WO2016127255A1 - Mediation of inflammatory response using inhibitors of netosis - Google Patents

Mediation of inflammatory response using inhibitors of netosis Download PDF

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Publication number
WO2016127255A1
WO2016127255A1 PCT/CA2016/050123 CA2016050123W WO2016127255A1 WO 2016127255 A1 WO2016127255 A1 WO 2016127255A1 CA 2016050123 W CA2016050123 W CA 2016050123W WO 2016127255 A1 WO2016127255 A1 WO 2016127255A1
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netosis
disease
inhibitor
inflammatory response
neutrophil
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PCT/CA2016/050123
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French (fr)
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Palaniyar Nadesalingam
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Palaniyar Nadesalingam
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    • G01N33/5047—Cells of the immune system

Definitions

  • the present disclosure relates to the treatment of diseases associated with an inflammatory response, such as inflammatory diseases and autoimmune diseases.
  • NETs help to trap infectious agents, they may have several negative effects on the host. Hence, the NETs formed in the process of NETosis may be responsible for some of the detrimental effects of inflammatory conditions.
  • WO2012/065720 A1 to Martinez et al. describes a neutrophil viability assay to screen compounds for NET inhibition.
  • US2013/0183662 A1 to Zychlinsky et al. uses NET degradation as a determinant in a diagnostic method for subjects with systemic lupus erythematosus to determine risk of developing renal manifestations.
  • WO2014/144572 A2 to Jin et al. describes a patient screening method to identify the occurrence of NETosis using biomarkers.
  • a commercial package comprising an inhibitor of NETosis together with instructions for use in treating a disease associated with an inflammatory response in a subject having the disease.
  • PHA767491 DNA helicase inhibitor
  • Etoposide Topicoisomerase II inhibitor
  • ACTD Actinomycin D
  • Figure 5 shows NETotic Index over time for PMA/PMA+DPI (panel A);
  • LPS/LPS+SP6 panel B
  • H2O2/H2O2+SP6 indicating that LPS and H 2 0 2 -induced NETosis were significantly suppressed by the JNK inhibitor SP6 in the plate reader assay.
  • DMSO vehicle control did not exert a significant effect on extracellular DNA release.
  • Figure 8 shows confocal images of NETs under different pH induced by
  • X-axis time (min).
  • Y-axis Sytox Green Fluorescence units indicating NETosis.
  • Figure 10 shows that nucleases present in the BAL fluid of LPS-instilled mice require cations such as calcium and magnesium for activity.
  • Figure 11 shows that nucleases of inflamed airways have two pH optimums.
  • the present disclosure relates to mediating the inflammatory response, and treating diseases associated with an inflammatory response, such as inflammatory diseases and autoimmune diseases.
  • diseases associated with an inflammatory response such as inflammatory diseases and autoimmune diseases
  • Many diseases associated with an inflammatory response such as inflammatory diseases and autoimmune diseases, have negative effects relating to or resulting from the inflammatory response of neutrophil infiltration. These negative effects can be mediated according to the methods described herein.
  • a subject having the disease associated with an inflammatory response such as an inflammatory disease or autoimmune disease, can benefit.
  • NOX NADPH oxidase, ROS, reactive oxygen species
  • CGD chronic granulomatous disease
  • DPI diphenyleneiodonium
  • PMA phorbol 12-myristate 13- acetate
  • MPO myeloperoxidase
  • FCCP carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone
  • DNP dinitrophenol
  • PAD peptidyl arginine deiminase
  • ERK extracellular-signal-regulated kinase
  • Akt Protein kinase B or PKB.
  • a method of treating a disease associated with an inflammatory response such as an inflammatory disease or autoimmune disease, is described herein, which comprises administering an inhibitor of NETosis to a subject having the disease.
  • the method may permit inhibition of NETosis, while permitting one or more other neutrophil immune functions to be maintained.
  • NETs cause or exacerbate inflammation and tissue damage.
  • the treatment described herein targets the reduction or inhibition of NETosis, and thus can be used to address and treat inflammatory or autoimmune diseases.
  • diseases include infectious diseases, lung infection, pneumonia, cystic fibrosis, neutrophilic asthma, neutrophil-mediated anaphylaxis, chronic obstructive pulmonary disease (COPD), cardiovascular diseases, vasculitis, blood clot-related diseases such as thrombosis, acute respiratory distress syndrome, nephritis, autoimmune diseases such as lupus or arthritis, such as rheumatoid arthritis, and diseases resulting in inflammation.
  • JNK inhibitor SP600125 can suppress NETosis induced by LPS or H 2 0 2 or A23 or ionomycin. These and other inhibitors targeting these kinases may permit the suppression of NETosis while maintaining other important immune functions of the neutrophils.
  • Nucleic acid synthesis is important for NETosis. Nucleic acid synthesis may be rapid in NADPH-independent NETosis than the NADPH-dependent NETosis pathway. Inhibitors of nucleic acid synthesis, such as Actinomycin D, suppress NETosis.
  • compounds for inhibiting NETosis may include, but are not limited to, Actinomycin D (ACTD), or an inhibitor of a kinase such as JNK, or an analogue or derivative of these.
  • ACTD Actinomycin D
  • JNK an inhibitor of a kinase
  • SP600125 is an exemplary inhibitor of JNK. It is understood that such analogues or derivatives would encompass those modified or derived in a manner that approximately maintains or has a greater NETosis inhibiting function when compared with the compound from which it is derived.
  • FDA-approved drugs may be utilized and identified as described herein, for inhibiting NETosis.
  • a screening approach for both types of NETosis (NADPH- dependent and -independent) may be utilized. Selected candidates can be validated and quickly recommended for clinical "drug-repurposing" trials utilizing the methods described herein.
  • Derivatives and analogues of successful screening candidates may optimize activity in suppression of NETosis, while maintaining neutrophil viability and/or one or more other neutrophil function other than NETosis (e.g., reactive oxygen species generation, phagocytic killing).
  • Pathway-specific compounds may be used for treating several, but specific diseases associated with an inflammatory response, such as inflammatory diseases, infections, or autoimmune diseases.
  • Method of Identifying Inhibitors There is described herein a method of identifying inhibitors of NETosis comprising: inducing NETosis in neutrophils; providing the neutrophils with a candidate NETosis inhibitor, and observing inhibition of reactive oxygen species (ROS) formation in the neutrophils.
  • ROS reactive oxygen species
  • Inducing NETosis may comprise exposing the neutrophils to a NETosis agonist or other compound (generally referring to large or small molecules or other activating combinations) able to induce NETosis, or may involve simply working with a population of neutrophils that are already activated through a previous route, for example if isolated from an individual in the throes of a disease associated with an inflammatory response, such as an inflammatory disease or an autoimmune disease.
  • a NETosis agonist or other compound generally referring to large or small molecules or other activating combinations
  • the compounds to be tested and identified as candidate inhibitors may be selected from among the categories of inhibitors mentioned above, and any analogues or derivative thereof, as well as from heretofore previously unknown categories of inhibitors which are otherwise identified using screening for libraries or collections of compounds.
  • NETosis may be induced by agonists such as PMA, calcium ionophores A23187 ("A23"), ionomycin, LPS, and immune complexes.
  • agonists such as PMA, calcium ionophores A23187 ("A23"), ionomycin, LPS, and immune complexes.
  • Immune complexes can induce Nox-independent NETosis. The kinetics of immune complexes may differ from that of ionophore-induced NETosis.
  • Uses are described herein for inhibitors of NETosis in treating a disease associated with an inflammatory response, such as an inflammatory disease or an autoimmune disease, in a subject having the disease. Further, a use of an inhibitor of NETosis is described for preparation of a medicament for treating a diseases associated with an inflammatory response, such as an inflammatory disease or an autoimmune disease, in a subject having the disease.
  • a composition comprising an inhibitor of NETosis and a pharmaceutically acceptable excipient is provided, for treating a disease associated with an inflammatory response, such as an inflammatory disease or an autoimmune disease, in a subject having the disease.
  • a disease associated with an inflammatory response such as an inflammatory disease or an autoimmune disease
  • Such a composition may be manufactured together with, or prescribed or used together with another active ingredient for use in addressing an inflammatory disease.
  • An inhibitor of NETosis is described for treating a disease associated with an inflammatory response, such as an inflammatory disease or an autoimmune disease, in a subject having the disease.
  • a commercial package comprising an inhibitor of NETosis together with instructions for use in treating a disease associated with an inflammatory response, such as an inflammatory disease or an autoimmune disease, in a subject having the disease.
  • an ELISA may be used to measure NETs.
  • Methods of evaluating NETs with myeloperoxidase present thereon can be used to quantify the amount of NETs DNA by incubating the reaction with a DNA binding fluorescence dye (for example: picogreen) and measuring fluorescence.
  • a DNA binding fluorescence dye for example: picogreen
  • NETosis analysis NETosis was monitored by a plate reader assay in the presence of SytoxTM Green cell-impermeable nucleic acid stain (5 ⁇ ). NETosis was confirmed by imaging the colocalization of myeloperoxidase, and DNA stained with Sytox Green after the fixation and permeablization. For some studies, Western blots were used for determining CitH3, the activation states of kinases (e.g., JNK). Inhibitors were preincubated with cells for 1 h prior to the activation of cells for NETosis.
  • FIG. 1 Figure 2 to Figure 4 show that molecules such as Actinomycin D (ACTD)
  • Figure 2 illustrates differences in the number of transcript formation
  • ROS Reactive Oxygen Species
  • PHA767491 DNA helicase inhibitor
  • Etoposide Topoisomerase II inhibitor
  • ACTD Actinomycin D
  • Figure 2 the results of a Sytox Green assay are provided, showing the degree of mRNA production (panels A and B), ROS production (panels C and D) and inhibition of NADPH-dependent (panel E) and NADPH-independent (panel F) NETosis by ACTD, Etoposide and PHA76749.
  • Figure 4 reveals additional classes of nucleotide binding and synthesis inhibitors which inhibits NETosis. These data however show that specific classes of nucleic acid binding molecules can be an effective therapeutic method for suppressing NETosis in inflammatory diseases.
  • Figure 4 provides a grey-scale representation of a color-coded imaging of screened candidates in a NETosis inhibitory heatmap of candidate drugs.
  • This greyscale representation was originally generated with blue and red scale representations (from 0 to 15), as shown in the side bar to the right.
  • the heatmap is determined by neutrophils differentiated from human HL-60 cells.
  • Nucleic acid metabolism inhibitors and nucleic acid synthesis inhibitors such as cyclocystidine hydrochloride (e.g., Cyclo-C or Idarubicin or Idamycin) inhibits NETosis.
  • cyclocystidine hydrochloride e.g., Cyclo-C or Idarubicin or Idamycin
  • the testing results obtained from the method conducted was able to identify inhibitors of NETosis by inducing NETosis in neutrophils; providing the neutrophils with a candidate NETosis inhibitor, and observing inhibition of Nox-dependent and Nox- independent NET formation in the neutrophils.
  • Serotonin pathway regulators were found to inhibit NETosis. These examples include (a) 5-HT 1 D serotonin receptor antagonist 4-(3-Chlorophenyl)-alpha- (diphenylmethyl)-l-piperazineethanol hydrochloride (e.g., BRL 15572), and (b) serotonin uptake inhibitor Methyl reserpate or 3,4,5-Trimethoxybenzoic acid ester (e.g., Reserpine).
  • 5-HT 1 D serotonin receptor antagonist 4-(3-Chlorophenyl)-alpha- (diphenylmethyl)-l-piperazineethanol hydrochloride e.g., BRL 15572
  • serotonin uptake inhibitor Methyl reserpate or 3,4,5-Trimethoxybenzoic acid ester e.g., Reserpine
  • a potent orally active D1 dopamine receptor agonist [(1 R, 3S)-3-(1-
  • a P2 purinoceptor agonist 2-(Methylthio)adenosine 5'-diphosphate trisodium salt hydrate was found to inhibit NETosis.
  • a Vanilloid receptor-1 (VR1) antagonist [(E)-3-(4-Chlorophenyl)-N-(3- methoxyphenyl)prop-2-enamide] (e.g., SB-366791) was found to inhibit NETosis.
  • kinase inhibitors were found to suppress NETosis: (a) a protein tyrosine kinase inhibitor 4,5-Dianilinophthalimide (e.g., CGP 5241 1) specific for epidermal growth factor receptor (EGFR), (b) a potent and selective vascular endothelial growth factor protein tyrosine kinase (VEGFR PTK) inhibitor 1 ,3-Dihydro-3-[(3,5-dimethyl-1 H- pyrrol-2-yl)methylene]-2H-indol-2-one (e.g., SU 5416), and (c) a diacylglycerol Kinase Inhibitor II (3-[2-[-[bis(4-Fluorophenyl)methylene]-1-piperidinyl]ethyl]-2-3-dihydro-2-thioxi- 4(1 H)-quinazolinone (e.g., R5994
  • the method of identifying inhibitors of NETosis located the following compounds from among the candidate inhibitors tested, having NETosis inhibitory activity: N-[5-(aminosulfonyl)-1 ,3,4-thiadiazol-2-yl]acetamide (Acetazolamide); N-(1-adamantyl)-N'-cyclohexyl-4-morpholinecarboxamidine hydrochloride (PNU-37883A); 4-(3-chlorophenyl)-alpha-(diphenylmethyl)-1-piperazineethanol hydrochloride (BRL 15572); reserpine; methyl reserpate; (1 R, 3S)-3-(1-adamantyl)-1-(aminomethyl)-3- 4dihydro-1 H-isochromene-5,6-diol; 2-(methylthio)adenosine 5'-diphosphate trisodium salt hydrate; 3-(4
  • JNK inhibitor SP6 inhibits NETosis
  • Figure 5 and Figure 6 indicate that LPS, H 2 0 2 , and A23-induced NETosis were significantly suppressed by the JNK inhibitor SP6 in the plate reader assay.
  • Figure 5 shows NETotic Index over time for PMA/PMA+DPI (panel A);
  • Figure 6 shows is a greyscale representation of confocal images, originally viewed in color, and illustrating the number of NETotic cells being significantly lower in the presence of the JNK inhibitor after 3 hours of stimulation for LPS, A23, ionomycin-mediated NETosis.
  • the presence or absence of SP-6 is revealed for Nox- dependent NETosis (PMA and LPS) as well as Nox-independent NETosis (A23 and ionomycin).
  • NETosis induced by PMA or LPS, Pseudomonas aeruginosa and Staphylococcus aureus
  • Nox-independent NETosis induced by A23187 (A)
  • X-axis time (min).
  • Y-axis Sytox Green Fluorescence units indicating NETosis.
  • Figure 7 illustrates the influence of various factors on NETotic Index.
  • Figure 8 shows confocal images of NETs under different pH induced by

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Abstract

A method and use is described for treating a disease associated with an inflammatory response by administering an inhibitor of NETosis to a subject having the disease. The inhibitor of NETosis may be a nucleic acid synthesis inhibitor such as Actinomycin D (ACDT) or inhibitor of a kinase such as JNK, a pH regulator, or an analogue or derivative of these. Disease associated with an inflammatory response include an infection, an infectious disease, lung infection, pneumonia, cystic fibrosis, neutrophilic asthma, neutrophil-mediated anaphylaxis, chronic obstructive pulmonary disease (COPD), cardiovascular diseases, vasculitis, a blood clot-related disease, thrombosis, acute respiratory distress syndrome, nephritis, Lupus, or arthritis, such as rheumatoid arthritis. A method of identifying inhibitors of NETosis is also described.

Description

MEDIATION OF INFLAM MATORY RESPONSE USING
INHIBITORS OF NETOSIS
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of priority of U.S. Provisional Patent
Application No. 62/1 14,396 filed February 10, 2015, which is hereby incorporated by reference.
FIELD
[0002] The present disclosure relates to the treatment of diseases associated with an inflammatory response, such as inflammatory diseases and autoimmune diseases.
BACKGROUND
[0003] Neutrophils are the first responders to invading pathogens, and play a pivotal role in the innate immune defense. Neutrophils are present in large numbers in circulation, and transmigrate to the sites of infection in response to chemotactic signals. However, upon infiltrating a site, neutrophils can exacerbate inflammation, and can damage inflamed tissues and organs. Once at the site of infection, activated neutrophils fight pathogens by various means, including oxidative burst, phagocytosis and the formation and release of neutrophil extracellular traps (NETs). Formation and release of NETs has been termed NETosis, and can ultimately result in cell death for neutrophils. NETs are made of neutrophil DNA, and are coated with anti-microbial and/or cytotoxic proteins and peptides. For a review of NETs and NETosis, see Cheng OZ & Palaniyar N (2013) Frontiers in Immunology 4, 1-13.
[0004] Although NETs help to trap infectious agents, they may have several negative effects on the host. Hence, the NETs formed in the process of NETosis may be responsible for some of the detrimental effects of inflammatory conditions.
[0005] Two distinct forms of NETosis have been described based on the requirement for NADPH oxidases 2 (NOX2). In the NOX-dependent pathway, pharmacological inhibition of NOX2 results in the inhibition of reactive oxygen species (ROS) and subsequent NET formation. Furthermore, neutrophils isolated from patients with chronic granulomatous disease (CGD) fail to form NETs for certain stimuli because these cells have deficiencies in NOX-mediated ROS production (Bianchi et al, 2009). The generation of ROS by the NOX enzyme complex is important for NETosis. A form of NETosis that is NOX-independent may be induced by certain other stimuli including calcium ionophores and immune complexes (Pilsczek et al, 2010; Parker et al, 2012). The molecular pathways and mechanisms governing NOX-independent pathway of NETosis are not well understood.
[0006] WO2012/065720 A1 to Martinez et al. describes a neutrophil viability assay to screen compounds for NET inhibition.
[0007] WO2012/16661 1 A2 to Wagner et al, describes a device for blood collection that is coated in a manner that reduces NET-related thrombosis.
[0008] US2013/0183662 A1 to Zychlinsky et al. uses NET degradation as a determinant in a diagnostic method for subjects with systemic lupus erythematosus to determine risk of developing renal manifestations.
[0009] WO2014/144572 A2 to Jin et al. describes a patient screening method to identify the occurrence of NETosis using biomarkers.
[0010] Identification of inhibitors of NETosis can improve treatments of inflammatory diseases.
SUMMARY
[0011] It is an object of the present disclosure to obviate or mitigate at least one undesirable effect of NETosis, or of diseases associated with an inflammatory response.
[0012] There is described herein a method of treating a disease associated with an inflammatory response, by administering an inhibitor of NETosis to a subject having the disease.
[0013] Further, there is described herein a method of identifying inhibitors of
NETosis comprising: inducing NETosis in neutrophils; providing the neutrophils with a candidate NETosis inhibitor, and observing inhibition of Nox-dependent and Nox- independent NET formation in the neutrophils.
[0014] There is also provided herein a use of an inhibitor of NETosis for treating a disease associated with an inflammatory response in a subject having the disease.
[0015] There is further provided a use of an inhibitor of NETosis for preparation of a medicament for treating a disease associated with an inflammatory response in a subject having the disease.
[0016] A composition is provided comprising an inhibitor of NETosis and a pharmaceutically acceptable excipient for treating a disease associated with an inflammatory response in a subject having the disease. [0017] Additionally, an inhibitor of NETosis is provided for treating a disease associated with an inflammatory response in a subject having the disease.
[0018] A commercial package is provided comprising an inhibitor of NETosis together with instructions for use in treating a disease associated with an inflammatory response in a subject having the disease.
[0019] Other aspects and features of the present disclosure will become apparent to those ordinarily skilled in the art upon review of the following description of specific embodiments in conjunction with the accompanying figures.
[0020] The following description, while indicating various aspects and
embodiments of the invention and numerous specific details thereof, is not exhaustive. Aspects and embodiments described herein are given by way of illustration and not of limitation. Substitutions, modifications, additions and/or rearrangements may be made, and are hereby encompassed.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] The following drawings form part of the present specification and are included to further demonstrate certain aspects and/or embodiments of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific aspects and/or embodiments presented herein.
[0022] Figure 1 is a flow chart showing transcription-based assays for drug screening involving mRNA isolation, transcriptomic evaluation and cluster analysis for control (C30/C60); PMA (P30/P60); and ionophore A23187 (A30/A60) induction.
[0023] Figure 2 shows transcript formation in panels A (PMA) and B (A23187);
ROS production in panels C (PMA), and D (A23187) with and without ACTD; and NETosis inhibition in panels E (PMA) and F (A23187) for PHA767491 (DNA helicase inhibitor), Etoposide (Topoisomerase II inhibitor), and Actinomycin D (ACTD, an antineoplastic antibiotic) during NADPH-dependent and NADPH-independent NETosis.
[0024] Figure 3 shows confocal images illustrating the inhibition of both Nox- dependent and Nox-independent NETosis by Actinomycin D either unstimulated (row A), or with exposure to PMA (row B) or A23187 (row C).
[0025] Figure 4 illustrates the drug screening procedure (panel A). Panel B provides a grey-scale representation of a color-coded imaging of screened candidates in a NETosis inhibitory heatmap of candidate drugs. This greyscale representation originally generated with blue and red scale representations (from 0 to 15), as shown in the side bar to the right.
[0026] Figure 5 shows NETotic Index over time for PMA/PMA+DPI (panel A);
LPS/LPS+SP6 (panel B); and H2O2/H2O2+SP6, indicating that LPS and H202-induced NETosis were significantly suppressed by the JNK inhibitor SP6 in the plate reader assay. DMSO vehicle control did not exert a significant effect on extracellular DNA release.
[0027] Figure 6 shows is a greyscale representation of confocal images, originally viewed in color, and illustrating the number of NETotic cells being significantly lower in the presence of the JNK inhibitor after 3 hours of stimulation for LPS, A23, ionomycin-mediated NETosis.
[0028] Figure 7 shows the influence of various factors on NETotic Index. Lower pH suppresses both Nox-dependent NETosis induced by PMA (panels A and B); or LPS (panels C and D), Pseudomonas aeruginosa (panels E and F) or Staphylococcus aereus (panels G and H). The X-axis shows time (min). The Y-axis shows Sytox Green
Fluorescence units indicating NETosis.
[0029] Figure 8 shows confocal images of NETs under different pH induced by
PMA (panel A) and LPS (panel B).
[0030] Figure 9 illustrates that lower pH suppresses Nox-independent NETosis induced by PMA (P) in top panel, LPS (L) in mid-panel, and A23187 (A) in bottom panel.
X-axis, time (min). Y-axis, Sytox Green Fluorescence units indicating NETosis.
[0031] Figure 10 shows that nucleases present in the BAL fluid of LPS-instilled mice require cations such as calcium and magnesium for activity.
[0032] Figure 11 shows that nucleases of inflamed airways have two pH optimums.
DETAILED DESCRIPTION
[0033] Generally, the present disclosure relates to mediating the inflammatory response, and treating diseases associated with an inflammatory response, such as inflammatory diseases and autoimmune diseases. Many diseases associated with an inflammatory response, such as inflammatory diseases and autoimmune diseases, have negative effects relating to or resulting from the inflammatory response of neutrophil infiltration. These negative effects can be mediated according to the methods described herein. By mediating the inflammatory response, a subject having the disease associated with an inflammatory response, such as an inflammatory disease or autoimmune disease, can benefit.
[0034] The following abbreviations may be used herein. NETs, Neutrophil
Extracellular Traps; NOX, NADPH oxidase, ROS, reactive oxygen species; CGD, chronic granulomatous disease; DPI, diphenyleneiodonium; PMA, phorbol 12-myristate 13- acetate; MPO, myeloperoxidase; FCCP, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone; DNP, dinitrophenol; PAD4, peptidyl arginine deiminase; ERK, extracellular-signal-regulated kinase; Akt, Protein kinase B or PKB.
[0035] As used herein, the singular forms "a," "and," and "the" include the option of plural unless otherwise specified or unless the context dictates otherwise.
[0036] The term "about", as used herein encompasses +/- 10%. As would be understood by a skilled person rarely can an absolute result be obtained or replicated because of variables impacting testing, production, and storage of biological and chemical materials, and because of the errors inherent error in test equipment.
[0037] All technical and scientific terms used herein have the same meaning as commonly understood to a person of ordinary skill in the art.
[0038] A method of treating a disease associated with an inflammatory response, such as an inflammatory disease or autoimmune disease, is described herein, which comprises administering an inhibitor of NETosis to a subject having the disease. The method may permit inhibition of NETosis, while permitting one or more other neutrophil immune functions to be maintained.
[0039] Diseases Associated With an Inflammatory Response, Such As
Inflammatory and Autoimmune Diseases. NETs cause or exacerbate inflammation and tissue damage. The treatment described herein targets the reduction or inhibition of NETosis, and thus can be used to address and treat inflammatory or autoimmune diseases. Such diseases include infectious diseases, lung infection, pneumonia, cystic fibrosis, neutrophilic asthma, neutrophil-mediated anaphylaxis, chronic obstructive pulmonary disease (COPD), cardiovascular diseases, vasculitis, blood clot-related diseases such as thrombosis, acute respiratory distress syndrome, nephritis, autoimmune diseases such as lupus or arthritis, such as rheumatoid arthritis, and diseases resulting in inflammation. Diseases resulting in inflammation may also include appendicitis, streptococcus pyogenes infection, fasciitis, Aspergillus infection, pre-eclampsia, small vessel vasculitis (SW), sepsis, Crohn's disease, Schistosoma infection, periodontitis, tuberculosis, mastitis, malaria, or systemic lupus erythematosus (SLE) among other forms of lupus.
[0040] Further diseases associated with an inflammatory response, including diseases resulting in inflammation, which are not listed herein can benefit from the inhibition of NETosis as described herein. The disease may be a disease that exhibits upregulated NETosis, or may be a disease conventionally considered to fall within the category of an "infection", and "inflammation", or an "autoimmune disease".
[0041] Inhibitors of NETosis. Compounds are described herein which may inhibit or suppress one or more forms (or routes) of NETosis. Such a compound may be referred to herein as an inhibitor of NETosis, or a NETosis inhibitor.
[0042] The different routes of NETosis may utilize different kinases, such as ERK,
Akt, JNK, or p38. Inhibitors of these kinases, for example JNK inhibitor SP600125 can suppress NETosis induced by LPS or H202 or A23 or ionomycin. These and other inhibitors targeting these kinases may permit the suppression of NETosis while maintaining other important immune functions of the neutrophils.
[0043] Nucleic acid synthesis is important for NETosis. Nucleic acid synthesis may be rapid in NADPH-independent NETosis than the NADPH-dependent NETosis pathway. Inhibitors of nucleic acid synthesis, such as Actinomycin D, suppress NETosis.
[0044] In general, compounds for inhibiting NETosis may include, but are not limited to, Actinomycin D (ACTD), or an inhibitor of a kinase such as JNK, or an analogue or derivative of these. The compound SP600125 is an exemplary inhibitor of JNK. It is understood that such analogues or derivatives would encompass those modified or derived in a manner that approximately maintains or has a greater NETosis inhibiting function when compared with the compound from which it is derived.
[0045] To inhibit NETosis, pH-regulators may be used. Such pH-regulators may have two effects: a reduction in pH can suppress NETosis and also increase the clearance of NETs by DNAses in the presence of calcium and magnesium. Thus, pH- lowering agents can suppress NETosis thereby reducing NETs formed by the neutrophils.
[0046] FDA-approved drugs may be utilized and identified as described herein, for inhibiting NETosis. A screening approach for both types of NETosis (NADPH- dependent and -independent) may be utilized. Selected candidates can be validated and quickly recommended for clinical "drug-repurposing" trials utilizing the methods described herein. Derivatives and analogues of successful screening candidates may optimize activity in suppression of NETosis, while maintaining neutrophil viability and/or one or more other neutrophil function other than NETosis (e.g., reactive oxygen species generation, phagocytic killing). Pathway-specific compounds may be used for treating several, but specific diseases associated with an inflammatory response, such as inflammatory diseases, infections, or autoimmune diseases.
[0047] Method of Identifying Inhibitors. There is described herein a method of identifying inhibitors of NETosis comprising: inducing NETosis in neutrophils; providing the neutrophils with a candidate NETosis inhibitor, and observing inhibition of reactive oxygen species (ROS) formation in the neutrophils.
[0048] Inducing NETosis may comprise exposing the neutrophils to a NETosis agonist or other compound (generally referring to large or small molecules or other activating combinations) able to induce NETosis, or may involve simply working with a population of neutrophils that are already activated through a previous route, for example if isolated from an individual in the throes of a disease associated with an inflammatory response, such as an inflammatory disease or an autoimmune disease.
[0049] The compounds to be tested and identified as candidate inhibitors may be selected from among the categories of inhibitors mentioned above, and any analogues or derivative thereof, as well as from heretofore previously unknown categories of inhibitors which are otherwise identified using screening for libraries or collections of compounds.
[0050] In screening, the selection of a candidate NETosis inhibitor may include those that permit other neutrophil immune functions, bearing in mind that an exemplary candidate for testing as a NETosis inhibitor would not exhibit neutrophil toxicity.
[0051] Compounds for Inducing NETosis. Compounds are described herein that induce or regulate NETosis through upregulation or induction, and may be used herein in the methods described, and specifically in methods of identifying inhibitors of Netosis. For example, NETosis may be induced by agonists such as PMA, calcium ionophores A23187 ("A23"), ionomycin, LPS, and immune complexes. Immune complexes can induce Nox-independent NETosis. The kinetics of immune complexes may differ from that of ionophore-induced NETosis. Compounds that suppress immune complex-mediated NETosis and/or ionophore-induced NETosis are suitable for treating autoimmune diseases. The compounds to be tested and identified as candidate inhibitors may be selected from among the categories of agonists mentioned above, and any analogues or derivative thereof, as well as from heretofore previously unknown categories of inhibitors which are otherwise identified using screening for libraries or collections of compounds. [0052] Figure 1 shows the overall experimental design for a study of transcriptomics. A global gene expression profile indicates that a large number of genes are transcribed at early time points in ionophore (A23187) induction of NETosis, while transcription occurs at later time points in PMA mediated NETosis. This indicates the faster NETosis kinetics shown by A23187 induction versus PMA inductions. Control samples are more closely clustered with PMA early time points, while ionophore samples are clustered with PMA late time points. Using such assays, drug screening can be conducted to identify additional candidate drugs capable of acting to block or induce NETosis, for use in mediating inflammation in a disease associated with an inflammatory response, such as an inflammatory disease or an autoimmune disease.
[0053] Uses are described herein for inhibitors of NETosis in treating a disease associated with an inflammatory response, such as an inflammatory disease or an autoimmune disease, in a subject having the disease. Further, a use of an inhibitor of NETosis is described for preparation of a medicament for treating a diseases associated with an inflammatory response, such as an inflammatory disease or an autoimmune disease, in a subject having the disease.
[0054] A composition comprising an inhibitor of NETosis and a pharmaceutically acceptable excipient is provided, for treating a disease associated with an inflammatory response, such as an inflammatory disease or an autoimmune disease, in a subject having the disease. Such a composition may be manufactured together with, or prescribed or used together with another active ingredient for use in addressing an inflammatory disease.
[0055] An inhibitor of NETosis is described for treating a disease associated with an inflammatory response, such as an inflammatory disease or an autoimmune disease, in a subject having the disease.
[0056] A commercial package is described, comprising an inhibitor of NETosis together with instructions for use in treating a disease associated with an inflammatory response, such as an inflammatory disease or an autoimmune disease, in a subject having the disease.
[0057] Numerous methods may be employed to evaluate NETs and/or NETosis.
For example, an ELISA may be used to measure NETs. Methods of evaluating NETs with myeloperoxidase present thereon can be used to quantify the amount of NETs DNA by incubating the reaction with a DNA binding fluorescence dye (for example: picogreen) and measuring fluorescence. [0058] EXAMPLE 1
[0059] Materials and Methods
[0060] Human peripheral neutrophils. The protocol was approved by the
Hospital for Sick Children (Toronto, Canada) ethics committee and signed informed consent was obtained from each subject enrolled. Peripheral blood from healthy donors was collected in K2 EDTA blood collection tubes (BD, Franklin Lakes, NJ), and neutrophils were isolated from the whole blood using PolymorphPrep™ (Axis-Shield PoC, Oslo, Norway). Some assays were conducted in dHL-60 neutrophil-like cells (e.g., drug screening).
[0061] NETosis analysis. NETosis was monitored by a plate reader assay in the presence of Sytox™ Green cell-impermeable nucleic acid stain (5 μΜ). NETosis was confirmed by imaging the colocalization of myeloperoxidase, and DNA stained with Sytox Green after the fixation and permeablization. For some studies, Western blots were used for determining CitH3, the activation states of kinases (e.g., JNK). Inhibitors were preincubated with cells for 1 h prior to the activation of cells for NETosis.
[0062] Statistical analysis. All data are presented as mean ± SEM. Statistical analysis was performed using GraphPad Prism™ statistical analysis software (Version 5.0a for Mac OS X). Student's t-test was used for comparing two groups. When comparing more than two groups, ANOVA with Bonferroni post test or Dunnett's test was used where appropriate. A p-value of 0.05 or less was considered to be statistically significant.
[0063] Molecules that interact with nucleic acid or inhibit nucleic acid synthesis
(e.g Actinomycin D or ACTD, Etoposide, PHA767491) inhibit both NADPH-dependent and NADPH-independent NETosis.
[0064] In this Example, human neutrophils are obtained and assessed for the effect of different nucleic acid binding molecules or inhibitor of nucleic acid synthesis.
[0065] Figure 2 to Figure 4 show that molecules such as Actinomycin D (ACTD)
Etoposide and PHA767491 that interact with nucleic acid or its copying inhibit both NADPH-dependent and NADPH-independent NETosis.
[0066] Figure 2 illustrates differences in the number of transcript formation,
Reactive Oxygen Species (ROS) production and inhibition of NETosis by several nucleic acid binding molecules or nucleic acid synthesis inhibitors such as PHA767491 (DNA helicase inhibitor), Etoposide (Topoisomerase II inhibitor), Actinomycin D (ACTD, an antineoplastic antibiotic) during NADPH-dependent and NADPH-independent NETosis. [0067] In Figure 2, the results of a Sytox Green assay are provided, showing the degree of mRNA production (panels A and B), ROS production (panels C and D) and inhibition of NADPH-dependent (panel E) and NADPH-independent (panel F) NETosis by ACTD, Etoposide and PHA76749. Inhibitors are assessed at the indicated level in the presence of PMA or calcium ionophore (lo), using a DMSO control. NETosis was assessed by determining the change in extracellular DNA as a proportion of total lysis over 240 minutes.
[0068] In Figure 3, it is shown that ACTD inhibits decondensing neutrophil chromatin DNA. Results are shown for PMA and A23187 ("A23") +/- ACTD.
[0069] Figure 4 reveals additional classes of nucleotide binding and synthesis inhibitors which inhibits NETosis. These data however show that specific classes of nucleic acid binding molecules can be an effective therapeutic method for suppressing NETosis in inflammatory diseases.
[0070] Figure 4 illustrates the drug screening procedure (panel A), which involves deposition of candidate drugs to be screened; addition of dHL-60 cells; addition of agonist (such as PMA/ionophore) at hour 1 , then taking readings at subsequent intervals of 5 hours. With use of the plate reader, Syntox-based NETosis kinetics are determined. ROS is determined by DHR123 and Mitosox, and fluorescent imaging is undertaken.
[0071] Figure 4 (panel B) provides a grey-scale representation of a color-coded imaging of screened candidates in a NETosis inhibitory heatmap of candidate drugs. This greyscale representation was originally generated with blue and red scale representations (from 0 to 15), as shown in the side bar to the right. The heatmap is determined by neutrophils differentiated from human HL-60 cells. Nucleic acid metabolism inhibitors and nucleic acid synthesis inhibitors such as cyclocystidine hydrochloride (e.g., Cyclo-C or Idarubicin or Idamycin) inhibits NETosis. The data obtained on the original drug candidates tested, leading to the results represented in Figure 4 are summarized below, revealing certain drug candidates showing positive results.
[0072] The testing results obtained from the method conducted was able to identify inhibitors of NETosis by inducing NETosis in neutrophils; providing the neutrophils with a candidate NETosis inhibitor, and observing inhibition of Nox-dependent and Nox- independent NET formation in the neutrophils.
[0073] It was revealed that carbonic anhydrase inhibitor N-[5-(Aminosulfonyl)-
1 ,3,4-thiadiazol-2-yl]acetamide (e.g., Acetazolamide) that regulates pH also was found to suppress NETosis. [0074] A potassium channel Kir 6.1 inhibitor N-(1-Adamantyl)-N'-cyclohexyl-4- morpholinecarboxamidine hydrochloride (e.g., PNU-37883A) was found to inhibit NETosis.
[0075] Serotonin pathway regulators were found to inhibit NETosis. These examples include (a) 5-HT 1 D serotonin receptor antagonist 4-(3-Chlorophenyl)-alpha- (diphenylmethyl)-l-piperazineethanol hydrochloride (e.g., BRL 15572), and (b) serotonin uptake inhibitor Methyl reserpate or 3,4,5-Trimethoxybenzoic acid ester (e.g., Reserpine).
[0076] A potent orally active D1 dopamine receptor agonist [(1 R, 3S)-3-(1-
Adamantyl)-1-(aminomethyl)-3-4dihydro-1 H-isochromene-5,6-diol] was found to inhibit NETosis.
[0077] A P2 purinoceptor agonist 2-(Methylthio)adenosine 5'-diphosphate trisodium salt hydrate was found to inhibit NETosis.
[0078] It was found that (a) L-type Ca2+ channel blocker 3-(4-Hydroxyphenyl)-1-
(2,4,6-trihydroxyphenyl)-1-propanone (e.g., Phloretin) and (b) a Store-operated calcium (SOC) channel inhibitor N-Propargylnitrendipene (e.g., MRS 1845) inhibit NETosis.
[0079] A Vanilloid receptor-1 (VR1) antagonist [(E)-3-(4-Chlorophenyl)-N-(3- methoxyphenyl)prop-2-enamide] (e.g., SB-366791) was found to inhibit NETosis.
[0080] Three kinase inhibitors were found to suppress NETosis: (a) a protein tyrosine kinase inhibitor 4,5-Dianilinophthalimide (e.g., CGP 5241 1) specific for epidermal growth factor receptor (EGFR), (b) a potent and selective vascular endothelial growth factor protein tyrosine kinase (VEGFR PTK) inhibitor 1 ,3-Dihydro-3-[(3,5-dimethyl-1 H- pyrrol-2-yl)methylene]-2H-indol-2-one (e.g., SU 5416), and (c) a diacylglycerol Kinase Inhibitor II (3-[2-[-[bis(4-Fluorophenyl)methylene]-1-piperidinyl]ethyl]-2-3-dihydro-2-thioxi- 4(1 H)-quinazolinone (e.g., R59949).
[0081] In summary, the method of identifying inhibitors of NETosis located the following compounds from among the candidate inhibitors tested, having NETosis inhibitory activity: N-[5-(aminosulfonyl)-1 ,3,4-thiadiazol-2-yl]acetamide (Acetazolamide); N-(1-adamantyl)-N'-cyclohexyl-4-morpholinecarboxamidine hydrochloride (PNU-37883A); 4-(3-chlorophenyl)-alpha-(diphenylmethyl)-1-piperazineethanol hydrochloride (BRL 15572); reserpine; methyl reserpate; (1 R, 3S)-3-(1-adamantyl)-1-(aminomethyl)-3- 4dihydro-1 H-isochromene-5,6-diol; 2-(methylthio)adenosine 5'-diphosphate trisodium salt hydrate; 3-(4-hydroxyphenyl)-1-(2,4,6-trihydroxyphenyl)-1-propanone (Phloretin); N- propargylnitrendipene (MRS 1845); (E)-3-(4-chlorophenyl)-N-(3-methoxyphenyl)prop-2- enamide (SB-366791); 4,5-dianilinophthalimide (CGP 5241 1); 1 ,3-dihydro-3-[(3,5- dimethyl-1 H-pyrrol-2-yl)methylene]-2H-indol-2-one (SU 5416); and 3-[2-[-[bis(4- fluorophenyl)methylene]-1-piperidinyl]ethyl]-2-3-dihydro-2-thioxi-4(1 H)-quinazolinone
(R59949).
[0082] EXAMPLE 2
[0083] JNK inhibitor SP6 inhibits NETosis
[0084] In this Example, human neutrophils are obtained and assessed for the effect of JNK inhibitor SP6. To induce NETosis, H202, LPS, and A23187 were utilized, and inhibition of NETosis was shown. These data affirm that JNK inhibitors can be effective in inhibition of NETosis and addressing diseases associated with an
inflammatory response.
[0085] Figure 5 and Figure 6 indicate that LPS, H202, and A23-induced NETosis were significantly suppressed by the JNK inhibitor SP6 in the plate reader assay.
[0086] Figure 5 shows NETotic Index over time for PMA/PMA+DPI (panel A);
LPS/LPS+SP6 (panel B); and H202/H202+SP6, indicating that LPS and H202-induced NETosis were significantly suppressed by the JNK inhibitor SP6 in the plate reader assay. DMSO vehicle control did not exert a significant effect on extracellular DNA release.
[0087] Figure 6 shows is a greyscale representation of confocal images, originally viewed in color, and illustrating the number of NETotic cells being significantly lower in the presence of the JNK inhibitor after 3 hours of stimulation for LPS, A23, ionomycin-mediated NETosis. The presence or absence of SP-6 is revealed for Nox- dependent NETosis (PMA and LPS) as well as Nox-independent NETosis (A23 and ionomycin).
[0088] EXAMPLE 3
[0089] pH Lowering Suppresses NETosis
[0090] In this Example, human neutrophils are obtained and assessed for the effect of pH lowering. To induce NETosis, PMA(P) or LPS (L) were used for Nox- dependent NETosis, and A23187 was utilized for Nox-independent NETosis induction. Inhibition of NETosis was shown. These data affirm that pH regulators, and in particular pH lowering compounds can be effective in inhibition of NETosis and addressing diseases associated with an inflammatory response. [0091] Figure 7 to Figure 9 show that lower pH suppresses both Nox-dependent
(induced by PMA or LPS, Pseudomonas aeruginosa and Staphylococcus aureus) and Nox-independent NETosis (induced by A23187 (A)). X-axis, time (min). Y-axis, Sytox Green Fluorescence units indicating NETosis.
[0092] Figure 7 illustrates the influence of various factors on NETotic Index.
Lower pH suppresses both Nox-dependent NETosis induced by PMA (panels A and B); or LPS (panels C and D), Pseudomonas aeruginosa (panels E and F) or Staphylococcus aereus (panels G and H).
[0093] Figure 8 shows confocal images of NETs under different pH induced by
PMA (panel A) and LPS (panel B), at pH 6.6, 7.4, and 7.8.
[0094] Figure 9 illustrates that lower pH suppresses Nox-independent NETosis induced by PMA (P) in top panel, LPS (L) in mid-panel, and A23187 (A) in bottom panel. X-axis, time (min). The Y-axis shows Sytox Green Fluorescence units, which are indicative of NETosis.
[0095] EXAMPLE 4
[0096] Optimal acidic pH Degrades NETs and Suppresses NETosis
[0097] In this Example, it is shown that airway nucleases degrade neutrophil extracellular traps (NETs) in a calcium-dependent manner and function optimally at an acidic pH.
[0098] Neutrophil extracellular traps (NETs) are unique defense structures. NETs are made of neutrophil DNA and antimicrobial peptides, which help to trap and kill pathogens. Although seemingly beneficial at first, excessive NETs or the inability to clear NETs has been shown to contribute to the pathogenesis of several inflammatory and autoimmune diseases. Conditions necessary for NETs degradation are evaluated in this example. Here it is shown that nucleases are present in murine airway mucosa of healthy and inflamed lungs, and these nucleases degrade genomic DNA (gDNA) in a Mg2+ and Ca2+-dependent manner. NETs present in the bronchoalveolar lavage fluid (BALF) of LPS-instilled mouse airways are fragmented by BALF nucleases in a Ca2+-dependent manner. This is different from the degradation of gDNA, which occurs in a Mg2+ and Ca2+- dependent manner. Optimal activity of BALF nucleases occurs at two pHs, one at near neutral pH (6.8-7.0) and another at more acidic pH (6.6 or less). These nuclease activities assist with DNA hydrolysis and eventual NET clearance. Therefore, modulating divalent cation content and pH can regulate NET function and clearance. Extracellular pH transiently drops during inflammation which can regulate NETs in various tissues and mucosal surfaces.
[0099] For the analysis of pulmonary NETs in vivo, BALB/c mice aged 4 - 6 weeks were sedated with a ketamine (150 mg/kg)/xylazine (5 mg/kg) mixture via intraperitoneal injection, and 1 μg LPS (E. coli 01 1 1 :B4) in 50 μΙ PBS or a PBS control was intranasally instilled for up to 1 day as described previously [1 1]. Mice were sacrificed with 0.05 ml Euthanyl (Bimeda-MTC, ON, CA) prior to performing the BAL procedure. BAL was performed with 1 ml of chilled calcium- and magnesium-free Hanks Balanced Salt Solution (HBSS) (Invitrogen) three times to a total of 3 ml. BAL fluid (BALF) was centrifuged at 400 μg for 10 min to pellet the cells. The cell-free supernatant was analysed for DNA using a Quant-iT™ PicoGreen dsDNA reagent kit (Invitrogen). Red blood cells (RBCs) in the cell pellet were lysed with a hypotonic saline solution (0.2 % (w/v) NaCI) for 30 seconds, followed by the addition of an equal volume of buffered hypertonic saline solution (1.6 % (w/v) NaCI, 20 mM Hepes, pH 7.2) to achieve an isotonic equilibrium. The resulting RBC-free cell pellet was resuspended in calcium- and magnesium-free HBSS for cell counting with a haemocytometer. Cytospin preparations were made and further stained with Hemacolor™ histology staining kit (EMD Chemicals, Gibbstown, NJ) for cell differential analysis. Histology images were taken with a light microscope and images were randomly chosen for determining percent populations of each cell type. A minimum of 100 cells were counted for each condition.
[00100] Figure 10 shows that nucleases present in the BAL fluid of LPS-instilled mice require cations for activity. Agarose gels showing gDNA degradation following the incubation with LPS-BALF, MgCI2, and/or CaCI2 at 37 °C for 3 h. (a) BALF nuclease activity is dose-dependent and time-dependent. LPS-BALF alone and controls show background DNA which are described as NETs. gDNA alone and gDNA with Mg2+ and Ca2+ remain as high molecular weight (m.w.) structures (10 kb). gDNA is incubated with LPS-BALF in the presence of both Mg2+ and Ca2+, gDNA becomes degraded into low m.w. gDNA (-0.25 - 0.3 kb). (b) Addition of Mg2+ leads to gDNA degradation by LPS- BALF nucleases, whereas Ca2+ does not. Nuclease activity is synergistically enhanced by the presence of both Mg2+ and Ca2+. EGTA and EDTA protect gDNA being degraded. Image of agarose gels are representative of three independent experiments.
[00101] Nucleases present in the airways of LPS-instilled mice were found to require cations for activity. After establishing that the airways of non-inflamed naive and PBS-instilled mice contained a basal level of nuclease activity, we next investigated whether the inflamed airways of LPS-instilled mice contained additional nucleases. Cell- free BALF supernatant was incubated with intact gDNA for 1 h and 3 h at 37 °C. gDNA controls remain as high molecular weight structures (Figure 10, part a) and that BALF alone controls have small amounts of NETs. Therefore, any additional increase in intensity on the agarose gel can be attributed to the degradation gDNA by LPS-BALF nucleases. Similar to the findings of non-inflamed lungs, gel electrophoretic assays show that gDNA is degraded in a dose- and time-dependent manner. Also, as reaction volumes and loading volumes were kept the same, a larger signal from the intensity plot indicates that more gDNA is being degraded by LPS-BALF compared to naive- and PBS-BALF. Therefore, inflamed airways contain more nucleases.
[00102] Next, it was examined whether these nucleases have similar cation preferences to nucleases found in naive and PBS-BALF. Indeed, LPS-BALF nucleases also become active in the presence of Mg2+ but not Ca2+ (Figure 10 part b).
Furthermore, nuclease activity is synergistically enhanced by adding both Mg2+ and Ca2+ together, resulting in the mass cleavage of gDNA to ~0.25 - 2 kb fragments. EGTA or EDTA was able to block nuclease activity as observed by the high molecular weight peak of gDNA. Therefore, cation supplementation is required for nuclease activity in the airways of mice instilled with LPS. Taken together, these data suggest that nucleases present in both inflamed and non-inflamed lungs are similar enzymes with the need for cations to initiate DNA hydrolysis.
[00103] Figure 11 shows that nucleases of inflamed airways have two pH optimums. PIPES buffer. Using the HEPES, PIPES and MES buffers, nuclease activity from the airways of LPS-instilled mice were assessed on an agarose gel. Samples were prepared as described above, (a, b) High molecular weight (m.w.) bands for gDNA controls, (c) NET-DNA in LPS-BALF control is degraded in the absence of cations at pH 6.0 to 6.4. (d) Cleavage of gDNA by BALF nucleases occurs near acidic pHs 6.0 to 6.4 and also specifically at pH 6.8 (arrow). Addition of Mg2+ causes slight gDNA degradation (e) compared to the null effect of Ca2+, except at acidic pH 6.0 to 6.4 (f). (g) The presence of both ions synergistically enhances the activity of BALF nucleases based on the disappearance of high m.w. bands and the abundance of low m.w. bands. Image of agarose gels are representative of three independent experiments. Abbreviations used are: gDNA, genomic DNA; LPS BALF, BAL fluid from LPS-instilled mice; and D, non- buffered DEPC-H20. [00104] Since the buffering capacity of HEPES and MES is limited to near pH 6.8 and 6.6, respectively, PIPES and MOPS were used to assess nuclease activity in these ranges. In a similar experiment using PIPES, it was noticeable again that without the addition of any cations, BALF controls containing NETs show some degradation near acidic pH (i.e. 6.0 to 6.4) (Figure 11 , part c). Once again, without the addition of cations, BALF show a significant degradation of gDNA at acidic pHs (6.0 to 6.4) (Figure 11 , part d). The effect of adding Mg2+ to the reaction initiates gDNA degradation at and above pH 6.6 (Figure 11 , part e). On the other hand, the nucleases consistently remain mostly inactive in the presence of Ca2+ (Figure 11 , part f). Addition of both Mg2+ and Ca2+ ions together enhance the function of LPS-BALF nucleases when buffered with PIPES (Figure 11 , part g).
[00105] In the preceding description, for purposes of explanation, numerous details are set forth in order to provide a thorough understanding of the embodiments. However, it will be apparent to one skilled in the art that these specific details are not required.
[00106] The above-described embodiments are intended to be examples only. Alterations, modifications and variations can be effected to the particular embodiments by those of skill in the art. The scope of the claims should not be limited by the particular embodiments set forth herein, but should be construed in a manner consistent with the specification as a whole.
[00107] References:
[00108] All publications and patents mentioned herein, including the documents noted below, are hereby incorporated by reference. These documents describing or disclosing information and methodologies which might assist in understanding or utilizing the aspects and embodiments are described herein.
[00109] Bianchi M, Hakkim A, Brinkmann V, Siler U, Seger RA, Zychlinsky A, & Reichenbach J (2009) Blood 1 14, 2619-2622.
[00110] Cheng OZ & Palaniyar N (2013) Frontiers in immunology 4, 1-13.
[00111] Douda DN, Yip L, Khan MA, Grasemann H, & Palaniyar N (2014) Blood 123, 597-600.
[00112] Douda DN, Khan MA, Grasemann H & Palaniyar N (2015) Proceedings of the National Academy of Sciences of the United States of America 1 12, 2817-2822.
[00113] Fay AJ, Qian X, Jan YN, & Jan LY (2006) Proceedings of the National
Academy of Sciences of the United States of America 103, 17548-17553. [00114] Neeli I & Radic M (2013) Frontiers in immunology 4, 38.
[00115] Parker H, Dragunow M, Hampton MB, Kettle AJ, & Winterbourn CC
(2012) Journal of leukocyte biology 92, 841-849.
[00116] Pilsczek FH, Salina D, Poon KK, Fahey C, Yipp BG, Sibley CD, Robbins SM, Green FH, Surette MG, Sugai M, et al. (2010) J Immunol 185, 7413-7425.
[00117] WO2012/065720 A1 to Martinez et al.
[00118] WO2012/16661 1 A2 to Wagner et al.
[00119] US2013/0183662 A1 to Zychlinsky et al.
[00120] WO2014/144572 A2 to Jin et al.

Claims

CLAIMS:
1. A method of treating a disease associated with an inflammatory response, by administering an inhibitor of NETosis to a subject having the disease.
2. The method claim 1 , wherein the inhibitor of NETosis interferes with or regulates nucleic acid synthesis.
3. The method of claim 1 or 2, wherein the inhibitor of NETosis is selected from the group consisting of Actinomycin D (ACDT), a pH regulator, a JNK inhibitor, and analogues and derivatives thereof.
4. The method of claim 3, wherein the inhibitor of NETosis comprises JNK inhibitor SP600125, or an analogue or derivative thereof.
5. The method of claim 1 or 2, wherein the inhibitor of NETosis is:
N-[5-(aminosulfonyl)-1 ,3,4-thiadiazol-2-yl]acetamide (Acetazolamide);
N-(1-adamantyl)-N'-cyclohexyl-4-morpholinecarboxamidine hydrochloride (PNU-37883A); 4-(3-chlorophenyl)-alpha-(diphenylmethyl)-1-piperazineethanol hydrochloride (BRL 15572);
reserpine;
methyl reserpate;
(1 R, 3S)-3-(1-adamantyl)-1-(aminomethyl)-3-4dihydro-1 H-isochromene-5,6-diol;
2- (methylthio)adenosine 5'-diphosphate trisodium salt hydrate;
3- (4-hydroxyphenyl)-1-(2,4,6-trihydroxyphenyl)-1-propanone (Phloretin);
N-propargylnitrendipene (MRS 1845);
(E)-3-(4-chlorophenyl)-N-(3-methoxyphenyl)prop-2-enamide (SB-366791);
4,5-dianilinophthalimide (CGP 5241 1);
1 ,3-dihydro-3-[(3,5-dimethyl-1 H-pyrrol-2-yl)methylene]-2H-indol-2-one (SU 5416); or 3-[2-[-[bis(4-fluorophenyl)methylene]-1-piperidinyl]ethyl]-2-3-dihydro-2-thioxi-4(1 H)- quinazolinone (R59949).
6. The method of any one of claims 1 to 5, wherein the disease associated with an inflammatory response exhibits upregulated NETosis.
7. The method of any one of claims 1 to 6, wherein the disease associated with an inflammatory response comprises an inflammatory disease or an autoimmune disease.
8 The method of claim 7, wherein the autoimmune disease is Lupus or rheumatoid arthritis.
9. The method of any one of claims 1 to 6, wherein the disease associated with an inflammatory response comprises an infection, an infectious disease, lung infection, pneumonia, cystic fibrosis, neutrophilic asthma, neutrophil-mediated anaphylaxis, chronic obstructive pulmonary disease (COPD), cardiovascular diseases, vasculitis, a blood clot- related disease, thrombosis, acute respiratory distress syndrome, nephritis, Lupus, or arthritis.
10. The method of any one of claims 1 to 9 wherein NETosis is inhibited while one or more other neutrophil immune function is maintained.
1 1. A method of identifying inhibitors of NETosis comprising:
inducing NETosis in neutrophils;
providing the neutrophils with a candidate NETosis inhibitor, and
observing inhibition of Nox-dependent and Nox-independent NET formation in the neutrophils.
12. The method of claim 1 1 , wherein inducing NETosis comprises exposing the neutrophils to a NETosis agonist.
13. The method of claim 1 1 , wherein inducing NETosis comprises exposing neutrophils to PMA, A23187, LPS, an immune complex, calcium ionophores, or inomycin.
14. The method of any one of claims 1 1 to 13, additionally comprising selecting a candidate NETosis inhibitor that permits neutrophil immune functions other than NETosis, wherein the candidate NETosis inhibitor does not exhibit neutrophil toxicity.
15. Use of an inhibitor of NETosis for treating a disease associated with an inflammatory response in a subject having the disease.
16. Use of an inhibitor of NETosis for preparation of a medicament for treating a disease associated with an inflammatory response in a subject having the disease.
17. The use of claim 15 or 16, wherein the inhibitor of NETosis interferes with or regulates nucleic acid synthesis.
18. The use of any one of claims 15 to 17, wherein the inhibitor of NETosis is selected from the group consisting of Actinomycin D (ACDT), a pH regulator, a JNK inhibitor, and analogues and derivatives thereof.
19. The use of claim 18, wherein the inhibitor of NETosis comprises JNK inhibitor SP600125, or an analogue or derivative thereof.
20. The use of any one of claims 15 to 17, wherein the inhibitor of NETosis is:
N-[5-(aminosulfonyl)-1 ,3,4-thiadiazol-2-yl]acetamide (Acetazolamide);
N-(1-adamantyl)-N'-cyclohexyl-4-morpholinecarboxamidine hydrochloride (PNU-37883A); 4-(3-chlorophenyl)-alpha-(diphenylmethyl)-1-piperazineethanol hydrochloride (BRL 15572);
reserpine;
methyl reserpate;
(1 R, 3S)-3-(1-adamantyl)-1-(aminomethyl)-3-4dihydro-1 H-isochromene-5,6-diol;
2- (methylthio)adenosine 5'-diphosphate trisodium salt hydrate;
3- (4-hydroxyphenyl)-1-(2,4,6-trihydroxyphenyl)-1-propanone (Phloretin);
N-propargylnitrendipene (MRS 1845);
(E)-3-(4-chlorophenyl)-N-(3-methoxyphenyl)prop-2-enamide (SB-366791);
4,5-dianilinophthalimide (CGP 5241 1);
1 ,3-dihydro-3-[(3,5-dimethyl-1 H-pyrrol-2-yl)methylene]-2H-indol-2-one (SU 5416); or 3-[2-[-[bis(4-fluorophenyl)methylene]-1-piperidinyl]ethyl]-2-3-dihydro-2-thioxi-4(1 H)- quinazolinone (R59949).
21. The use of any one of claims 15 to 20, wherein the disease associated with an inflammatory response comprises an inflammatory disease or an autoimmune disease.
22. The use of claim 21 , wherein the autoimmune disease is Lupus or rheumatoid arthritis.
23. The use of any one of claims 15 to 20, wherein the disease associated with an inflammatory response comprises an infection, an infectious disease, lung infection, pneumonia, cystic fibrosis, neutrophilic asthma, neutrophil-mediated anaphylaxis, chronic obstructive pulmonary disease (COPD), cardiovascular diseases, vasculitis, a blood clot- related disease, thrombosis, acute respiratory distress syndrome, nephritis, Lupus, or arthritis.
24. A composition comprising an inhibitor of NETosis selected from the group consisting of Actinomycin D (ACDT), a pH regulator, a JNK inhibitor, analogues and derivatives thereof; and a pharmaceutically acceptable excipient, for treating a disease associated with an inflammatory response in a subject having the disease.
25. The composition of claim 24, wherein the disease associated with an inflammatory response comprises an infection, an infectious disease, lung infection, pneumonia, cystic fibrosis, neutrophilic asthma, neutrophil-mediated anaphylaxis, chronic obstructive pulmonary disease (COPD), cardiovascular diseases, vasculitis, a blood clot-related disease, thrombosis, acute respiratory distress syndrome, nephritis, Lupus, or arthritis.
26. A commercial package comprising an inhibitor of NETosis selected from the group consisting of Actinomycin D (ACDT), a pH regulator, a JNK inhibitor, analogues and derivatives thereof, together with instructions for use in treating a disease associated with an inflammatory response in a subject having the disease, wherein the disease is selected from the group consisting of an infection, an infectious disease, lung infection, pneumonia, cystic fibrosis, neutrophilic asthma, neutrophil-mediated anaphylaxis, chronic obstructive pulmonary disease (COPD), cardiovascular diseases, vasculitis, a blood clot- related disease, thrombosis, acute respiratory distress syndrome, nephritis, Lupus, and arthritis.
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