Method of treating inflammation

Abstract

The present invention relates to a method of suppressing or inhibiting TNF-a-mediated proinflammatory signaling in a mammalian cell or tissue, the method comprising contacting the mammalian cell or tissue with an agent capable of upregulating the expression or activity of miR-335. In particular, the agent is an miR-335 agonist, such as belinostat, or an miR-335 mimic. Also disclosed is a method of treating, reducing or inhibiting TNF-a-mediated inflammation in a subject, the method comprising administering an agent capable of regulating the expression or activity of miR-335 to the subject. In particular, the inflammation is characterized by an increased expression and / or secretion of caspase 7, CCL3 and / or CCL5 as compared to a reference.

Description

[0001] METHOD OF TREATING INFLAMMATION Technical field

[0002] The present invention relates, in general terms, to the field of molecular biology. In particular, the present invention relates to methods of treating inflammation and inflammatory diseases.

[0003] Background

[0004] Inflammation is a body’s natural defence response to external or internal stimuli such as mechanical injury, infection, allergen exposure, chemical irritation, or immune dysregulation leading to symptoms such as redness, heat, swelling and pain. Inflammation is a hallmark of many diseases including atopic dermatitis, psoriasis, inflammatory bowel disease, rheumatoid arthritis of asthma. During inflammation, immune and non-immunc cells, including neutrophils, eosinophils, basophils, mast cells, macrophages, endothelial cells, and platelets, respond to inflammatory stimuli and foreign substances by producing bioactive mediators. These mediators act in autocrine and paracrine fashions to amplify and sustain the inflammatory response through interactions with diverse cell types. Numerous classes of mediators can drive inflammation.

[0005] TNFa (Tumor Necrosis Factor alpha) is a cytokine which promotes an inflammatory response and is involved in several inflammatory and autoimmune diseases or disorders. Therapeutics have been successfully developed to target TNF-a signalling in autoimmune and inflammatory disorders like rheumatoid arthritis and psoriasis. However, TNF-a-targeted therapies can paradoxically result in inflammatory conditions such as atopic dermatitis (AD)-like eczema in other cases due to cytokine or immune imbalance.

[0006] Atopic dermatitis (AD), commonly known as eczema, is a multifactorial disease characterized by ry. itchy and inflamed skin. While the exact cause is unknown, it is believed that genetics, immune system deficiencies and defects in skin barrier function are major contributing factors. Clinical management of AD is currently limited to controlling inflammation to mitigate pain and itching associated with skin lesions. Topical application of corticosteroids or calcineurin inhibitors and IgE-neutralizing antibodies is sufficient tomanage mild disease. However, moderate to severe cases of AD would require systemic treatment, which may incur the risk of adverse effects. There is therefore a need for safer and more efficacious therapies

[0007] It would be desirable to overcome or alleviate at least one of the above-described problems, or at least to provide a useful alternative.

[0008] Summary

[0009] Disclosed herein is a method of suppressing or inhibiting TNFa-mediated proinflammatory signaling in a mammalian cell or tissue, the method comprising contacting the mammalian cell with an agent capable of upregulating the expression or activity of miR-335.

[0010] Disclosed herein is a method of treating, reducing or inhibiting TNFa-mediated inflammation in a subject, the method comprising administering an agent capable of up-regulating the expression or activity of miR-335 to a subject.

[0011] Disclosed herein is a method of treating, reducing or inhibiting TNFa-mediated inflammation in a subject, the method comprising: a) detecting an increased level of a biomarker associated with TNFa-mediated inflammation in a biological sample obtained from the subject, wherein the increased level of biomarker as compared to a reference indicates that the subject has TNFa-mediated inflammation, and b) administering an agent capable of upregulating the expression or activity of miR-335 to the subject found to have TNFa-mediated inflammation.

[0012] Disclosed herein is an agent capable of up-regulating the expression or activity of miR-335 for use in suppressing or inhibiting TNFa-mediated proinflammatory signaling in a mammalian cell or tissue.

[0013] Disclosed herein is an agent capable of up-regulating the expression or activity of miR-335 to a subject for use in treating, reducing or inhibiting TNFa-mediated inflammation in a subject.

[0014] Disclosed herein is a use of an agent capable of uprcgulating the expression or activity ofmiR-335 in the manufacture of a medicament for the treatment of TNFa-mediated inflammation in a subject.

[0015] Brief description of the drawings

[0016] Embodiments of the present invention will now be described, by way of non-limiting example, with reference to the drawings in which:

[0017] Figure 1. CASP7 is a direct target of miR-335. A) Heatmap shows CASP7 as a bona fide target of miR-335. Up-regulated genes are shown in black box. B) The miR-335 binding site in the CASP73’-UTR (sequence shown in the top row). Mutant seed sequence to disrupt the binding site (underlined). C) Bar graph showing normalized luciferase activity for cells cotransfected with wild-type or mutant luciferase reporter constructs in the presence of miR-335 or control. Renilla luciferase was used as a normalization control. Luciferase activity is expressed as the mean relative to the controls (n = 3). ****P < .001. The student t-test was used to calculate P value; error bars denote means ±SEMs. D) Relative transcript abundance of CASP7 in keratinocytes transfected with miR-335 mimics or control. E) Western blot analysis of CASP7 protein levels in keratinocytes transfected with miR-335 mimics or control, p-actin served as a loading control. F) In situ hybridization sho 'ing miR-335 expression (left panels) on sections from healthy human skin (top) and AD lesional skin (AD-LS) (bottom) (n=5). Immunohistochemical analysis of CASP7 (right panels) on sections from healthy skin (top) and AD lesional skin (bottom). Scale bars = 100 pm.

[0018] Figure 2. CASP7 enhances the expression of pro-inflammatory chemokines: A) Validation of doxycycline (DOX)-inducible CASP7 expression by RT-qPCR. B) Confirmation of doxycycline (DOX)- inducible CASP7 expression by Western blot analysis.

[0019] C) Cytokine profiling reveals differentially expressed chemokines CCL5 (light grey circle) and CCL3 (dark grey circle). D) siRNA knockdown of CASP7 significantly downregulates TNFa-mediated CCL5 expression assessed by RT-qPCR. E) siRNA knockdown of CASP7 significantly downregulates TNFa-mediated CCL3 expression assessed by RT-qPCR. F) Significant downregulation of CCL5 transcript abundance in cells expressing miR-335. G) Significant downregulation of CCL3 transcript abundance in cells expressing miR-335. H) CCL5 protein expression analysis by ELISA confirms the role of miR-335. 1) CCL3 protein expression analysis by ELISA confirms the role of miR-335.Figure 3. Belinostat restores miR-335 expression and suppresses pro-inflammatory pathways: A) Bar graph depicts a dose-dependent effect of Belinostat on induction of miR-335 in keratinocytes by RT-qPCR. B, C) Bar graph shows a significant decrease in CCL5 and CCL3 transcript abundance upon treatment with 250 nM Belinostat compared to treatment with DMSO control. D, E) Violin plots depict CCL5 and CCL3 protein quantification in the conditioned medium from keratinocytes treated with 250 nM Belinostat or DMSO control. **p < 0.05, *** p < 0.01, ****p < 0.001, Student’s / -test. F) Heatmap represents cytokine expression data from a Luminex assay on conditioned medium from the co-culture of keratinocytes and PBMCs. PBMC stimulation using PMA / ionomycin significantly upregulated expression of all chemokines and cytokines. Belinostat treatment downrcgulatcd expression of most cytokines, relative to their levels in the DMSO control.

[0020] G) Belinostat treatment resulted in significant downregulation of chemokines CCL3, CCL5, CCL13, and CCL18 expression, relative to their levels in the DMSO control. H) Belinostat treatment resulted in significant downregulation of cytokines IL-4, IL-13, IL-31 and TSLP expression, relative to their levels in the DMSO control.

[0021] Figure 4. Belinostat suppresses inflammation and restores barrier function in an AD mouse model: A) Photographs of mouse cars after 7 days of treatment with vehicle (ethanol)-only control, MC903, and MC903 + Belinostat. MC903 induces AD-like phenotype which is counteracted by Belinostat. B) Changes in ear thickness across a 7-day course of treatment with MC903 or MC903 + Belinostat. Ear swelling is amarker of ear skin inflammation. C) TEWL measurements on mouse ear skin across a 7-day course of treatment with MC903 or MC903 + Belinostat. D) H&E staining of mouse ear sections following 7-day treatment with vehicle-only control, MC903 or MC903 + Belinostat. E) Immunohistochemical staining for the TSLP in mouse car sections following 7-day treatment with vehicle-only control, MC903 or MC903 + Belinostat.

[0022] Detailed description

[0023] The specification teaches a method of suppressing or inhibiting proinflammatory signaling in a mammalian cell or tissue.

[0024] Provided herein is a method of suppressing or inhibiting proinflammatory signaling in amammalian cell or tissue, the method comprising contacting the mammalian cell with an agent capable of upregulating the expression or activity of miR-335.

[0025] Disclosed herein is a method of suppressing or inhibiting TNFa-mediated proinflammatory signaling in a mammalian cell or tissue, the method comprising contacting the mammalian cell with an agent capable of upregulating the expression or activity of miR-335.

[0026] The methods as defined herein may reduce inflammation, in particular TNFa-mediated inflammation, for the treatment of a disease, such as atopic dermatitis (AD). This may be done by restoring the expression of miR-335 and / or rescue the barrier defect by suppressing cytokine and chemokine expression.

[0027] Without being bound by theory, the inventors have found that the expression of miR-335 may be suppressed by histone deacetylases (HDACs), such as HDAC2. The treatment with histone deacetylase inhibitors (HDACi’s) may facilitate restoration of miR-335 expression. The inventors have identified a specific HDACi, Belinostat, -which may restores the expression of miR-335. Furthermore, by analysing the role of miR-335 in the resolution of inflammation it was discovered that miR-335 is a potent suppressor of pro-inflammatory pathways implicated in diseases such as AD. The therapeutic efficacy of Belinostat in the resolution of TNFa-mediated inflammation, was validated in pre-clinical MC903 mouse models. It was found that treatment with Belinostat significantly downregulate pro-inflammatory chemokines and cytokines, and may be used as a prophylactic and a therapeutic to alleviate AD, in particular TNFa-mediated AD.

[0028] In some embodiments, the agent is an miR-335 agonist or an miR-335 mimic. The miR-335 mimic may be a synthetic double-stranded RNA to replicate endogenous miR-335 function. The agent may be one that is described in WO2021133260A1.

[0029] In one embodiment, the miR-335 agonist is a HDAC inhibitor such as a HDAC2 inhibitor. HDAC inhibitors are well known in the ait. The HDAC2 inhibitor may, for example, be Mocetinostat (PubChemCID: 9865515), Quisinostat, Scriptaid, LMK-235 or belinostat. The miR-335 agonist may comprise Belinostat or a derivative thereof.Belinostat may be used in its native from, or as a salt, hydrate, or solvate. It may therefore be convenient or desirable to prepare, purify, and / or handle a corresponding salt of belinostat, for example, a pharmaceutically acceptable salt. Examples of pharmaceutically acceptable salts are discussed in Berge et at., 1977, “Pharmaceutically Acceptable Salts” J. Pharm. ScL. Vol. 66, pp. 1-19.

[0030] Analogues and derivatives of belinostat are known in the art. Such analogues and derivatives preferably comprise histone deacetylase (HD AC) inhibitor activity. They may also comprise a N-hydroxycinnamamide moiety. For example, analogues and derivatives of belinostat are described in Zhang et al (2019) Design, synthesis and evaluation of belinostat analogs as histone deacetylase inhibitors. Future Medicinal Chemistry, 11(21) and Li et al (2019) Design, synthesis, and biological evaluation of target water-soluble hydroxamic acid-based HDACi derivatives as prodrugs. Chemical Biology & Drug Design 94(4), 1760-1767. An example of a belinostat analogue / derivative is compound 7e, described in Zhang et al (2019), which has been shown to exhibit an IC50 value of 11.5 nM in an HDAC inhibition assay.

[0031] The term “agonist” as defined herein refers to an agent such as a compound, a molecule, or the like, that is capable of increasing the expression or activity of the target.

[0032] The term “miR-335” as used herein refers to microRNA-335, which is a small non-coding RNA that may comprise a nucleic acid sequence of SEQ ID NO: 1. The miR-335 is a master gene regulator, with diverse functions in cancer regulation, metastasis control, and tumor microenvironment modulation. The function of miR-335 varies by tissue type and cancer context. miR-335 may participate in controlling molecular pathways affecting growth, cellular differentiation, apoptosis and tumor biology. miR-335 may also be involved in skin kcratinocytc differentiation and cornification (which arc processes essential for forming a healthy and watertight epidermal barrier), skin development, cell proliferation, differentiation, immune response, wound healing and / or extracellular matrix (ECM) maintenance.

[0033]

[0034] The term “miR-335 mimic” or “microRNA-335 mimic” refers to a synthetic nucleic acid that mimics the action of endogenous miR-335. The miR-335 mimic may be used to generate a downstream effect that corresponds to an upregulation of miR-335. The miR-335 mimic may comprise nucleotide analogues or modified bases, hi one embodiment, the miR-335 mimic comprises a nucleic acid sequence according to SEQ ID No: 7. In one embodiment, the mature miR-335 sequence comprises a nucleic acid sequence according to SEQ ID No: 8.

[0035] Nucleotide analogues include nucleotides having modifications in the chemical structure of the base, sugar and / or phosphate, including, but not limited to, 5-position pyrimidine modifications, 8-position purine modifications, modifications at cytosine exocyclic amines, and substitution of 5-bromo-uracil; and 2’-position sugar modifications, including but not limited to, sugar-modified ribonucleotides in which the 2’-OH is replaced by a group such as an H, OR, R, halo, SH, SR, NH2, NHR, NR2, or CN, wherein R is an alkyl moiety. Nucleotide analogs are also meant to include nucleotides with bases such as inosine, queuosine, xanthine, sugars such as 2’-methyl ribose, non-natural phosphodiester linkages such as methylphosphonates, phosphorothioates and peptides.

[0036] Modified bases refer to nucleotide bases such as, for example, adenine, guanine, cytosine, thymine, uracil, xanthine, inosine, and queuosine that have been modified by the replacement or addition of one or more atoms or groups. Some examples of types of modifications that can comprise nucleotides that are modified with respect to the base moieties include but are not limited to, alkylated, halogenated, thiolated, aminated, amidated, or acetylated bases, individually or in combination. More specific examples include, for example, 5-propynyluridine, 5-propynylcytidine, 6-methyladenine, 6-mcthylguaninc, N,N,-dimcthyladcninc, 2-propyladcninc, 2-propylguaninc, 2-aminoadcninc, 1 -methylinosine, 3 -methyluridine, 5-methylcytidine, 5 -methyluridine and other nucleotides having a modification at the 5 position, 5-(2-amino)propyl uridine, 5-halocytidine, 5-halouridine, 4-acetylcytidine, 1 -methyladenosine, 2 -methyl adenosine, 3-methylcytidine, 6-methyluridine, 2-methylguanosine, 7-methylguanosine, 2,2-dimethylguanosine, 5-methylaminoethyluridine, 5-methyloxyuridine, deazanucleotides such as 7-deaza-adenosine, 6-azouridine, 6-azocytidine, 6-azothymidine, 5-methyl-2-thiouridine, other thio bases such as 2-thiouridinc and 4-thiouridinc and 2-thiocytidinc, dihydrouridinc, pseudouridine, queuosine, archaeosine, naphthyl and substituted naphthyl groups, any O-and N-alkylated purines and pyrimidines such as N6-methyladenosine, 5-methylcarbonylmethyluridine, uridine 5-oxyacetic acid, pyridine-4-one, pyridine-2-one, phenyl and modified phenyl groups such as aminophenol or 2,4,6-trimethoxy benzene, modified cytosines that act as G-clamp nucleotides, 8-substituted adenines and guanines, 5-substituted uracils and thymines, azapyrimidines, carboxyhydroxyalkyl nucleotides, carboxyalkylaminoalkyl nucleotides, and alkylcarbonylalkylated nucleotides. Modified nucleotides also include those nucleotides that are modified with respect to the sugar moiety, as well as nucleotides having sugars or analogs thereof that are not ribosyl. For example, the sugar moieties may be, or be based on, mannoses, arabinoses, glucopyranoses, galactopyranoses, 4 ’-thioribose, and other sugars, heterocycles, or carbocycles.

[0037] In one embodiment, the proinflammatory signaling is associated with Tumor Necrosis Factor alpha (TNFa) or a TNFa-mediated pathway. Tn some embodiments, the TNFa-mediated proinflammatory signaling is further associated with a TNFa-mediated inflammation or a TNFa-mediated inflammatory disease.

[0038] Tumor Necrosis Factor alpha (TNFa) is a proinflammatory cytokine produced by macrophages, monocytes, dendritic cells, and activated T cells. It plays a role in initiating and amplifying inflammation by promoting immune-cell activation, increasing vascular permeability, and driving the recruitment of leukocytes to sites of tissue injury or infection. TNFa is recognized as one of the principal mediators of the acute inflammatory response and functions alongside other major cytokines such as IL-1 and IL-6 to coordinate host defense. Through binding to TNF receptors, TNFR1 and TNFR2, TNFa regulates diverse processes including inflammatory' signaling, apoptosis, and immune-cell proliferation. Dysregulated or chronic TNFa production contributes to a variety of pathological conditions, including autoimmune disorders, cancer, and chronic inflammatory diseases. TNFa may also be upregulated in a chronic inflammatory environment, such as a tumor microenvironment, and act as a pro-tumor factor in many cancers. In some embodiments, the proinflammatory signaling is associated with inflammation at the skin of a subject.

[0039] As used herein, the terms “suppress” or “inhibit” may be used interchangeably and refers to reducing, limiting, preventing, or otherwise decreasing the activity, expression, function, or progression of a gene (such as mRNA), protein, molecule, pathway, or condition. In some embodiments, the inhibition may be partial or complete and encompasses any decrease ininflammatory markers as compared to a reference level. The decrease may be, for example, a decrease of at least about 1%, at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90% or up to and including a 100% decrease or any decrease between 1-100% as compared to a reference level. In some embodiments, the inflammation is detected by measuring the levels of inflammatory markers including, but not limited to, acute-phase proteins (such as C-reactive protein (CRP) and high-sensitivity CRPs), cytokines, proinflammatory cytokines (such as IL-17, IL-22, IL-23, CASP7 and TNFa), interleukin-1 (IL-1), chemokines (such as CXCL8, IL-8, MCP-1, RANTES, CCL3 and CCL5), ccll-adhcsion molecules (such as ICAM-1 and VCAM-1), infection-linked inflammatory markers (such as procalcitonin), or a combination thereof.

[0040] As used herein, the terms “upregulate”, “promote” or “stimulate” may be used interchangeably and refers to increasing, enhancing, elevating, or otherwise augmenting the expression, activity, production, or functional output of a gene, protein, pathway, cellular process, or biological response. Upregulation may be partial or substantial and encompasses any increase as compared to a reference. In some embodiments, an agent is capable of upregulating the expression or activity of miR-335. In another embodiment, the agent is capable of upregulating the expression or activity of miR-335 from about 1% to about 100%. In another embodiment, the upregulation is from about 2-fold to about 10-fold.

[0041] The increase may be, for example, an increase of at least about 1%, at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90% or up to and including a 100% increase or any increase between 1-100% as compared to a reference level. The increase may be from about 2-fold to about 10-fold, such as about 2-fold, about 3 -fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, or more.Provided herein is a method of suppressing or inhibiting inflammation in a mammalian cell or tissue, wherein the method comprising contacting the mammalian cell with an agent capable of upregulating the expression or activity of miR-335.

[0042] Disclosed herein is a method of suppressing or inhibiting TNFa-mediated inflammation in a mammalian cell or tissue, wherein the method comprising contacting the mammalian cell with an agent capable of upregulating the expression or activity of miR-335.

[0043] In one embodiment, the inflammation is associated with an inflammatory disease.

[0044] The terms “inflammation” or “inflammatory” as defined herein refers to a physiological or pathological response of tissue to internal or external stimuli, including but not limited to mechanical injury, infection, allergen exposure, chemical irritation, or immune dysregulation. The inflammatory response or inflammatory signaling is characterized by the activation of resident and infiltrating immune cells, modulation of cytokine and chemokine networks, increased vascular permeability, alterations in tissue architecture, or a combination thereof. In some embodiments, the inflammation is an acute or a chronic inflammation. In some embodiments, the inflammation is a condition characterized by dysrcgulatcd immune or skin barrier responses, contributing to erythema, edema, pruritus, pain, functional impairment of the affected tissue, or a combination thereof.

[0045] Provided herein is a method of treating an inflammatory disease, the method comprising administering an effective amount of an inhibitor of HD AC to a subject.

[0046] Disclosed herein is a method of treating a TNFa-mediated inflammatory disease, the method comprising administering an effective amount of an inhibitor of HD AC to a subject.

[0047] Provided herein is a method of treating an inflammatory disease, the method comprising administering an effective amount of an inhibitor of HDAC to a subject, for increasing the expression or activity of miR-335, and for further suppressing or inhibiting a proinflammatory signaling in the subject.

[0048] Disclosed herein is a method of treating a TNFa-mediated inflammatory disease, the method comprising administering an effective amount of an inhibitor of HDAC to a subject, forincreasing the expression or activity of miR-335, and for further suppressing or inhibiting a proinflammatory signaling in the subject.

[0049] Histone deacetylase (HD AC) is an enzyme that removes acetyl groups from the lysine amino acid present in histones and proteins. The removal of acetyl groups on histones may cause the double helix deoxyribonucleic acid (DNA) strands to wind tighter around the histones, leading to condensed chromatins and gene silencing, thereby reducing the expression of the affected genes. In some embodiments, the histone deacetylase is also known as lysine deacetylase (KDAC). The inhibitor of histone deacetylase may inhibit the action of the enzyme, histone deacetylase, to allow an expression of a target gene, or to prevent the suppression of a target gene.

[0050] In some embodiments, the inhibitor of HDAC or the miR-335 agonist is Belinostat.

[0051] In one embodiment, inhibitor of HDAC or the miR-335 agonist has a chemical structure of

[0052]

[0053] Provided herein are methods of suppressing or inhibiting proinflammatory signaling in a mammalian cell or tissue, wherein the cell or the tissue is in a subject suffering from an inflammatory disease.

[0054] In some embodiments, the inflammatory disease is a TNFa-mediated inflammatory disease or TNFa-mediated inflammation. TNFa-mediated inflammatory disease or TNFa-mediated inflammation refers to an inflammatory response that is initiated, amplified, or sustained by the activity of Tumor Necrosis Factor-alpha (TNFa), a pro-inflammatory cytokine produced primarily by macrophages and other immune cells.

[0055] Upon recognition of microbial components or endogenous danger signals, immune cells secrete TNFa in a soluble or membrane-bound form. TNFa subsequently binds TNFR1 and / or TNFR2, triggering downstream cascades characterized by NF-KB activation, MAPK signaling, apoptotic or necroptotic signaling, endothelial activation and leukocyte adhesion,systemic effects such as sustained TNFa release induces fever, cachexia, acute -phase responses, and systemic inflammatory syndromes, and a combination thereof. MAPK signaling is characterized by the recruitment of TRADD, TRAF2, and RIPK1 leads to activation of the IKK complex and subsequent nuclear translocation of NF-KB, resulting in transcription of proinflammatory genes, including cytokines (IL-10, IL-6), chemokines (CXCL8 / IL-8, CCL2), adhesion molecules (ICAM-1, VCAM-1), survival factors, and a combination thereof. The activation of JNK, ERK, and p38 MAPK pathways may also promote cytokine expression, leukocyte recruitment, and modulation of cellular stress responses. TNFR1 may also engage caspase-8, leading to apoptosis, or R1PK1 / R1PK3 / MLKL pathways, resulting in necroptosis. TNFa may also upregulate endothelial adhesion molecules and increases vascular' permeability, facilitating rapid leukocyte extravasation into inflamed tissues.

[0056] Caspase-7 (CASP7) is an executioner caspase involved in apoptosis. CASP7 may act downstream of initiator caspases such as caspase-8 and caspase-9. Upon activation, CASP7 may cleave intracellular substrates, leading to controlled cell dismantling. CASP7 may be activated in extrinsic (via death receptors) and intrinsic (via mitochondria) apoptotic pathways. The expression and / or activity of CASP7 may be mediated by TNFa.

[0057] In some embodiments, the inflammatory disease is a skin-related inflammatory disease or a skin inflammatory disease. In another embodiment, the skin inflammatory disease is atopic dermatitis. In one embodiment, the inflammatory disease is one that is associated with an increased expression of CASP7 or is a C AS P7 -mediated inflammatory disease. In one embodiment, the inflammatory disease is a atopic dermatitis associated with increased expression of CASP7 or a CASP7-mediated atopic dermatitis.

[0058] In some embodiments, the inflammatory disease is an autoimmune and / or systemic inflammatory disease. In some embodiments, the inflammatory disease is a gastrointestinal inflammatory disease. In some embodiments, the inflammatory disease is a respiratory and / or allergy-related inflammation. In some embodiments, the inflammatory disease is a metabolic and / or obesity-related inflammation. In some embodiments, the inflammatory disease is a cardiovascular' and / or vascular inflammatory disorder. In some embodiments, the inflammatory disease is an infectious and / or immune activation-related disease. In some embodiments, the inflammatory disease is a neurological and / or neuroinflammatory disease.In some embodiments, the inflammatory disease is a dermatological inflammatory condition. In some embodiments, the inflammatory disease is a musculoskeletal or tissue inflammation.

[0059] Examples of TNFa-mediated inflammation or inflammatory disease include but not limited to rheumatoid arthritis, psoriasis, psoriatic arthritis, ankylosing spondylitis, systemic lupus erythematosus, Behcet's disease, juvenile idiopathic arthritis, Crohn’s disease, ulcerative colitis, celiac disease, chronic pancreatitis, asthma (severe, steroid-resistant forms), chronic obstructive pulmonary disease (COPD), sarcoidosis, allergic rhinitis (hay fever), obesity-induced chronic low-grade inflammation, diabetes mellitus (type I and type II), TNFa-associated metabolic dysfunction, non-alcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), atherosclerosis, vasculitis, ischcmia-rcpcrfusion injury, heart failure with inflammation-associated remodeling, sepsis, septic shock, chronic infections with excessive inflammatory response (such as tuberculosis-associated inflammation, and viral hepatitis-induced inflammation), systemic inflammatory response syndrome (SIRS), multiple sclerosis, Alzheimer’s disease, Parkinson’s disease, neuropathic pain, atopic dermatitis, hidradenitis suppurativa, urticaria, chronic urticaria, osteoarthritis, tendonitis and enthesitis.

[0060] In some embodiments, the inflammatory disease is a skin-related and TNFa-mediated inflammatory disease. Examples of skin-related TNFa-mediated inflammatory disease include but are not limited to psoriasis, atopic dermatitis, hidradenitis supprativa, chronic urticaria, cutaneous sarcoidosis, pyoderma gangrenosum, acute febrile neutrophilic dermatosis (Sweet’s syndrome), erythema nodosum, lupus-related cutaneous inflammation, cutaneous inflammation, and contact dermatitis.

[0061] Atopic dermatitis (AD), commonly referred to as eczema, is a chronic, inflammatory skin condition characterized by episodes of itchy, red, and inflamed skin. It is one of the most prevalent dermatological conditions worldwide, affecting individuals of all ages but most commonly beginning in childhood. Atopic dermatitis is part of the "atopic triad," often associated with other allergic conditions such as asthma and allergic rhinitis (hay fever). AD is characterized by periodic flares, intense pruritus and eczematous lesions, which vary depending on the stage of the disease. AD arises from a complex interplay of genetic, immunological, and environmental factors. Mutations in the filaggrin gene (FLG), which iscritical for skin barrier function, are commonly associated with AD. Impaired skin barrier increases trans-epidermal water loss, susceptibility to allergens, and infections. Immune dysregulation, involving type 2 T-helper (Th2) cells, leads to chronic inflammation and exacerbates barrier dysfunction.

[0062] In some embodiments, the atopic dermatitis is characterized by an activation of TNFa-mediated proinflammatory pathway.

[0063] In some embodiments, the atopic dermatitis and / or the activation of TNFa-mediated proinflammatory pathway is characterized by an elevated level of biomarker as compared to a reference, selected from TNFa, CASP7, TARC, CTACK, MDC, EDN, SCCA2, LDH, IL-18, NOS2, hBD-2, MMP-8, MMP-9, IL-17, IL-23, IL-33, IDO1, pcriostin, PARC, IL-22, eotaxin-1, eotaxin-3, IL-8, an IL4RA polymorphism, a TSLP variant, FABP5, Staphylococcus aureus abundance, and a combination thereof. In some embodiments, AD is characterized by the lack or decrease in level of biomarker as compared to a reference, selected from involucrin, small proline rich proteins (SPRRs), SOX6, or a combination thereof.

[0064] In some embodiments, the atopic dermatitis is characterized by an increased level of TNFa in a biological sample obtained from the subject as compared to a reference. In some embodiments, the atopic dermatitis is characterized by an increased level of CASP7 in a biological sample obtained from the subject as compared to a reference. In some embodiments, the atopic dermatitis is characterized by an increased level of CCL3 in a biological sample obtained from the subject as compared to a reference. In some embodiments, the atopic dermatitis is characterized by an increased level of CCL5 in a biological sample obtained from the subject as compared to a reference. In some embodiments, the atopic dermatitis is characterized by increased levels of CASP7, CCL3 and CCL5 in a biological sample obtained from the subject as compared to a reference. In some embodiments, the atopic dermatitis is characterized or further characterized by an increased level of miR335 in a biological sample obtained from the subject as compared to a reference.

[0065] The level of the biomarkcr may be compared to a reference to determine changes in expression (such as overexpression or upregulation). In one embodiment, the reference isthe level of the biotnarker in a healthy cell or tissue (i.e., a cell / tissue without an inflammatory disease or condition such as AD) from the same subject or from a different subject of the same species. In another embodiment, the reference is an average gene / protein expression level in samples from a population of subjects of the same species (e.g., of varying ages, ethnic backgrounds and genders) without an inflammatory disease or condition such as AD The reference values can be stored in a database and used as a reference in subsequent analyses. In some embodiments, the reference is the level of biomarker(s) in a healthy subject, or skin samples without an inflammatory disease or condition such as AD, or skin samples from a healthy subject.

[0066] Provided herein are methods of suppressing or inhibiting proinflammat ory signaling in a mammalian cell or tissue, wherein the cell or the tissue is in a subject suffering from a skin inflammatory disease.

[0067] In some embodiments, the cell is a skin cell. In some embodiments, the cell is a keratinocyte, melanocyte, Langerhans cell, Merkel cell, epithelial cell, fibroblast, macrophage, Mast cell, adipocytes, dermal stem cell, hypodermal cell, or a combination thereof. In some embodiments, the inflammatory disease is a skin inflammatory disease. In some embodiments, the skin inflammatory disease is atopic dermatitis, eczema, psoriasis, seborrheic dermatitis, contact dermatitis, acne vulgaris, rosacea, urticaria, hives, lichen planus, or a combination thereof. In another embodiment, the skin inflammatory disease is atopic dermatitis.

[0068] In some embodiments, the inflammatory' disease is diabetes mellitus. In some embodiments, the inflammatory disease is a type II diabetes mellitus. In some embodiments, the inflammatory disease is a sepsis-induced myocardial injury. In some embodiments, the inflammatory disease is a renal fibrosis. In some embodiments, the inflammatory disease is an osteoarthritis.

[0069] Type II diabetes mellitus is a symptom wherein the body of a subject is unable to use insulin correctly, or unable to produce enough insulin, and leading to build up of sugar in the blood. In some embodiments, the subject has an elevated level of blood glucose level as compared to a healthy individual under the same circumstances. In some embodiments, the subject has hyperglycemia. In some embodiments, the subject may experience more thirst, moreurination, more hungry, weight loss, tiredness, blurred vision, slow-healing sores, frequent infections, or numbness or tingling in the hands or feet. In some embodiments, the subject may have other metabolic diseases.

[0070] In some embodiments, the inflammatory disease is myocardial injury. In some embodiments, the inflammatory disease is sepsis-induced myocardial injury. Sepsis-induced myocardial injury (SIMI) is a condition of damage to the heart, heart muscles, cardiomyocytes, or myocardial cells, which may be due to the body’s response to an infection. This may cause inflammation, cellular' dysfunction, and microvascular problems, leading to a weakened heart muscle. In some embodiments, the subject is suffering from cardiomyopathy. In some embodiments, the sepsis-induced myocardial injury may be caused by immune dysrcgulation, mitochondrial dysfunction, dysrcgulation in the nitric oxide pathways, microvascular issues (such as endothelial damage, and fluid leakage), metabolic reprogramming, or autonomic dysregulation.

[0071] Renal fibrosis is a condition of excessive scarring and accumulation of extracellular matrix (ECM) proteins in the kidney. In some embodiments, the subject may be suffering from chronic kidney disease (CKD), progressive loss of kidney function, and / or renal failure requiring dialysis or kidney transplant.

[0072] Osteoarthritis is a degenerative joint disease whereby the protective cartilage, generally anatomically located at the ends of long bones, wears down, and osteoarthritis may cause pain, stiffness, swelling and reduced mobility, typically in the hands, hips, knees and spine. In some embodiments, the osteoarthritis in a subject may also be caused by gradual wear-and-tear of the cartilage, injury to the cartilage, or aging.

[0073] In some embodiments, the agent reduces expression and / or secretion of proinflammatory cytokines. In some embodiments, the proinflammatory cytokines comprises CASP7. In another embodiment, the proinflammatory cytokines comprises CCL3 and / or CCL5.

[0074] Provided herein are methods of treating TNFa-mediated diseases.

[0075] In some embodiments, the TNFa-mediated disease is a cancer. In some embodiments, the TNFa promotes the inflammation in the cancer or tumor microenvironment. Examples ofcancers that are mediated by or associated with TNFa includes but not limited to , breast cancer, prostate cancer, ovarian cancer, cervical cancer, pancreatic cancer, colorectal cancer, lung cancer, hepatocellular cancer, gastric cancer, liver cancer, bladder cancer, cancer of the urinary tract, thyroid cancer, renal cancer, carcinoma, melanoma, brain cancer, non-small cell lung cancer, squamous cell cancer of the head and neck, endometrial cancer, multiple myeloma, rectal cancer, and esophageal cancer. In some embodiments, the cancer is a metastatic cancer.

[0076] In some embodiments, the agent capable of upregulating the expression or activity of miR-335 further reduces expression and / or secretion of proinflammatory cytokines. In some embodiments, the proinflammatory cytokines comprises TNFa, Casp7, CCL3, CCL5, or a combination thereof.

[0077] Provided herein is a method of treating, reducing or inhibiting inflammation in a subject, the method comprising administering an agent capable of upregulating the expression or activity of miR-335 to a subject.

[0078] Disclosed herein is a method of treating, reducing or inhibiting TNFa-mediated inflammation in a subject, the method comprising administering an agent capable of upregulating the expression or activity of miR-335 to a subject.

[0079] In some embodiments, the inflammation is characterized by sustained expression and / or expression of TNFa. In some embodiments, the inflammation is characterized by sustained expression and / or expression of Caspase-7 (Casp7). In some embodiments, the inflammation is characterized by sustained expression and / or secretion of CCL3 and / or CCL5.

[0080] Disclosed herein is a method of treating, reducing or inhibiting TNFa-mediated inflammation in a subject, the method comprising: a) detecting an increased level of a biomarker associated with TNFa-mediated inflammation in a biological sample obtained from the subject, wherein the increased level of biomarker as compared to a reference indicates that the subject has TNFa-mediated inflammation, and b) administering an agent capable of upregulating the expression or activity of miR-335 to the subject found to have TNFa-mediated inflammation.In some embodiments, the biomarker is a cell, mRNA, cDNA, protein or peptide that is associated with TNFa inflammation. Examples of biomarkers include but are not limited to TNFa, TNF receptor 1 (TNFR1), TNFR2, IL-6, IL-10, IFN-y, CXCL8, IL-8, C-reactive protein (CRP), serum amyloid A (SAA), activated monocytes, macrophages, NF-KB activation markers, ICAM-1, VCAM-1, TNFa, Caspase-7 (Casp7), CCL3 and CCL5.

[0081] The biomarkers and the corresponding level of expression or activity may be detected or measured using methods known in the art. Examples of the methods of detecting the biomarkers including but are not limited to Western blot, RT-qPCR, RNA-seq, transcriptome panels, enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, immunocytochemistry, immunofluorescence, in situ hybridisation, tissue sectioning, multiplex immunoassays, flowcytomctry, and mass spectrometry.

[0082] In one embodiment, the biomarker is selected from the group consisting of TNFa, Caspase-7 (Casp7), CCL3 and CCL5.

[0083] In one embodiment, the biomarker is miR-335. This may be detected by using well known techniques such as in situ hybridization or qPCR.

[0084] In one embodiment, the subject is suffering from atopic dermatitis. In another embodiment, the subject is suffering from TNFa-mediated atopic dermatitis.

[0085] The TNFa-mediated atopic dermatitis may be characterized by an increased in TNF-a, IFN-y, IL-17, IL-22, TNFR1 and / or TNFR2 expression on lymphocytes (including T cells, B cells and monocytes), increased innate immune cells (such as myeloid cells) at the site of atopic dermatitis, increased Thl, Thl7, and / or Th22 cytokines, or a combination thereof.

[0086] The non-TNFa-mediated atopic dermatitis may be characterized by an increased in Th2 cytokines, IL-4, IL-13, IL-5, IL-31, IgE, Oncostatin M (OSM), FccRI expression, eosinophil cationic protein (ECP), eosinophil-derived neurotoxin (EDN), CCL17, CCL22, or a combination thereof.

[0087] In one embodiment, there is provided a method of treating, reducing or inhibiting TNFa-mediated inflammation in a subject, the method comprising: a) selecting a subject found thave an increased level of a biomarker associated with TNFa-mediated inflammation in a biological sample obtained from the subject, wherein the increased level of biomarker as compared to a reference indicates that the subject has TNFa-mediated inflammation, and b) administering an agent capable of upregulating the expression or activity of miR-335 to the subject found to have TNFa-mediated inflammation.

[0088] In one embodiment, there is provided a method of stratifying a patient population for treating with an agent capable of upregulating the expression or activity of miR-335 (such as belinostat), the method comprising (a) detecting the level of a biomarker associated with TNFa-mediated inflammation in a biological sample obtained from the subject and (b) classifying patients into a responder group and a non-responder group based on the level of the biomarkcr, wherein an increased level of the biomarkcr classifies patients into a responder group, wherein a lack of increase in the level of the biomarker classifies patients into a non-responder group.

[0089] Disclosed herein is a composition comprising an agent capable of upregulating the expression or activity of miR-335. In some embodiments, the agent is an inhibitor of HD AC.

[0090] In some embodiments, the composition is a pharmaceutical composition comprises an agent capable of upregulating the expression or activity of miR-335.

[0091] The pharmaceutical compositions may be in a variety of forms. These include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, liposomes and suppositories. The preferred form depends on the intended mode of administration and therapeutic application. Active agents herein may be administered in a variety of routes including oral, transdcrmal, topical, parenteral (including subcutaneous, intravenous, intramuscular and intraperitoneal), vaginal, rectal, oropharyngeal, intrapulmonary and intranasal routes. In some embodiments, the compositions are in the form of injectable or infusible solutions. In some embodiments, the administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular, intranasal, topical or transdcrmal). Pharmaceutical compositions can be in the form of sterile aqueous solutions or dispersions. They can also be formulated in a micro-emulsion, liposome, or other ordered structure suitable to high drug concentration.The pharmaceutical composition may be a controlled release formulation, including implants, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, poly orthoesters, and poly lactic acid.

[0092] By “pharmaceutically acceptable carrier” is meant a pharmaceutical vehicle comprised of a material that is not biologically or otherwise undesirable, i.e., the material may be administered to a subject along with the selected active agent without causing any or a substantial adverse reaction. The compositions may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general, the compositions arc prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product. Carriers may include excipients and other additives such as diluents, detergents, colouring agents, wetting or emulsifying agents, pH buffering agents, preservatives, and the like.

[0093] Representative pharmaceutically acceptable carriers include any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated herein by reference). Except insofar as any conventional carrier is incompatible with the active ingrcdicnt(s), its use in the pharmaceutical compositions is contemplated.

[0094] The pharmaceutical compositions may be in a variety of forms. These include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, liposomes and suppositories. The preferred form depends on the intended mode of administration and therapeutic application. Suitable pharmaceutical compositions may be administered intravenously, subcutaneously or intramuscularly. In some embodiments, the compositions arc in the form of injectable orinfusible solutions. In some embodiments, the administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular, intranasal, topical or transdermal).

[0095] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. In the subject invention, pharmaceutically acceptable earners include, but are not limited to, 0.01-0. IM and preferably 0.05M phosphate buffer or 0.8% saline. Other common parenteral vehicles include sodium phosphate solutions, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose, and the like. Preservatives and other additives can also be present such as for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like.

[0096] In some embodiments, the composition is a topical composition. In some embodiments, the composition is a transdermal composition.

[0097] Modes of administration including topical and transdermal administration may be possible.

[0098] The pharmaceutical composition of the present invention may be suitable for topical administration to the skin may comprise an agent capable of up-regulating the expression or activity of miR-335 dissolved or suspended in any suitable carrier or base and may be in the form of lotions, gel, creams, pastes, ointments and the like. Suitable carriers include mineral oil, propylene glycol, polyoxyethylene, polyoxypropylcnc, emulsifying wax, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water. Transdermal patches may also be used to administer the pharmaceutical composition of the invention.

[0099] Compositions of the invention may take many different forms, depending upon the way in which it is intended that they are to be used. Merely by way of example, a composition of the invention may take the form of a composition for topical administration. A composition of the invention in accordance with this embodiment may be used to provide localizedadministration of an effective amount of an inhibitor thereof to a site where it is desired to inhibit inflammation.

[0100] Topical compositions may be formulated as is known in the art for direct application to a target area, for example the scalp, nails, hair or skin. Formulations for topical application may take the form, for example, of shampoos, conditioners, other hair products, lotions, serums, creams, pastes, jellies, gels, powders, dispersions, microemulsions, ointments, gel sticks, aerosol formulations (e.g., sprays or foams), soaps. Other conventional forms for this purpose include impregnated patches or pads, dressings and coated bandages or other polymer coverings. In some embodiments, the active agents are formulated for application to the scalp, for example, in the form of a shampoo, conditioner, serum, gel or spray.

[0101] These formulations can contain pharmaceutically and / or cosmetically acceptable carriers, vehicles and adjuvants that are well-known in the art.

[0102] Suitable carrier materials for topical administration include any carrier or vehicle commonly used as a base for creams, ointments, gels, emulsions, lotions, pastes, jellies, sprays, foams, powders, or paints for topical administration, including but not limited to emulsifying agents, inert carriers including hydrocarbon bases, emulsifying bases, water-soluble bases, or combinations thereof. Suitable solvents, emollients and emulsifiers for hydrophobic topical formulations include lanolin, paraffin, beeswax, emulsifying waxes, dimethicones, mineral oils, silicone oils, vegetable oils, fatty acids and alkyl esters of fatty acids or dicarboxylic acids, triglyceride esters, fatty alcohols and fatty alcohol ethers and sterols. Other suitable solvents, emollients and emulsifiers include polyhydric alcohols and polyether derivatives such as glycerol, sorbitol, polyethylene glycols, polypropylene glycols, ionic and zwitterionic surfactants, amphoteric surfactants and non-ionic surfactants.

[0103] Suitable examples of forms of compositions of the invention include those selected from the group consisting of creams, pastes, lotions, serums, ointments, gels, oils, sprays, and foams. A composition of the invention may comprise an inhibitor, in combination with an acceptable carrier such as polyethylene glycol (PEG).In another embodiment, an agent can be provided to the individual as a composition comprising emulsifying ointment BP, isopropyl myristate, hydroxyethylcellulose, glycerol, phenoxyethanol, propylene glycol and water.

[0104] In another embodiment, an agent can be formulated with a delivery vehicle. For example, in a suitable embodiment, an inhibitor of the invention may comprise liposomes in which at least some of the inhibitor, is incorporated.

[0105] By way of further example, suitable delivery vehicles include those selected from the group consisting of: liposomes, amphoteric and cationic liposomes, niosomes, lipophilic formulations, aqueous-alcoholic solutions, hydrophilic formulations, water-in-oil nanocmulsions, nanoparticlcs of various sizes, microparticles of various sizes, polystyrene microspheres of various sizes, titanium dioxide particles and solid lipid particles.

[0106] Suitably liposomes employed in the compositions or methods of the invention may be ones that have a particular affinity for skin or keratinocytes. By way of example suitable liposomes include, amphoteric and cationic liposomes and niosomes.

[0107] In a suitable embodiment an agent capable of up-regulating the expression or activity of miR-335 may be formulated for use in combination with a device which enhances transdermal penetration of the composition.

[0108] In some embodiments, the agent capable of up-regulating the expression or activity of miR-335 is provided at a concentration of about 0.1% w / w to about 5% w / w, such as about 0.1% w / w, about 0.5% w / w, about 1% w / w, about 1.5% w / w, about 2% w / w, about 2.5% w / w, about 3% w / w, about 3.5% w / w, about 4% w / w, about 4.5% w / w, or about 5% w / w. In one embodiment, the inhibitor is provided at a concentration of about 1% w / w to about 3% w / w.

[0109] In some embodiments, the agent capable of up-regulating the expression or activity of miR-335 is provided at a concentration of about 20 nM to about 500 nM, such as about 20 nM, about 30 nM, about 40 nM, about 50 nM, about, about 65 nM, about 80 nM, about 100 nM, about 125 nM, about 150 nM, about 200 nM, about 250 nM, about 300 nM, about 400 nM, about 500 nM. In one embodiment, the inhibitor is provided at a concentration of about 200 nM to about 300 nM.The term “nucleic acid” refers to a deoxyribonucleotide or ribonucleotide polymer in either single- or double- stranded form, and unless otherwise limited, encompasses known analogues of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally occurring nucleotides.

[0110] As used herein, the term “nucleic acid”, and equivalent terms such as “polynucleotide”, refer to a polymeric form of nucleotides of any length, such as ribonucleotides, deoxyribonucleotides or peptide nucleic acids (PNAs), that comprise purine and pyrimidine bases, or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. The nucleic acid may be double stranded or single stranded. References to single stranded nucleic acids include references to the sense or antisense strands. The backbone of the polynucleotide can comprise sugars and phosphate groups, as may typically be found in RNA or DNA, or modified or substituted sugar or phosphate groups. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. The sequence of nucleotides may be interrupted by non-nucleotide components. The terms nucleoside, nucleotide, deoxynucleoside and deoxynucleotide generally include complements, fragments and variants of the nucleoside, nucleotide, deoxy nucleoside and deoxy nucleotide, or analogs thereof.

[0111] The term “nucleotide” refers to a ribonucleotide or a deoxyribonucleotide or modified form thereof, as well as an analogue thereof. Nucleotides include species that comprise purines, e.g., adenine, hypoxanthine, guanine, and their derivatives and analogues, as well as pyrimidines, e.g., cytosine, uracil, thymine, and their derivatives and analogues.

[0112] An “oligonucleotide” as used herein is a single stranded molecule which may be used in hybridization or amplification technologies. In general, an oligonucleotide may be any integer from about 15 to about 100 nucleotides in length but may also be of greater length.

[0113] As used herein, the term “subject” includes any human or non-human animal. In one embodiment, the subject is a human. The term “non-human animal” includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dog, cow, chickens, amphibians, reptiles, etc.The terms “patient”, “subject”, “host” or “individual” used interchangeably herein, refer to any subject, particularly a vertebrate subject, and even more particularly a mammalian subject, for whom therapy or prophylaxis is desired. Suitable vertebrate animals that fall within the scope of the invention include, but are not restricted to, any member of the subphylum Chordata including primates (e.g., humans, monkeys and apes, and includes species of monkeys such from the genus Macaca (e.g., cynomologus monkeys such as Macaca fascicularis, and / or rhesus monkeys (Macaca mulatto)) and baboon (Papio ursinus), as well as marmosets (species from the genus Callithrix), squirrel monkeys (species from the genus Saimiri) and tamarins (species from the genus Saguinus), as well as species of apes such as chimpanzees (Pan troglodytes)), rodents (e.g., mice rats, guinea pigs), lagomorphs (e.g., rabbits, hares), bovines (e.g., cattle), ovines (e.g., sheep), caprines (e.g., goats), porcincs (e.g., pigs), equines (e.g., horses), canines (e.g., dogs), felines (e.g., cats), avians (e.g., chickens, turkeys, ducks, geese, companion birds such as canaries, budgerigars etc.), marine mammals (e.g., dolphins, whales), reptiles (snakes, frogs, lizards etc.), and fish.

[0114] As used herein, the terms “treating”, “treatment” and the like refer to partially or completely alleviating, ameliorating, improving, relieving, reducing, delaying onset of, inhibiting progression of, reducing severity of, and / or reducing incidence of one or more symptoms or features of a particular infection, disease, disorder, and / or condition for at least a period of time. It is also to be understood that terms “treating”, “treatment” and the like do not imply that the condition, or a symptom thereof, is permanently relieved, reduced, alleviated, ameliorated or otherwise inhibited and therefore also encompasses the temporary relief, reduction, alleviation, amelioration or otherwise inhibition of the condition, or of a symptom thereof.

[0115] The term “administering” refers to contacting, applying, injecting, transfusing or providing an inhibitor as referred to herein to a subject.

[0116] By “effective amount”, in the context of treating or preventing a condition is meant the administration of an amount of a depigmentation agent to an individual in need of such treatment or prophylaxis, either in a single dose or as part of a series, that is effective for the prevention of incurring a symptom, holding in check such symptoms, and / or treating existing symptoms, of that condition. The effective amount will vary depending upon the health and physical condition of the individual to be treated, the taxonomic group of individual to betreated, the formulation of the composition, the assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.

[0117] Dosing may occur at intervals of minutes, hours, days, weeks, months or years or continuously over any one of these periods. Suitable dosages may lie within the range of about 0.1 ng per kg of body weight to 1 g per kg of body weight per dosage, such as is in the range of 1 mg to 1 g per kg of body weight per dosage. In one embodiment, the dosage may be in the range of 1 mg to 500 mg per kg of body weight per dosage. In another embodiment, the dosage may be in the range of 1 mg to 250 mg per kg of body weight per dosage. In yet another embodiment, the dosage may be in the range of 1 mg to 100 mg per kg of body weight per dosage, such as up to 50 mg per body weight per dosage.

[0118] Suitable dosage amounts and dosing regimens can be determined by the attending physician and may depend on the severity of the condition as well as the general age, health and weight of the patient to be treated.

[0119] The terns “detecting”, “determining”, “measuring”, “evaluating”, “assessing” and “assaying” arc used interchangeably herein to refer to any form of measurement, and include determining if an element is present or not. These terms include both quantitative and / or qualitative determinations. Assessing may be relative or absolute. The methods defined herein may comprise RT-qPCR, luciferase assays, Western blotting, immunohistochemistrybased assays, phenotypic observations, microscopy, flow cytometry, and enzyme-linked immunosorbent assay (ELISA).

[0120] As used herein, the term “reference” refers to a subject or a testing condition that is not administered with an agent. In some embodiments, the reference is a subject or a testing condition that is not administered with the agent. In some embodiments, the reference is a healthy subject.

[0121] The term “sample” herein is used in its broadest sense. In one sense, it is meant to include a specimen or culture obtained from any source, including both biological and environmental sources. A “biological sample” includes within its scope a collection of similar fluids, cells, or tissues isolated from a biological source, such as a whole organism or in vitro culture.Samples include but are not limited to tissue biopsies, tissue resections, tissue aspirates, swabs (e.g., buccal swabs), whole blood, plasma, serum, urine, saliva, cerebrospinal fluid, and cell cultures, and may be obtained using any suitable method known in the art. Archival tissues, such as those having treatment or outcome history may also be used for sample extraction. The sample may be pooled from multiple aliquots. Samples include untreated, treated, diluted and concentrated samples.

[0122] A “biological fluid” herein includes, but is not limited to, intravascular fluid (e.g., blood, plasma, serum, lymph), urine, saliva, sputum, cerebrospinal fluid, pleural fluid, fluid of the respiratory, intestinal, and genitourinary tracts, synovial fluid, vaginal secretion, tear fluid, pus, breast milk, semen, fluid from ascites, cyst or tumour, amniotic fluid, or combinations thereof.

[0123] Provided herein is an agent capable of up-regulating the expression or activity of miR-335 for use in suppressing or inhibiting proinflammatory signaling in a mammalian cell or tissue.

[0124] Disclosed herein is an agent capable of up-regulating the expression or activity of miR-335 for use in suppressing or inhibiting TNFa-mediated proinflammatory signaling in a mammalian cell or tissue.

[0125] Provided herein is an agent capable of up-regulating the expression or activity of miR-335 to a subject for use in treating, reducing or inhibiting inflammation in a subject.

[0126] Disclosed herein is an agent capable of up-regulating the expression or activity of miR-335 to a subject for use in treating, reducing or inhibiting TNFa-mediated inflammation in a subject.

[0127] Provided herein is a use of an agent capable of upregulating the expression or activity of miR-3335 in the manufacture of a medicament for the treatment of inflammation in a subject.

[0128] Disclosed herein is a use of an agent capable of upregulating the expression or activity of miR-3335 in the manufacture of a medicament for the treatment of TNFa-mediated inflammation in a subject.As used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (or).

[0129] As used in this application, the singular form “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “an agent” includes a plurality of agents, including mixtures thereof.

[0130] Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0131] The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

[0132] Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications, which fall within the spirit and scope. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

[0133] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0134] Certain embodiments of the invention will now be described with reference to the following examples which are intended for the purpose of illustration only and are not intended to limit the scope of the generality hereinbefore described.EXAMPLES

[0135] Example 1

[0136] CASP7 is a direct target of miR-335: MicroRNAs (miRNA) are non-coding RNAs, that function as tiny molecular rheostats regulating multiple cellular pathways and are often found dysregulated in diseases. The data highlights the role of miR-335 in controlling inflammation. With this background the objective is to restore miR-335 expression and rectify the dysregulated pro-inflammatory pathway using small molecule modulators of miR-335 expression.

[0137] As depicted in the heatmap, CASP7 has been identified as a bona fide target of miR-335 (Fig. 1A). An analysis of the CASP7 3’UTR revealed miR-335 binding site (Fig. IB). To determine whether CASP7 is a direct target of miR-335, the 3’-UTR was cloned into a luciferase reporter construct. Co-transfection of miR-335 mimics with CASP7 wild-type 3’-UTRs, resulted in significant reduction of the luciferase reporter activity, while mutation of miR-335 binding sites blocked miR-335-mediated regulation of CASP73’-UTR confirming that CASP7 is a direct target of miR-335 (Fig. 1C). CASP7 was downregulated in N / TERTs expressing miR-335 compared to scrambled control. The results were validated by RT-qPCR and confirmed by western blot analysis (Fig. ID, IE). A clear inverse correlation in the expression pattern of miR-335 and CASP7 was reflected in normal healthy skin sections (Fig. IF upper panel). Immunohistochemistry analysis of healthy skin sections demonstrated intense cytoplasmic staining of C ASP7, which was limited to basal layer of epidermis, which expresses little or no miR-335 (Fig. IF upper panel). However, in the suprabasal layers of epidermis with abundant miR-335 expression, CASP7 could not be detected. On the contrary in AD lesional skin sections in the absence of miR-335, significant expression of CASP7 throughout the epidermis was observed (Fig. IF lower panel).

[0138] miR-335 mediated suppression of pro-inflammatory pathways: To understand the underlying mechanism by which CASP7 mediates the pro-inflammatory pathway in AD, a stable keratinocyte cell line with doxycycline (DOX)-inducible CASP7 expression was developed. Upregulation of CASP7 transcript abundance and protein upon doxycycline treatment were apparent (Fig. 2A, 2B). To determine the downstream effect of CASP7 on pro -inflammatory cytokines, cells were treated with DOX, and the conditioned medium was subjected to cytokine array analysis. Analysis of the cytokine array data indicates CCL5(Fig.2C, light grey circle) and CCL3 (Fig.2C, dark grey circle) as the two cytokines, which are significantly upregulated upon DOX treatment, suggesting that CASP7 is an upstream regulator of these cytokines. RANTES / CCL5 and Macrophage inflammatory protein (MIP)-la / CCL3 are members of the CC family of chemoattractant cytokines (chemokines). Increased expression of CCL5 and CCL3 were observed in AD patients compared to healthy controls and suggests that CCL5 and CCL3 are important in the recruitment of the mononuclear cell infiltrate.

[0139] Having identified CCL5 and CCL3 as keratinocyte-intrinsic chemokines that are induced by CASP7 overexpression, their response to inflammatory cytokine stimulation was investigated. Keratinocytes from AD patients are known to upregulate CCL5 in response to TNFa. To evaluate the effect of CASP7 on TNFa-mcdiatcd CCL5 and CCL3 expression, siRNA knockdowns of CASP7 in keratinocytes (Fig. 2D, 2E) was performed. CASP7 knockdown reduces both baseline and stimulated levels of CCL5 and CCL3 expression, relative to those obtained using a control siRNA (Fig. 2D, 2E). As CASP7 is suppressed by miR-335 in healthy skin (Fig. 1), it was expected that miR-335 transfection should phenocopy the effects of siCASP7. Indeed, miR-335 also suppressed CCL5 and CCL3 transcript expression as measured by RT-qPCR (Fig. 2F, 2G). Further quantification of CCL5 and CCL3 protein expression by enzyme-linked immunosorbent assay (ELISA) reflected the same trend in the transcript abundance (Fig. 2H, 21). In summary, miR-335 in epidermal keratinocytes limits secretion of proinflammatory chemokines CCL5 and CCL3. This effect is mediated by CASP7, which is a direct target of miR-335. In AD, the loss of miR-335 from the epidermis results in sustained expression of CASP7. The resultant increase in CCL5 and CCL3 creates a pro-inflammatory environment conducive for enhanced immune cell recruitment and infiltration.

[0140] Belinostat restores miR-335 expression and suppresses pro-inflammatory pathways:

[0141] The epigenetic regulation of miR-335 by histone deacetylases (HDACs) was previously demonstrated. Belinostat, a pan-HDAC inhibitor, induces miR-335 expression in keratinocytes and in a dry skin ex vivo model. A dose dependent increase in the expression of miR-335, upon treatment with Belinostat, was observed. The treatment with 250 nM of Belinostat results in a significant increase in miR-335 expression (Fig.3A) and a significant downregulation of the expression of CCL5 and CCL3 relative to the DMSO control (Fig.

[0142] 3B-3E). To further evaluate Belinostat as a candidate therapeutic for the resolution ofinflammation in AD, its effects on both cell-intrinsic chemokine secretion by keratinocytes, as well as the overall cytokine milieu were investigated in a co-culture model with human primary keratinocytes and activated peripheral blood mononuclear cells (PBMCs).

[0143] Initially the inventors analyzed the efficacy of Belinostat on cytokine secretion in the presence of both immune cells and keratinocytes, and set up a co-culture of HaCaT keratinocytes with PBMCs. Cytokine secretion was stimulated via the addition of phorbol 12-myristate 13-acetate (PMA) and ionomycin. qRT-PCR analysis of samples harvested 24 hours post- stimulation revealed a significant reduction of RANTES, MIP-la, and a panel of pro-inflammatory cytokines known to be important in the pathogenesis of AD in the presence of Belinostat, relative to the DMSO control (Fig, 3F). Mutiplex cytokine analysis using a Lumincx assay revealed that a large number of pro-inflammatory cytokines were downregulated upon treatment with Belinostat (Fig. 3G). The list of downregulated cytokines includes TSLP and IL-31, both of which are pruritogens that contribute to intense itching in AD. This preliminary work was followed up on HaCaT keratinocytes by studying the effects of Belinostat on a co-culture of human primary keratinocytes (HPKs) and PBMCs. Efficacy of Belinostat on the secretion of chemokines CCL3, CCL5, CCL13, CCL18 and cytokines IL-4, IL-13, IL-31 and TSLP were investigated by ELISA (Fig. 3H and 31). Significant downregulation of chcmokincs CCL3, CCL5, CCL13, CCL18 and cytokines IL-4, IL-13, IL-31 and TSLP upon treatment with Belinostat was apparent.

[0144] Belinostat suppresses inflammation and restores barrier function in an AD mouse model: AD lesional skin is characterized by a barrier defect, associated with significant transepidermal water loss (TEWL), as well as excessive local inflammation.

[0145] Topical application of MC903, a low-calccmic analog of vitamin D3, induces skin inflammation and changes in skin morphology, which resemble inflammation associated with human AD. Characteristics of AD, such as skin eczematous-like lesions with xerosis and pruritus, increased serum IgE levels, and inflammatory infiltrates in skin were also observed in mice treated with MC903. To evaluate the efficacy of Belinostat in vivo, the MC903-induced AD mouse model was used.

[0146] It was observed that 7 days of MC903 application to the cars induced visible flaking of the skin, relative to vehicle-only control. The condition was counteracted by simultaneousapplication of Belinostat (Fig. 4A). The improvement in macroscopic appearance of the ear skin in the presence of Belinostat was also associated with reduced ear swelling and a concomitant reduction in TEWL (Fig. 4B, 4C). H&E staining of mouse ear sections following 7-day treatment with vehicle-only control, MC903 or MC903 + Belinostat, indicates that Belinostat restores epidermal homeostasis and repairs the defective barrier (Fig. 4D). Immunohistochemical staining for TSLP indicates that MC903 treatment enhanced TSLP expression relative to vehicle control. Significant reduction in the expression of TSLP was apparent upon treatment with Belinostat (Fig. 4E). All these evidence highlight the role of Belinostat in the resolution of inflammation in AD.

[0147] It will be appreciated that many further modifications and permutations of various aspects of the described embodiments arc possible. Accordingly, the described aspects arc intended to embrace all such alterations, modifications, and variations that fall within the spirit and scope of the appended claims.

Claims

CLAIMS1. A method of suppressing or inhibiting TNFa-mediated proinflammatory signaiing in a mammalian cell or tissue, the method comprising contacting the mammalian cell with an agent capable of uprcgulating the expression or activity of miR-335.

2. The method of claim 1, wherein the agent is an miR-335 agonist or an miR-335 mimic.

3. The method of claim 2, wherein the miR-335 agonist comprises Belinostat or a derivative thereof.

4. The method of any one of claims 1 to 3, wherein the mammalian cell or tissue is in a subject suffering from a TNF-cc mediated skin inflammatory disease.

5. The method of claim 4, wherein the skin inflammatory disease is atopic dermatitis.

6. The method of any one of claims 1 to 5, wherein the agent reduces expression and / or secretion of proinflammatory cytokines.

7. The method of claim 6, wherein the proinflammatory cytokines comprises Caspase- 7 (Casp7).

8. The method of claim 7, wherein the proinflammatory cytokines further comprise CCL3 and / or CCL5.

9. A method of treating, reducing or inhibiting TNFa-mediated inflammation in a subject, the method comprising administering an agent capable of upregulating the expression or activity of miR-335 to a subject.

10. The method of claim 9, wherein the inflammation is characterized by an increased expression and / or secretion of Casp7 as compared to a reference.

11. The method of claims 9 or 10, wherein the inflammation is further characterized by an increased expression and / or secretion of CCL3 and / or CCL5 as compared to a reference.

12. The method of any one of claims 9 to 11, wherein the subject is suffering from a skin inflammatory disease.

13. The method of claim 12, wherein the skin inflammatory disease is atopic dermatitis.

14. The method of claim 13, wherein the atopic dermatitis is characterized by an activation of TNFa-mediated proinflammatory pathway.

15. A method of treating, reducing or inhibiting TNFa-mediated inflammation in a subject, the method comprising: a) detecting an increased level of a biomarker associated with TNFa-mediated inflammation in a biological sample obtained from the subject, wherein the increased level of biomarker as compared to a reference indicates that the subject has TNFa-mediated inflammation, and b) administering an agent capable of upregulating the expression or activity of miR-335 to the subject found to have TNFa-mediated inflammation.

16. The method of claim 15, wherein the biomarker is selected from the group consisting of TNFa, Casp7, CCL3 and CCL5.

17. The method of claim 15 or 16, wherein the biomarker is TNFa.

18. The method of claim 15 or 16, wherein the biomarkcr is Casp7.

19. The method of any one of claims 15 to 18, wherein the subject is suffering from atopic dermatitis.

20. An agent capable of upregulating the expression or activity of miR-335 for use in suppressing or inhibiting TNFa-mediated proinflammatory signaling in a mammalian cell or tissue.

21. An agent capable of upregulating the expression or activity of miR-335 to a subject for use in treating, reducing or inhibiting TNFa-mediated inflammation in a subject.

22. Use of an agent capable of upregulating the expression or activity of miR-335 in the manufacture of a medicament for the treatment of TNFa-mediated inflammation in a subject.

23. The use of claim 22, wherein the subject is suffering from a skin inflammatory disease.

24. The use of claim 22 or 23, wherein the skin inflammatory disease is atopic dermatitis.

Images