that the selective caspase- 8 inhibitor inhibits only caspase- 8 and not other di f ferent caspases , therefore it is not a pan-caspase .
Within the scope of the present invention, by tetrapeptide is meant an organic compound made up by four linearly arranged amino acids linked by peptide bonds .
Within the scope of the present invention, by cancer or tumor or cancer pathology or tumoral pathology is meant any type of cancer and tumor or cancer pathology or tumoral pathology described in "Encyclopedic Reference of Cancer" , Manfred Schwab, Editor, Springer-Verlag, ISBN : 978-3-540-30683-2 , incorporated herein by reference in its entirety . Within the scope of the present invention, by pathogen or pathogenic agent or microorganism is meant any type of microbial agent described in Topley and Wilson ' s Microbiology and Microbial Infections" , 8 Volume Set , Wiley, 10th Edition" , ISBN- 10- 0470 - 68638-3 , incorporated herein by reference in its entirety .
Within the scope of the present invention, by extracellular pathogen is meant a pathogenic agent causing infection by prevalently growing outside of host cells , or in which its virulence is at least partially mediated by capsule constituents .
Within the scope of the present invention, by antibacterial or bactericidal agent is meant any agent or molecule capable of killing bacteria or preventing or slowing or suppressing their growth or ability to reproduce in a direct or indirect manner in vi tro or in vi vo ( e . g . , by stimulating host defenses ) .
Within the scope of the present invention, by bacteria are meant either gram-positive or gram-negative bacteria .
Within the scope of the present invention, by antimicrobial agent is meant any agent or molecule belonging to the group of germicides , antibiotics , antiseptics , antibacterials , antivirals , anti fungals , antiprotozoarians , antiparasitic agents or any agent capable of preventing, slowing or suppressing microbial growth or ability to reproduce in a direct or indirect manner ( e . g . by stimulating host defenses ) in vitro or in vivo .
Within the scope of the present invention, by host-directed antimicrobial therapy is meant a form of antimicrobial therapy acting indirectly on the pathogenic agent and directly on host components by interfering with host factors necessary for pathogen' s growth or persistence or by potentiating host defenses against the pathogen .
The present invention concerns a selective caspase- 8 inhibitor, with the proviso that said caspase- 8 inhibitor is not a pancaspase inhibitor and does not inhibit other caspases , for use in treating infections .
The present invention concerns a selective caspase- 8 inhibitor, with the proviso that said caspase- 8 inhibitor is not a pancaspase inhibitor and does not inhibit other caspases , for use as an antimicrobial .
Preferably, the selective caspase- 8 inhibitor, with the proviso that said caspase- 8 inhibitor is not a pan-caspase inhibitor and does not inhibit other caspases , does not inhibit caspase- 1 and/or caspase 11 and/or caspase 4 and/or caspase-5 and there fore is not a caspase- 1 , 4 , 5 or 11 inhibitor .
Preferably, the selective caspase- 8 inhibitor, with the proviso that said caspase- 8 inhibitor is not a pan-caspase inhibitor and does not inhibit other caspases , is used as an in vivo antimicrobial .
Preferably, the selective caspase- 8 inhibitor, with the proviso that said caspase- 8 inhibitor is not a pan-caspase inhibitor and does not inhibit other caspases , is used as an antiviral agent , for use for the treatment of viral infections .
Preferably, the selective caspase- 8 inhibitor, with the proviso that said caspase- 8 inhibitor is not a pan-caspase inhibitor and does not inhibit other caspases is used as a host-directed antimicrobial . The present invention concerns a selective caspase-8 inhibitor, with the proviso that said caspase-8 inhibitor is not a pancaspase inhibitor and does not inhibit other caspases, for use in the treatment of cancer.
The present invention concerns a selective caspase-8 inhibitor, with the proviso that said caspase-8 inhibitor is not a pancaspase inhibitor and does not inhibit other caspases, for use in the prevention and/or treatment of septic shock.
The caspase-8 inhibitor pertaining to the present invention is selective for caspase-8, and therefore does not inhibit other caspases and is devoid of a generalized (or pan-caspase) effect on multiple caspases.
Preferably, the selective caspase-8 inhibitor, which is not a pan-caspase inhibitor and does not inhibit other caspases except for caspase-8, is any molecule, optionally displaying a peptide portion, that can bind to caspase-8 thereby preventing binding of this enzyme to its physiological substrates.
Preferably, the selective caspase-8 inhibitor, which is not a pan-caspase inhibitor and does not inhibit other caspases except for caspase-8, is selected from the group consisting of: small molecule, antibody, antibody-derived molecule, nucleic acid inhibitor, anti-sense mRNA, small interfering RNA, substance capable of decreasing caspase-8 production, substance capable of increasing caspase-8 degradation, substance capable of sequestering caspase-8, substance capable of preventing the correct folding of caspase-8.
Preferably, the selective caspase-8 inhibitor, which is not a pan-caspase inhibitor and does not inhibit other caspases except for caspase-8, is a small molecule with a molecular weight lower than 5.000 daltons, more preferably is a small molecule with a molecular weight comprised between 100 and 5,000 daltons.
Preferably, the selective caspase-8 inhibitor, which is not a pan-caspase inhibitor and does not inhibit other caspases except for caspase-8, is a tetrapeptide.
More preferably, the selective caspase-8 inhibitor, which is not a pan-caspase inhibitor and does not inhibit other caspases except for caspase-8, is selected in a group consisting of: z- lETD-fmk, molecules containing the IETD, DEVD, WEHD, or VDVAD peptide sequences, cFLIP short (CASH beta) , cFLIP long (CASH alpha) , caspase-8 associated RING proteins and caspase-10 associated RING proteins (CARPs) .
Preferably, infections are caused by pathogens selected from the group consisting of viruses, bacteria, mycetes, other eucariotic pathogens o by any other pathogen described in Topley and Wilson's Microbiology and Microbial Infections", 8 Volume Set, Wiley, 10th Edition", ISBN-10-0470 -68638-3, incorporated herein by reference in its entirety.
Preferably, infections are those described in Topley and Wilson's Microbiology and Microbial Infections", 8 Volume Set, Wiley, 10th Edition", ISBN-10-0470 -68638-3, incorporated herein by reference in its entirety.
In a preferred form, infections bacterial peritonitis, bacterial pneumonia, and sepsis.
In another preferred form, infections are caused by extracellular pathogens.
More preferably, extracellular pathogens are selected from a group consisting of: Staphylococcus aureus, Staphylococcus epidermidis , Staphylococcus spp , Streptococcus pyogenes , Streptococcus agalactiae , Streptococcus pneumoniae , Streptococcus spp, Enterococcus spp, Clostridium perfringens , Clostridium spp . , Actinomyces israelii , Actinomyces spp, Escherichia coli , Klebsiella pneumoniae , Pseudomonas aeruginosa, Neisseria gonorrheae , Serratia spp, Proteus spp, Campylobacter spp, Neisseria meningitidis , Neisseria spp, Moraxella spp, Haemophylus influenzae , Haemophylus spp, Acinetobacter spp, Helicobacter pylori .
Preferably, cancer is one of those described in "Encyclopedic Reference of Cancer" , Manfred Schwab, Editor, Springer-Verlag, ISBN : 978-3-540-30683-2 , incorporated herein by reference in its entirety .
In a preferred form, cancer is selected from the group consisting of : lymphoma, leukemia, carcinoma, adenocarcinoma, teratocarcinoma, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma .
The invention also concerns a pharmacological composition comprising as an active principle at least one selective inhibitor of caspase- 8 , which is not a pan-caspase inhibitor and does not inhibit other caspases except for caspase- 8 , and pharmaceutically acceptable vehicles and or excipients .
In a form of implementation of the herein described invention, the pharmaceutical composition may optionally comprise an additional active principle selected from a group consisting of an antimicrobial agent or a chemotherapeutic antineoplastic agent .
Pharmaceutical preparations applicable in the herein described invention can be made according to conventional methods and techniques that are common practice in the pharmaceutical industry, such as, for example, those illustrated in Remington's Pharmaceutical Science Handbook, Mack Pub. NY - last edition.
In some embodiments, pharmaceutically acceptable vehicles and/or excipients are included as useful formulation adjuvants, including for example solubilizing agents, dispersing agents, suspension agents and emulsifying agents.
In other embodiments, the pharmaceutical composition contains a pharmaceutically acceptable vehicles, suitable for administering the active principle. Said vehicles include antibodies and other polypeptides, genes, and other delivery agents such as liposomes, microparticles and nanoparticles on condition that such agents do not induce the production of harmful antibodies and can be administered without causing undue toxicity.
In some embodiments, suitable vehicles are selected from a group consisting of large slowly metabolized macromolecules including proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers and inactive virus particles .
For an in-depth discussion on pharmaceutically acceptable vehicles reference is made to Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991) .
In other embodiments, pharmaceutically acceptable vehicles include, in addition, liquids, such as water, saline, glycerol and ethanol.
Such compositions may contain auxiliary agents such as wetting agents emulsifying agents, pH buffers and similar. Such vehicles allow the formulation of pharmaceutical compositions such as tablets, pills, sugar-coated tables, capsules, liquids, gels, syrups, doughs, suspensions and similar forms suitable for ingestion .
The compounds and pharmaceutical compositions pertaining to the present inventions are administered in therapeutically effective doses .
Therapeutically effective doses will be generally determined by a doctor based on circumstances, including conditions to be treated, administration route, administered compound, drug combination, age, body weight, response to therapy, severity of symptoms and similar. For any given compound the therapeutic dose can be initially estimated in cell culture assays, in animal models including those involving mice, rats , guinea pigs, rabbits dogs and pigs. Animal models can be utilized to determine the appropriate dose range and administration route. Such information can then be used to determine doses and routes useful for administration to humans. In calculating human equivalent doses (HED) reference is made to the conversion table included in the document entitled: Guidance for Industry and Reviewers (2002, U.S. Food and Drug Administration, Rockville, Maryland, USA) . Preferably the therapeutically effective doses is between 3 and 12 mg/kg and more preferably is 6 mg/kg.
Pharmaceutical compositions can be administered by various routes, including but not limited to, oral, intravenous, intramuscular, intramedullary, intrathecal, intraventricular, transdermal, transcutaneous, subcutaneous, intraperitoneal, intranasal, enteral, topic, sublingual, intravaginal or rectal.
A pharmaceutical composition suitable for oral administration can take the form of loose liquid solutions or suspensions or loose powders. More commonly, compositions are presented in a unitary form to facilitate accurate dosage. The term unitary pharmaceutical dosage form refers to physically discrete units that are useful as unit doses for human individuals or other mammals, each unit containing a predetermined amount of active material calculated as to produce the desired therapeutic effect, in association with a suitable excipient. Typical unitary dosage forms include vials or pre-loaded syringes, predosed liquid compositions, or pills tablets, capsules and similar forms in case of solid compositions. The compound may be given in a single dose or in a multiple dose program.
EXAMPLES
Materials and methods
Six- to eight-week-old C57BL/6 and GDI wild-type (WT) female mice were obtained from Charles River Laboratories. Although data presented here were obtained with female mice only, sex- related differences in responses to caspase-8 inhibition were not detected in additional experiments. Caspl/11^^, Ripk3^^,
Figure imgf000026_0001
mice were all on a C57B1/6 background.
All mice were housed in individually ventilated cages under specific pathogen-free conditions in the animal facilities of the Department of Pathology of the University of Messina. The GBS WT strain H36B serotype lb was used in the present example. Streptococcus pneumoniae serotype 2 strain D39 and Klebsiella pneumoniae AC133, a carbapenem resistant strain isolated from a pneumonitis patient, were used to induce pneumonitis. GBS, S. pneumoniae and K. pneumoniae were grown in, respectively, Todd- Hewitt broth (THB) , THB supplemented with 1% (vol/vol) fetal calf serum and Luria broth to the mid-log phase at 37°C with 5% CO2, washed twice in nonpyrogenic PBS (0.01 M phosphate, 0.15 M NaCl [pH 7.4] ) , and resuspended to the desired concentration, z- lETD-fmk (caspase-8 inhibitor) , z-YVAD-fmk (caspase-1/11 inhibitor) and z-VAD-fmk (pan-caspase inhibitor) were dissolved in dimethysulfoxide (DMSO) to a 25 mM concentration. In these examples, z-IETD-fmk was given to mice i.p. at a dose of 6 mg/kg of body weight.
In preliminary experiments it was determined that this dose results in inhibition of caspase-8, but not caspase-1, as detected using luminescent enzyme assays (Promega) in peritoneal cells obtained from in vivo treated mice. Endotoxin contamination in inhibitor preparations was detected using the Pyrochrome amebocyte lysate test. To induce GBS infection, mice were challenged i.p. with 5xl07 CFU, or i.v. with 5xl06 CFU. Endotoxic shock was induced by an i.p. injection of LPS (40 mg/kg of body weight) .
To induce pneumonitis, mice were challenged with S. pneumoniae (1 x 108 CFU/mouse) or K. pneumoniae (5 x 107 CFU/mouse) by the intranasal route.
Peritoneal lavage fluid (PLF) , blood and organ homogenates were obtained and analyzed for CFU numbers, cell counts and cytokine determinations by commonly used methods. Peritoneal lavage fluid was obtained by injecting 2 ml of buffered saline in the peritoneal cavity and subsequently aspirating a total of 1.7- 1.9 ml of fluid. Unconcentrated PLF samples were used to measure cytokine levels. After challenge, animals were observed for the development of clinical signs.
Disease severity was assessed using a scoring system (mouse clinical assessment score for sepsis or M-CASS) based on predefined clinical criteria and humane endpoints. Animal showing signs of irreversible disease underwent euthanasia. Bone marrow derived neutrophils and macrophages were obtained from the femurs and tibias of 6-8-week-old female mice. Purity of neutrophil preparations was > 97%, as assessed by flow cytometry. Peritoneal cells were obtained from peritoneal lavage fluid (PLF) samples by centrifugation at 400 xg for 15min. Relative proportions of various cell types in a representative sample are reported in Table S2.
For in vitro stimulation experiments, cells were seeded in microtiter plates at a concentration of 5 x 105 per well in 0.2 ml of RPMI medium with 10% fetal calf serum. When indicated, peritoneal cells were pre-treated in vitro with recombinant IFN- p (10 pg/ml) at 2 h before the addition of z-IETD-fmk (50 pM) or vehicle. Cells were then cultured for the indicated length of time in the presence of z-IETD-fmk or vehicle.
Cytokine and mRNA levels were measured in culture supernatants and cell pellets, respectively, by ELISA and Real Time-PCR as described below. Samples were assayed using the Pierce LDH cytotoxicity assay kit (Thermo Fisher Scientific) or for cytokine/chemokine concentrations using the following assays (all from R&D Systems) : Proteome Profiler Mouse Cytokine Array Kit; CXCL1/KC DuoSet; CXCL2/MIP-2 DuoSet; TNF-a DuoSet; IL-ip DuoSet; IL-l" DuoSet; IL-18 DuoSet. The lower detection limits of these assays were 15.6 (IL-lp, IL-la, CXCL1 and 2) , 31.3 (TNF- a) and 46.9 pg/ml (IL-18) .
In selected experiments, cytokines were measured in PLF or peritoneal cell cultures obtained from neutrophil-depleted mice. Neutrophil depletion was achieved by i.v. injection of 100 pg of rat monoclonal anti-mouse Ly-6G Ab (clone 1A8 ) or rat IgG2a control (isotype control) at 24 h before z-IETD-fmk (6 mg/kg, i.p.) or vehicle treatment for 4 h in the absence of other stimuli. Under these conditions, anti-Ly6G was sufficient to reduce neutrophil blood and peritoneal counts to <1% at 24 h after treatment. For gene expression measurements, total RNA was extracted from 4 xlO6 PLF cells, bone marrow neutrophils or macrophages and retrotranscribed . Expression of the genes encoding IL-lp, Cxcll, Cxcl2, CxcllO, IL-6, IFN-p, IL12b and TNF-a was determined by qPCR using a CFX96 Touch Real-Time PCR Detection System (Bio-Rad) as described in: Biondo, C. et al. MyD88 and TLR2, but not TLR4, are required for host defence against Cryptococcus neo f ormans . European journal of immunology 35, 870-878, doi : 10.1002 /e j i .200425799 (2005) . For protein analysis peritoneal cell lysates were collected from mice at 4 h after i.p. injection with z-IETD-fmk or vehicle, washed three times with ice-cold PBS and lysed by vigorous vortexing in RIPA lysis buffer [50 mM Tris -HC1, pH 7.5, 100 mM NaCl, 1% Triton X- 100, 20% glycerol, lx protease inhibitor cocktail] . Lysates were then centrifuged at 13, 000 x g for 15 min at 4°C to eliminate cellular debris. Protein concentration in each sample was determined using the Micro BCA Protein Assay Kit. Protein samples (30 pg of protein per lane) were run on precast Bolt Bis-Tris 4- 12% gels with lx MOPS buffer and transferred on PVDF (polyvinylidene difluoride) membranes. Membranes were washed in TBS-T (Tris Buffered Saline with 0.1% Tween-20) and blocked with TBS-T containing 5% bovine serum albumin (BSA) for 2 h. Membranes were subsequently incubated with primary antibodies in TBS-T containing 1% BSA at 4 °C overnight. The following primary antibodies were used: phospho-RIP (Serl66) (E7G6O) rabbit mAb, RIP (D94C12) XP rabbit mAb, anti-mouse IL-1 beta/IL-lF2 antibody and anti-beta actin. After incubation, membranes were washed with TBS-T and incubated with secondary antibody (anti-rabbit or anti-goat IgG HRP-linked antibodies) for 2 h at room temperature in TBS-T containing 1% BSA. Protein bands were visualized by Immobilon Forte Western HRP substrate and detected using a BioRad's ChemiDoc XRS system. Beta-actin was used as loading control. For immuno-staining and flow cytometric analysis, peritoneal cells were collected from mice at the indicated times after various treatments, washed three times with DPBS and stained in the dark for 20 minutes with LIVE/DEAD Fixable Aqua Dead Cell Stain Kit, according to the manufacturer's instructions. Cells were then blocked with 0.5 pg Fc Block for 20 minutes at room temperature and stained for surface markers for 20 minutes in the dark with rat anti-mouse Ly-6G (clone 1A8 ) , rat anti-mouse F4/80 (Clone BM8) , anti-mouse pro-IL-1 beta (clone NJTEN3) or isotype control monoclonal antibodies. For intracellular staining, the Intracellular Fixation & Permeabilization Buffer Set ( eBioscience ) was used, following the manufacturer's instruction. Briefly, cells were incubated for 30 min in the dark with IC Fixation Buffer and washed twice with Permeabilization Buffer IX (Perm Buffer) . Cells were then stained with the anti-mouse IL-1 beta (Pro-form) Monoclonal Antibody (clone NJTEN3) APC for 30 min in the dark at 4°C. Following two washes with Perm Buffer, cells were resuspended in PBS and 100,000 events per sample were collected on a FACS Canto II flow cytometer (BD Biosciences) . Data analysis was performed using FlowJo version 10 software. For enumeration of peritoneal cells, BD Trucount Absolute Counting Tubes (BD Trucount Absolute Counting Tubes; BD Biosciences) . Survival data were analyzed by Kaplan-Meier survival plots. All other data were analyzed by the Mann-Whitney test using GraphPad Prism 5.0 (GraphPad Software, Inc., San Diego, CA) . Differences were considered significant when P values were less than 0.05.
Experimental results
Because genetic inactivation of caspase-8 can produce inflammation in various types of tissues, it was ascertained whether pharmacological caspase-8 inhibition can augment neutrophil responses in the context of bacterial infection and promote pathogen clearance. To test this, mice were given the caspase-8 inhibitor z-IETD-fmk at a dose that was found in preliminary experiments to inhibit in vivo activation of caspase-8, but not caspase-1 (see Methods) . After 4 h, mice were challenged i.p with a highly lethal dose of group B streptococcus (GBS) , an important agent of sepsis and meningitis that has been used over the years to model anti-bacterial innate immune responses. In control animals, which consisted of mice treated with saline or with the DMSO vehicle, GBS rapidly grew during the first hour post-challenge and persisted at elevated numbers in the peritoneal cavity (Fig. 1) . In contrast, bacterial numbers quickly declined in animals treated with z-IETD-fmk, reaching levels that were 4 to 5 orders of magnitude lower than those of control mice (Fig. 1) . Inhibitor-treated animals completely cleared infection by 5 h post-challenge and remained in good health thereafter, while all control animals showed signs of irreversible disease and were humanely euthanized. The protective effect of z-IETD-fmk was not due to direct antibacterial activity since this compound did not affect in vitro GBS growth at concentrations up to 2 mg/ml. The early decline in bacterial numbers observed in lETD-treated mice was coincident in timing with the influx of neutrophils into the peritoneal cavity (Fig. 2) and the release of proinf lammatory chemokines and cytokines, such as interleukin Ip (IL-lp; Fig. 3) , in the absence of increased cell death. Since IETD may crossinhibit other caspases in addition to caspase-8, we compared the z-IETD-fmk effects with those of other caspase inhibitors, such as the pan-caspase inhibitor z-VAD-fmk, and the caspase-1 inhibitor z-YVAD-fmk. However, both z-VAD-fmk and YVAD-fmk were ineffective at reducing bacterial burden or at increasing neutrophil influx. Collectively, these data indicate that treatment with the caspase-8 inhibitor z-IETD-fmk, but not with pan-caspase or caspase-1 inhibitors, potentiates the production of pro-inflammatory cytokines and chemokines in a cell deathindependent manner, resulting in increased neutrophil recruitment and containment of lethal bacterial infection. We next investigated whether z-IETD-fmk could produce proinf lammatory changes by itself, in the absence of infection or other external stimuli, by analyzing PLF samples obtained at different times after i.p. inoculation with the compound. Significant cytokine/chemokine elevations were measured as early as 1 (Cxcll and 2) , 2 (IL-lp and IL-18) and 3 h (IL-la) after treatment (for IL-lp levels, see Fig. 4) , while their levels were low or undetectable in samples from vehicle-treated mice or from mice treated with the pan-caspase inhibitor z-VAD-fmk. Cytokine production occurred concomitantly with neutrophil influx in the peritoneal cavity (Fig. 5) and in the absence of detectable cell death since no significant decrease in peritoneal cell viability occurred over 5 h after administration of z-IETD-fmk. Cytokine appearance was preceded by activation of selected cytokine genes and was concomitant in timing with neutrophil recruitment into the peritoneal cavity. These effects were not due to endotoxin contamination since endotoxin levels in the z-IETD-fmk preparations employed were <0.01 EU/ml. Further analysis of PLF supernatants using a protein array revealed that z-IETD-fmk induced the release of a range of chemokines, cytokines and growth factors, including CCL2, CCL12, Cxcll/2/10/12/13, IL-16, IL-17, G-CSF and M-CSF, as well as the complement component C5a and the metalloproteinase inhibitor TIMP-1 (Fig.6) . These data indicate that z-IETD-fmk administration is by itself sufficient to induce transcriptionally regulated inflammatory changes in vivo, including the production of a distinctive pattern of proinf lammatory cytokines and neutrophil-attracting chemokines, as well as neutrophil recruitment. Next, it was of interest to assess whether z-IETD-fmk could induce cytokine production not only in vivo but also in vitro. Indeed, significant Cxcll and IL-lp elevations were detected in unseparated peritoneal cells cultured overnight in the presence of 50 pM z-IETD-fmk and in bone marrow cells, albeit at lower levels than in vivo. However, Cxcll or IL-lp production could not be induced by z-IETD-fmk treatment in cultures of mouse macrophages isolated from a variety of sources, in macrophage cell lines or in mast cells. Notably, increased transcription of IL-lp and TNF-a genes and cytokine release was detected in murine bone marrow neutrophils, but not in macrophages, in the presence of z-IETD-fmk. Moreover, production of CXCL8 and MIP-la was detected in human blood after addition of z-IETD-fmk.
Since neutrophils spontaneously undergo apoptosis during in vitro culture, it was of interest to ascertain whether perturbed patterns of cell death might have contributed to the IETD effects. Culture for 24 h in the presence of z-IETD-fmk slightly increased spontaneous cell death in neutrophils. This increase in cell death was not due to necroptosis since it was not prevented by lack of the pseudokinase MLKL, the essential executioner of necroptosis. Increased cell death was not responsible for lETD-induced cytokine release, since the latter could be observed even at times (e.g., at 6-8 h after stimulation) at which cell viability was high and similar in z- lETD-fmk- and vehicle-treated neutrophils.
In further experiments, we found that neutrophils were required for lETD-induced in vivo production of cytokines and chemokines, since significant cytokine elevations could not be detected in mice depleted of neutrophils by anti-Ly6G antibody treatment prior to i.p. injection with z-IETD-fmk. To gain further insights into the cell types and mechanisms involved in z-IETD-fmk- induced cytokine production, we stained peritoneal cells obtained at various times after i.p. administration of z-IETD- fmk for intracellular immunoreactive IL-lp, used as a marker for cytokine-producing cells. Immunoreactive ILlp was detected in 1- 2% of peritoneal cells obtained from mice inoculated with vehicle (Fig. 7 and 8) , PBS or from untreated mice (data not shown) . After z-IETD-fmk treatment, the number of IL-lp-producing cells increased concomitantly with neutrophil influx, and at 4 h neutrophils and macrophages represented 60-70 and 10-20%, respectively, of the IL-lp-producing cells (Fig. 7) . Collectively these data indicate that neutrophils are activated both in vitro and in vivo by z-IETD-fmk and represent most of the cells producing IL-lp cells after i.p. treatment with the inhibitor. Next, we aimed at obtaining insights into the molecular mechanisms underlying z-IETD-fmk-induced inflammation using mice lacking key signaling proteins involved in cytokine responses and programmed cell death. First, we sought to formally confirm that IETD produces its effects by specifically acting on caspase-8. To this end, we used mice lacking both caspase-8 and MLKL, the essential executioner of necroptotic cell death, since the isolated absence of caspase 8 is embryonically lethal due to uncontrolled necroptosis. Notably, lETD-induced neutrophil recruitment and IL-lp production were completely abrogated in mice lacking both caspase-8 and MLKL, but not in those lacking just MLKL, confirming that the IETD effects are specifically linked to inhibition of caspase-8. Next, we showed that Tolllike receptors (TLRs) and their agonists, which can potently induce proinf lammatory changes, are not involved in the z-IETD- fmk effects, as evidenced by robust responses to the inhibitor in mice lacking the TLR adaptors Myd88 or TRIE. Since expression of enzymatically inactive caspase-8 is sufficient to activate a caspase-l/ll-dependent inflammasome in the intestine, we next investigated whether combined deletion of caspase-1 and caspase- 11 would affect z-IETD-fmk-induced responses. The absence of caspase-1 and 11, but not NLRP3, led to significant reductions in IL-lp and IL-18 levels as well as in the numbers of neutrophils and IL-lp-producing cells after z-IETD-fmk treatment. Moreover, transcription of pro-ILl-p mRNA was significantly decreased in peritoneal cells lacking caspase-1/11 after z-IETD-fmk treatment. Partially processed IL-lp was detected in peritoneal cells from wild type mice after i.p. administration of z-IETD-fmk and such processing was reduced in caspase-1/11 double KO animals. Residual IL-lp processing in these animals after z-IETD-fmk treatment was possibly related to the activities of neutrophil serine proteases. Notably, z-IETD administration was unable to reduce bacterial burden in GBS- challenged caspase-1/11 double KO mice, and co-administration of the caspase 1/11 inhibitor z-YVAD-fmk in wild type mice abrogated the beneficial effects on infection observed when z-IETD-fmk was given alone.
Collectively, these data indicate that caspase-1, 11 or both, but not NLRP3, make a significant contribution to the inflammatory changes induced by z-IETD-fmk in vivo. Moreover, abrogation of the protective effects of z-IETD-fmk by caspase-1 inhibition provided an explanation for the previously observed ineffectiveness of pan-caspase inhibition to promote bacterial clearance .
Next, we asked whether necroptosis, which requires RIPK1- dependent activation of RIPK3 and MLKL, was involved in the in vivo inflammatory response triggered by caspase-8 inhibition. Phosphorylation of RIPK1 was detected by western blot in lysates of peritoneal cells obtained after i.p. administration of z- lETD-fmk. Pre-treatment with the RIPK1 kinase-inhibitor necrostatin-1 significantly attenuated lETD-induced pro- inflammatory changes, while these were completely abrogated by the absence of RIPK3 (Fig. 8) .
In contrast, the number of IL-lp-positive cells (Fig. 8) and pro-IL-lp transcription were increased in necroptosis-deficient mice lacking MLKL . Because several type I interferon-dependent genes become activated synchronously with the IL-lp gene during neutrophil development, we asked whether z-IETD-induced IL-lp transcription depended on type I interferon production.
This seemed indeed to be the case, since transcription of IL- lp, as well as Cxcll, was totally abrogated in peritoneal cells obtained from IFN-p“/_mice after i.p. treatment with z-IETD-fmk and this effect was reversed by treatment with recombinant IFN- p. Moreover, baseline levels of mRNA transcripts encoding for caspase-8, caspase-1 and RIPK1, but not RIPK3, were reduced in cells lacking IFN-p and such levels were increased by IFN-p treatment .
Collectively these data indicate that the proinf lammatory effects of z-IETD-fmk are driven by a RIPK3-dependent mechanism that is partially kept under control by MLKL. Moreover, responsiveness to z-IETD-fmk depends on tonic IFN-p production, which might be required for maintaining baseline levels of RIPK1, caspase-8 and caspase-1 expression.
Excessive proinf lammatory cytokine production can have detrimental consequences for the outcome of infections, particularly in the context of sepsis. Since z-IETD-fmk treatment induced here robust proinf lammatory cytokine responses, we ascertained whether such treatment is detrimental in septic shock models. LPS, or endotoxin, is a crucial contributor to septic shock caused by gram negative bacteria in humans, and this shock form can be modeled in mice by high-dose LPS challenge.
Strikingly, z-IETD-fmk treatment largely prevented hypothermia and death induced by high-dose endotoxin, despite producing moderate elevations in TNF-a and IL-p blood levels (Fig. 9) . Similarly, z-IETD-fmk treatment significantly protected mice in a gram positive shock model involving the i.v. administration of live GBS bacteria, by inducing moderate elevations in cytokine blood levels and reducing bacterial burden.
Notably, z-IETD-fmk treatment did not ameliorate lethality or increase circulating cytokine levels in mice lacking Caspl/11 or RIPK3, indicating that these molecules are required for the therapeutic effects of IETD.
Exogenous administration of recombinant IFN-p moderately ameliorated GBS-induced sepsis in wild type mice, in general agreement with the notion that IFN-p promotes host defenses against GBS and other extracellular bacteria. However, IFN-p treatment was ineffective in Caspl/11 or RIPK3 KO mice, indicating that these proteins are involved the therapeutic activities of the cytokine.
Excessive inflammatory reactions can be dangerous not only in the context of septic shock but also during pneumonia, since the presence of exudate in the alveolar spaces can hinder gas exchange resulting in respiratory insufficiency. Therefore, we assessed whether z-IETD-fmk might be detrimental for the outcome of pneumonia using a model of infection by Streptococcus pneumoniae, the main cause of community-acquired pneumonia worldwide .
Mice were challenged withl xlO8 CFU of the serotype 2 D39 reference strain by the intranasal route and, after 24 h, were treated daily with z-IETD-fmk (6 mg/kg i.v.) or vehicle. Under these conditions, 62% of the mice receiving vehicle showed signs of irreversible disease and were humanely euthanized within 5 days, while only 12% of z-IETD-fmk-treated animals succumbed to infection (p<0.05; Fig. 10) . A significantly lower bacterial burden was detected in z-IETD-fmk-treated mice compared with control animals. Moreover, z-IETD-fmk had similar protective activities in a model of pneumonia caused by carbapenem-resistant Klebsiella pneumoniae . Collectively, our data indicate that z- lETD-fmk administration produces marked protective effects in models of septic shock or invasive pneumonia caused by partially or extremely antibiotic-resistant pathogens.
To summarize, data presented here suggest that caspase-8 suppresses a proinf lammatory program that is spontaneously activated in neutrophils, depends on RIPK3, and is sustained by tonic IFN-p production. Accordingly, it was found that exposure of bone marrow neutrophils to a caspase-8 inhibitor is sufficient to induce the production of pro-inflammatory cytokines at both the mRNA and protein level in the absence of other external stimuli .
Moreover, intraperitoneal injection of the inhibitor induced marked neutrophil recruitment and the appearance of a characteristic pattern of chemokines and cytokines known to be potentially released by neutrophils upon activation. This distinctive cytokine signature included several members of Cxcl chemokine family, CCL2, IL-17, TIMP-1, and IL-lp, while TNF- a/IL-12 responses were less pronounced. In addition, neutrophils were found to represent most of the IL-lp-producing cells and to be required for cytokine elevations in lETD-induced peritoneal exudates, as shown by, respectively, immunofluorescence and in vivo neutrophil depletion experiments.
The caspase-8 inhibitor z-IETD-fmk was carefully titrated here in comparison with other fmk-based inhibitors, to provide maximal selectivity. Under the conditions used, the proinf lammatory changes induced by z-IETD-fmk were due to specific caspase-8 inhibition, since they were completely absent in animals lacking caspase-8. Moreover, these effects depended on the kinase activity of RIPK1 and on RIPK3, which are both substrates of caspase-8 but not of other caspases. Therefore, evidence obtained from multiple approaches indicates that the effects of z-IETD-fmk at the used doses are due to selective inhibition of caspase-8. Moreover, although completely dependent on the presence of RIPK3, the mechanisms unleashed by selective caspase-8 inhibition are independent from MLKL, the essential executioner of necroptosis, and occur in the absence of other forms of cell death. Permanent deletion or inactivation of caspase 8 can produce detrimental effects in genetically modified mice. However, according to the present invention, temporary inhibition of caspase-8 had no detrimental effects and ameliorated the outcome of lethal infections due to augmented neutrophil-mediated bacterial clearance.
Indeed, during infection, host-protective neutrophil influx is triggered by Cxcll and Cxcl2 and sustained by neutrophil-derived Cxcl2 and IL-lp production in a sequence that could be recapitulated here by specific caspase-8 inhibition even in the absence of bacterial stimulation. According to the present invention, the ability of specific caspase-8 inhibition to prevent lethality in sepsis models is due not only to increased host defenses, but also to attenuation of the indirect toxic effects of bacterial agonists. Indeed, treatment with z-IETD- fmk prevented here lethality in mice challenged with high-dose endotoxin, suggesting that caspase-8 has an important role in driving lethality during sepsis. Thus, according to the present invention, selective caspase-8 inhibition augments the production of pro-inflammatory cytokines while, at the same time, preventing their toxic effects. Therefore, the proinf lammatory program described according to the present invention can be harnessed against dif f icult-to-treat bacterial diseases, such as those caused by antibiotic-resistant pathogens. Of note, according to the present example, the pancaspase inhibitor z-VAD-fmk was ineffective against bacterial infections that were effectively prevented by z-IETD-fmk under conditions resulting in selective caspase-8 inhibition. In conclusion, these data underscore the importance of selectively targeting caspase- 8 in anti-infectious strategies aiming at potentiating host defenses and demonstrate the ef ficacy of such an approach against potentially lethal infections .

Claims

1. A selective inhibitor of caspase-8, with the proviso that said inhibitor of caspase-8 is not a pan-caspase inhibitor and does not inhibit other caspases, for use as antimicrobic agent in the treatment of bacterial infections.
2. The selective inhibitor of caspase-8, with the proviso that said inhibitor of caspase-8 is not a pan-caspase inhibitor and does not inhibit other caspases, for use according to claim 1 wherein bacterial infection is selected from the group consisting of: bacterial peritonitis, bacterial pneumonia and sepsis.
3. A selective inhibitor of caspase-8, with the proviso that said inhibitor of caspase-8 is not a pan-caspase inhibitor and does not inhibit other caspases, for use as antimicrobic agent in the treatment of viral infections.
4. A selective inhibitor of caspase-8, with the proviso that said inhibitor of caspase-8 is not a pan-caspase inhibitor and does not inhibit other caspases, for use in the treatment of cancer.
5. A selective inhibitor of caspase-8, with the proviso that said inhibitor of caspase-8 is not a pan-caspase inhibitor and does not inhibit other caspases, for use for in the prevention and/or treatment of septic shock.
6. The selective inhibitor of caspase-8, with the proviso that said inhibitor of caspase-8 is not a pan-caspase inhibitor and does not inhibit other caspases, for use according to anyone of claims 1-5 being a host-directed agent.
7. The selective caspase-8 inhibitor for use according to any one of claims 1-6 wherein the selective caspase-8 inhibitor is not a caspase-11 inhibitor and is not a caspase-1 inhibitor .
8. The selective caspase-8 inhibitor for use according to any one of claims 1-6 wherein the selective caspase-8 inhibitor is any molecule, optionally having a peptide moiety, capable of binding to caspase-8 preventing its binding to physiological substrates.
9. The selective caspase-8 inhibitor for use according to claim 8 wherein the selective caspase-8 inhibitor is selected from the group consisting of: small molecule, antibody, antibody-derived molecule, nucleic acid inhibitor, Antisense mRNA, small interfering RNA, substance that can decrease the production of caspase-8, substance that can enhance the degradation of caspase-8, substance that can sequester caspase-8 or prevent its correct folding .
10. The selective caspase-8 inhibitor for use according to claim 9 wherein the selective caspase-8 inhibitor is a small molecule with a molecular weight of less than 5,000 daltons.
11. The selective caspase-8 inhibitor for use according to any one of claims 1-6 wherein the selective caspase-8 inhibitor is administered in a therapeutically effective dose between 3 and 12 mg/kg.
12. The selective caspase-8 inhibitor for use according to claim 11 wherein the selective caspase-8 inhibitor is administered in a therapeutically effective doses of 6 mg/kg .
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