WO2021243148A2 - Méthodes et compositions de traitement de la maladie alcoolique du foie - Google Patents

Méthodes et compositions de traitement de la maladie alcoolique du foie Download PDF

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WO2021243148A2
WO2021243148A2 PCT/US2021/034752 US2021034752W WO2021243148A2 WO 2021243148 A2 WO2021243148 A2 WO 2021243148A2 US 2021034752 W US2021034752 W US 2021034752W WO 2021243148 A2 WO2021243148 A2 WO 2021243148A2
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seq
liver
inflammation
inflammatory
disease
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WO2021243148A3 (fr
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Jack Jacek HAWIGER
Jozef ZIENKIEWICZ
Danya Liu
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Vanderbilt University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1825—Fibroblast growth factor [FGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals

Definitions

  • Alcoholic liver disease afflicts an estimated 2 million patients in the US with an astonishing 65% mortality rate over a 4-year period.
  • Acute or chronic alcohol abuse alters homeostatic balance that exists between intracellular mediators and suppressors of proinflammatory signaling. This signaling culminates in genomic reprogramming of the liver cells manifested by expression of proinflammatory and proapoptotic mediators.
  • ALD represents an example of metabolic inflammation caused by excessive and chronic use of ethanol-containing beverages.
  • Inflammation represents a fundamental mechanism of diseases caused by microbial, autoimmune, autoinflammatory, metabolic, and physical insults. Millions of people in the United States and globally suffer from inflammatory diseases. Inflammation is the body's response to harmful stimuli, and when limited, is beneficial and helps the body heal. However, when inflammation is unchecked it can lead to tissue destruction, necrosis, and fibrosis. For example, the action of microbial insults on microvascular endothelial cells in severe microbial infections evolving into their end stage, septic shock, leads to endothelial dysfunction that contributes to major organ failure, disseminated intravascular coagulation (DIC) involving liver microcirculation, acute respiratory distress syndrome (ARDS), acute kidney injury, and acute brain injury.
  • DIC disseminated intravascular coagulation
  • ARDS acute respiratory distress syndrome
  • autoimmune factors targeting the body's own cells and organs develop into rampant inflammation, destroying skin and joints in psoriasis, lupus, and rheumatoid arthritis, and insulin-producing beta cells in Type 1 diabetes.
  • Microbial and metabolic inflammation leads to insulin resistance, which underlies Type 2 diabetes.
  • Chronic microbial inflammation caused by the oral microbiota of periodontitis, and bronchitis contribute to coronary heart disease while Hepatitis C vims infecting 200 million people worldwide contributes to fatty liver (steatosis), cirrhosis and, ultimately liver cancer.
  • Steroidal anti-inflammatory drugs e.g., hydrocortisone, prednisone, and methylprednisolone
  • Steroidal anti-inflammatory drugs have significant side effects increasing blood glucose, blood lipids and body fat distribution, skin thinning and delayed wound healing, muscle weakness, osteoporosis, increased susceptibility to infections, cataract, increased in eye pressure, stomach ulcers, and psychiatric disturbances.
  • Methotrexate therapy is associated gastrointestinal and liver toxicity.
  • Non-steroidal anti-inflammatory drugs may cause fluid retention leading to edema, kidney failure (primarily with chronic use), liver failure, ulcers and prolonged bleeding after an injury or surgery.
  • Inhibitors of kinases that target Bruton Tyrosine kinase (ibrutinib, acalabrutinib) and the JAK family of kinases may cause serious opportunistic infections with Mycobacterium tuberculosis, Herpes zoster, Cytomegalovirus, and Pneumocystis jirovecii pneumonia.
  • monoclonal antibodies such as anti-TNFa monoclonal antibody carry the risk of the reactivation of latent infection with Mycobacterium tuberculosis and the monoclonal antibody natalizumab carries the risk of JC virus-caused progressive multifocal leukoencephalopathy in patients with multiple sclerosis.
  • natalizumab carries the risk of JC virus-caused progressive multifocal leukoencephalopathy in patients with multiple sclerosis.
  • a composition comprising one or more Nuclear Transport Modifier (NTM) such as, for example, an NTM that comprises the sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19; SEQ ID NO: 20;
  • NTM Nuclear Transport Modifier
  • SEQ ID NO: 27 SEQ ID NO: 28, SEQ ID NO: 29; SEQ ID NO: 30; SEQ ID NO: 31; SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39; SEQ ID NO: 40; and/or SEQ ID NO: 41.
  • Figure 1 displays proinflammatory signaling to the nucleus mediated by Stress- Responsive Transcription Factors (SRTFs).
  • Figure 2 shows metabolic signaling to the nucleus mediated by metabolic transcription factors that comprise Sterol Regulatory Element-Binding Proteins (SREBPs) and Carbohydrate-Responsive Element-Binding Proteins (ChREBPs).
  • SREBPs Sterol Regulatory Element-Binding Proteins
  • ChREBPs Carbohydrate-Responsive Element-Binding Proteins
  • FIG. 3 shows EtOH-Enhanced liver injury caused by Concanavalin A (Con A). Elevated levels of serum hepatic enzyme ALT, plasma cytokines, and liver tissue Caspase 3/7 activity.
  • Figure 4 shows liver sections from pair-fed control mouse that received a low dose of ConA. Single focus of necrosis is present. (Panel A, lower power; Panel B high power). Liver section from EtOH-fed mouse that received a low dose of Con A. Many foci of necrosis are present. Hematoxylin and eosin stain. (Panel C, low power; Panel D, high power).
  • Figure 5 shows LPS-Induced Liver injury: survival and liver apoptosis accompanied by hemorrhagic necrosis in control mice as compared to the cSN50 peptide- treated mice.
  • Figure 7 shows that Microbial Inflammation is Interwoven with Metabolic Inflammation.
  • Polymicrobial causes viruses, bacteria, fungi, and protozoa
  • SRTFs stress responsive transcription factors
  • MTFs metabolic transcription factors
  • ChREBPs transcription factors that regulate genes encoding proteins involved in glucose homeostasis
  • Inhibition of nuclear transport of SRTFs reduces expression of genes encoding mediators of inflammation, cytokines, chemokines, signal transducers, and cell adhesion molecules.
  • Inhibition of nuclear transport of SREBPs reduces expression of genes encoding proteins involved in synthesis of cholesterol, triglycerides and fatty acids. The accumulation of these metabolites can activate proinflammatory pathway mediated by NF-KB RelA and other SRTFs thereby aggravating microbial inflammation (see text for details; NPS: Nuclear Pore Complex).
  • Ranges can be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed.
  • “Optional” or “optionally” means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not. 19.
  • the terms "patient,” “subject” and “individual” are used interchangeably herein, and mean an animal (e.g., mammalian (such as human, equine, bovine, ovine, porcine, canine, etc.), reptilian, piscine, etc.) to be treated, diagnosed and/or to obtain a biological sample from.
  • bind means that one molecule recognizes and attaches/adheres to a particular second molecule in a sample or organism but does not substantially recognize or attaches/adhere to other structurally unrelated molecules in the sample.
  • a first molecule that "specifically binds" a second molecule has a binding affinity greater than about 10 8 to 10 12 moles/liter for that second molecule and involves precise "hand-in-a-glove” docking interactions that can be covalent and noncovalent (hydrogen bonding, hydrophobic, ionic, and van der Waals).
  • nuclear transport modifier and “NTM” is meant a cell-penetrating peptide that is capable of modulating entry of transcription factors into the nucleus.
  • An example of a nuclear transport modifier is a 26-29 amino acid peptide derived from human nuclear factor kappa B 1 nuclear localization sequence and from human Fibroblast Growth Factor 4 signal sequence hydrophobic region. This phrase is used interchangeably with the phrase “nuclear import inhibitor.”
  • any of the amino acid residues in the NTM sequence can be mutated and/or modified, i.e. to form mimetics or stapled peptides to stabilize their conformation (Moiola M et al 2019) so long as the modifications do not affect the cell membrane translocating function of the peptide.
  • the word "peptide” includes mimetics and the word “amino acid” includes modified amino acids, unusual amino acids, D- form amino acids, etc.
  • NTM nuclear Transport Modifier
  • Importin alpha inhibitor also known as “Importin alpha inhibitor”
  • importin alpha- selective NTM also known as importin alpha- selective inhibitor
  • NLS nuclear localization sequence
  • the importin alpha 5-selective NTM is the sequence of or a sequence derived from AAVALLPAVXLAXXAPCVQRKRQKLMPC (SEQ ID NO: 41), where X represents any amino acid from the group of hydrophobic or special amino acids (e.g., cysteine, glycine, and proline, non-natural amino acids) (e.g., cSN50.1 peptide).
  • AAVALLPAVXLAXXAPCVQRKRQKLMPC SEQ ID NO: 41
  • X represents any amino acid from the group of hydrophobic or special amino acids (e.g., cysteine, glycine, and proline, non-natural amino acids) (e.g., cSN50.1 peptide).
  • NTM nuclear Transport Modifier
  • Importin beta inhibitor also known as “Importin beta inhibitor”
  • importin beta- selective NTM also known as importin beta-selective inhibitor
  • the importin beta-selective NTM includes a peptide sequence that includes an SSHR domain derived from Signal Sequence Hydrophobic Region of Fibroblast Growth Factor 4 and a hydrophilic segment to counterbalance hydrophobic properties of SSHR.
  • nuclear import adaptor and “nuclear transport adaptor”, or “nuclear transport shuttle” mean a cell component capable of mediating transport of a protein usually larger than 45 kD (e.g., a transcription factor) or a complex of two proteins (e.g. a dimer comprising transcription factors cFos and cJun) into the nucleus.
  • a nuclear transport adaptor is an importin also known as karyopherin.
  • protein and “polypeptide” are used synonymously to mean any peptide-linked chain of amino acids, regardless of length or post-translational modification, e.g., glycosylation or phosphorylation.
  • gene is meant a nucleic acid molecule that codes for a particular protein, or in certain cases, a functional or structural RNA molecule.
  • nucleic acid or a “nucleic acid molecule” means a chain of two or more nucleotides such as RNA (ribonucleic acid) and DNA (deoxyribonucleic acid).
  • labeled with regard to a nucleic acid, protein, probe or antibody, is intended to encompass direct labeling of the nucleic acid, protein, probe or antibody by coupling (i.e., physically or chemically linking) a detectable substance (detectable agent) to the nucleic acid, protein, probe or antibody.
  • therapeutic and “therapeutic agent” are used interchangeably, and are meant to encompass any molecule, chemical entity, composition, drug, cell(s), therapeutic agent, chemotherapeutic agent, or biological agent capable of preventing, ameliorating, or treating a disease or other medical condition.
  • the term includes small molecule compounds, antisense reagents, siRNA reagents, antibodies, enzymes, peptides organic or inorganic molecules, cells, natural or synthetic compounds and the like.
  • treatment is defined as the application or administration of a therapeutic agent to a patient or subject, or application or administration of the therapeutic agent to an isolated tissue or cell line from a patient or subject, who has a disease, a symptom of disease or a predisposition toward a disease, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease, the symptoms of disease, or the predisposition toward disease.
  • a “decrease” can refer to any change that results in a smaller amount of a symptom, disease, composition, condition, or activity.
  • a substance is also understood to decrease the genetic output of a gene when the genetic output of the gene product with the substance is less relative to the output of the gene product without the substance.
  • a decrease can be a change in the symptoms of a disorder such that the symptoms are less than previously observed.
  • a decrease can be any individual, median, or average decrease in a condition, symptom, activity, composition in a statistically significant amount.
  • the decrease can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60,
  • “Inhibit,” “inhibiting,” and “inhibition” mean to decrease an activity, response, condition, disease, or other biological parameter. This can include but is not limited to the complete ablation of the activity, response, condition, or disease. This may also include, for example, a 10% reduction in the activity, response, condition, or disease as compared to the native or control level. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels.
  • An "increase” can refer to any change that results in a greater amount of a symptom, disease, composition, condition or activity.
  • An increase can be any individual, median, or average increase in a condition, symptom, activity, composition in a statistically significant amount.
  • the increase can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% increase so long as the increase is statistically significant.
  • Increases can also be referenced in terms of fold increases.
  • an increase can comprise a 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 s , 10 9 , 10 10 -fold increase.
  • compositions, kits, cells, and methods similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable compositions, kits, cells, and methods are described below. All publications, patent applications, and patents mentioned herein are incorporated by reference in their entirety.
  • Small transcription factors ( ⁇ 40 kD), usually those regulating the housekeeping genes that encode cell survival factors, have free passage from the cytoplasm to the nucleus.
  • SRTFs Stress-Responsive Transcription Factors
  • SRTFs Stress-Responsive Transcription Factors
  • This nuclear import machinery recognizes nuclear localization sequence (NLS) exposed on SRTFs and Carbohydrate Regulatory Element Binding Proteins (ChREBPs). NLSs are then recognized by nuclear transport adaptor proteins, importins/karyopherins alpha (Imp a) (see Figure 1).
  • SRTFs The stimulus -induced formation of SRTF and importins a complexes (or ChREBPs and importin a complexes) also encompasses importin beta 1 (Imp b ⁇ ), which is recognized by nuclear pore proteins to allow translocation of the cargo to the nucleus.
  • Imp b ⁇ importin beta 1
  • SRTFs activate a myriad of genes that encode cytokines, chemokines, cell adhesion molecules, signal transducers, and other mediators of inflammation and apoptosis. In turn these mediators evoke leukocyte migration, cell adhesion, and tissue injury.
  • Metabolic transcription factors such as ChREBPs, regulate glucose homeostasis, glycogen catabolism, and synthesis of triglycerides.
  • ChREBPs In cooperation with SREBPlc, ChREBPs induce glycolytic and lipogenic genes. Metabolic transcription factors, such as SREBP1 and SREBP2 are solely transported to the nucleus by importin b ⁇ that recognizes highly conserved basic-helix-loop-helix that forms a dimer and binds to this nuclear transport shuttle (see Figure 2). Similar recognition mechanism exists in ChREBPs albeit it plays a lesser role than a distinct NLS motif responsible for interaction of ChREBPs with importin a complexes.
  • SRTFs proinflammatory SRTFs
  • IkBa the degradation-resistant inhibitor of NF-KB
  • NF-KB is only one of multiple SRTFs that mediate signaling to the nucleus in response to infection.
  • Other SRTFs such as AP-1, STAT1 and NFAT, are also transported to the nucleus during the inflammatory response yet their nuclear transport is not impeded by IkBa; contrarily, the AP-1 pathway is enhanced (Hawiger and Zienkiewicz 2019 Scand. J. Immunol).
  • NTMs Nuclear Transport Modifiers
  • NTMs target the nuclear transport shuttles, Imp a5 and Imp b ⁇ , that translocate SRTFs to the nucleus and control signal transduction pathways, which culminate in genomic reprogramming.
  • NTMs target nuclear transport adaptors.
  • NTMs modulate signaling to the nucleus mediated by transcription factors that include but are not limited to NF-KB, AP-1, NFAT, STAT1, NRF2, and Carbohydrate Responsive Element-Binding Proteins (ChREBPs) that utilize importins alpha and beta heterodimer, SREBPla, SREBPlc, and SREBP2, that utilize solely importin beta 1 for nuclear transport whereas ChREBP can utilize both importins alpha and beta for nuclear translocation.
  • ChREBPs Carbohydrate Responsive Element-Binding Proteins
  • SRTFs such as NF-KB, AP-1, NFAT, STAT1 and STAT3 are transported to the nucleus in response to proinflammatory stimuli.
  • SRTFs activate genes that encode mediators of inflammation ⁇ Examples of NTMs include SN50, cSN50 and cSN50.1 described in more detail in the following paragraphs, as well as the sequences set forth in Table 1.
  • SN50, cSN50, cSN50.1 and their derivatives inhibit nuclear import of SRTFs by binding to their target, importin alpha 5 (Imp a5) known also as karyopherin al (KPNA1, SRP1).
  • Imp a5 importin alpha 5
  • KPNA1, SRP1 karyopherin al
  • Adaptor proteins targeted by these peptides constitute a recognition arm of nuclear import machinery. They recognize nuclear localization sequence (NLS) on SRTFs, ChREBPs or other transcription factors, e.g. NRF2, and carry this cargo across the nuclear pore.
  • Imp a5 (KPNAl/ SRP1) forms a complex with importin b ⁇ (Imp b ⁇ ) which docks at the cytoplasmic rim of the nuclear pore (Fig.l).
  • SN50, cSN50, and cSN50.1 and their derivatives also bind Imp b ⁇ and compete with its transporting of SREBP 1 and 2 to the nucleus. As these transcription factors do not display NLS recognized by importins a, their transport is independent of Imp a5. The latter recognizes NLS motif on SRTFs and ChREBPs.
  • NTM signal-sequence hydrophobic region
  • NTMs have been shown to inhibit nuclear translocation of SRTFs and metabolic transcription factors, Sterol Regulatory Element-Binding Proteins (SREBPs) and Carbohydrate-Responsive Element- Binding Proteins (ChREBPs).
  • SREBPs Sterol Regulatory Element-Binding Proteins
  • ChREBPs Carbohydrate-Responsive Element- Binding Proteins
  • a novel form of immunotherapy that targets nuclear import as described herein can arrest inflammation-driven destruction of microbe- infected tissue and surrounding area of a given organ.
  • inflammation such as, for example, acute inflammation, subacute inflammation, chronic inflammation, organ-specific inflammation, systemic inflammation, and/or the end stage of microbial inflammation, sepsis and septic shock
  • pro- inflammatory signaling initiated through stimulation of the principal receptors of innate immunity, Toll-like receptors (TLRs) is one mechanism that activates non-immune and immune cells.
  • Non-immune cells comprise skin keratinocytes, mucosal epithelial cells, andvascular endothelial cells. They serve as an organ- specific barrier while acting as the first line sentinels.
  • NK Natural Killer
  • ILC innate lymphoid cells
  • non-immune cells alert immune system to the activity of inflammation-causing irritants while they modulate the inflammatory response.
  • Inhibiting nuclear transport at a common “checkpoint” located downstream of TLRs and cytokine receptors globally suppresses expression of inflammatory genes thereby calming the genomic storm and averting multiple organ injury (Fig.l).
  • SRTF Stress-Responsive Transcription Factor
  • SREBPs Sterol Regulatory Element-Binding Proteins
  • ChREBP Carbohydrate-Responsive Element-Binding Proteins
  • the disclosed NTM can reduce, inhibit, and/or prevent inflammation causing genomic reprogramming (such as, for example, acute inflammation, subacute inflammation, chronic inflammation, organ-specific inflammation, systemic inflammation, and/or end stage of microbial inflammation, sepsis), and consequently, the occurrence of endothelial dysfunction, multi-organ failure and ultimately fatal septic shock associated with sepsis, as the end-stage of microbial inflammation ⁇
  • genomic reprogramming such as, for example, acute inflammation, subacute inflammation, chronic inflammation, organ-specific inflammation, systemic inflammation, and/or end stage of microbial inflammation, sepsis
  • endothelial dysfunction multi-organ failure and ultimately fatal septic shock associated with sepsis
  • elevated blood cholesterol, triglycerides, and other lipids comprising hyperlipidemia cause metabolic inflammation producing a fatty liver (steatosis) that evolves into steatohepatitis and end-stage liver disease (a primary indication for liver transplantation
  • a method of treating, inhibiting, decreasing, reducing, ameliorating, and/or preventing inflammatory liver injury due to a liver disease mediated by inflammation comprising administering to the subject with the inflammation a composition comprising NTM.
  • the inflammation causing said liver disease being caused by external forces including, but not limited to microbial disease (such as, for example, viral, bacterial, fungal, and/or parasitic infections), liver injury (such as, for example, alcoholic liver disease), autoimmune disease, allergic disease, autoinflammatory disorder, metabolic disorder, neoplastic disorders, chemical exposure (including, but not limited to cocaine, synthetic estrogens, retinoids, vitamin A, cytotoxic drugs, and/or azathioprine), and/or physical insults (such as trauma, burns, radiation),
  • microbial disease such as, for example, viral, bacterial, fungal, and/or parasitic infections
  • liver injury such as, for example, alcoholic liver disease
  • autoimmune disease allergic disease
  • autoinflammatory disorder such as, metabolic disorder, neoplastic disorders
  • chemical exposure including, but not limited to cocaine, synthetic estrogens, retinoids, vitamin A, cytotoxic drugs, and/or azathioprine
  • physical insults such as trauma, burns, radiation
  • the NTM can
  • the method for reducing levels of SRTF, SREBPs (such as, for example, SREBPla, SREBPlc, SREBP2) and ChREBPs (such as ChREBPa and ChREBP ) in a cell methods treating, inhibiting, reducing, and/or preventing diseases mediated by inflammation (such as, for example, microbial, allergic, autoimmune, metabolic, physical, and constitutive inflammation in their acute, subacute , chronic stages or organ-specific , systemic inflammation, and/or sepsis as the end-stage of microbial inflammation ) include administering a therapeutically effective amount of a composition comprising one or more nuclear transport modifier (NTM) to the mammalian subject.
  • NTM nuclear transport modifier
  • the composition decreases inflammation by attenuating expression of at least one Stress- Responsive Transcription Factor-regulated gene and/ or at least one Sterol-Regulatory Element Binding Proteins (SREBPs)-regulated gene, and/or at least one Carbohydrate- Responsive Element-Binding Proteins (ChREBPs)-regulated gene.
  • the effective dose is an amount effective for reducing importin alpha-mediated nuclear translocation of at least one stress response Stress-Responsive Transcription Factors (SRTF) or one metabolic transcription factor, Carbohydrate-Responsive Element-Binding Proteins (ChREBPs), and reducing inflammation caused by infection and/or metabolic insult in the mammalian subject.
  • SRTF stress response Stress-Responsive Transcription Factors
  • ChREBPs Carbohydrate-Responsive Element-Binding Proteins
  • the effective dose is an amount effective for reducing importin beta-mediated nuclear translocation of at least one metabolic transcription factors, Sterol-Regulatory Element Binding Protein (SREBP) and reducing inflammation caused by infection and/or metabolic insult in the mammalian subject.
  • SREBP Sterol-Regulatory Element Binding Protein
  • the NTM may bind to importin alpha, to importin beta, or to both importin alpha and importin beta.
  • cSN50.1 peptide and its congeners are their ability to reach the site of inflammation caused by an infectious agent, i.e. the infected host cell, as well as cells in other myeloid, lymphoid, and non-lymphoid organs.
  • infectious agent i.e. the infected host cell
  • MTM membrane-translocating motif
  • SSHR signal sequence hydrophobic region
  • the amphipathic helix-based structure of SSHR facilitates its insertion directly into the plasma membrane and the tilted transmembrane orientation permits the translocation of the Nuclear Transport Modifier through the phospholipid bilayer of the plasma membrane directly to the interior of the cell without perturbing membrane integrity.
  • This mechanism explains the efficient delivery of SSHR-guided cargo across the plasma membrane of multiple cell types involved in microbial, allergic, autoimmune, metabolic, physical, and constitutive inflammation (Hawiger J and Zienkiewicz J (2019) Scand. J. Immunology, 00:el2812).
  • the NTMs disclosed herein are derived from N50-containing NTMs (SN50, cSN50, and cSN50.1) that are comprised of a hydrophilic N50 motif patterned on the nuclear localization sequence (NLS) region of the NF-KBl/p50 subunit (see Table 1) fused to a motif from the signal sequence hydrophobic region (SSHR) of human fibroblast growth factor 4.
  • the SSHR allows peptides to cross the plasma membrane by an ATP- and endosome- independent mechanism, and the N50 motif was designed to bind to importins a during stimulus -initiated signaling and thereby limit docking of NLS-bearing SRTFs to their adaptor proteins and reduce nuclear import of activated STRFs.
  • Any mimetics, derivatives, or homologs of SN50, cSN50, and cSN50.1 may be used in the compositions, methods, and kits disclosed herein.
  • Hydrophobic regions of the SSHR domain are distinguished from the cluster of basic amino acids (NLS).
  • NTM indicates nuclear transport modifier; SSHR, signal sequence hydrophobic region; NLS, nuclear localization sequence.
  • SN50 is a fragment- linked peptide combining the signal sequence hydrophobic region (SSHR) of the Kaposi fibroblast growth factor (K-FGF) and the nuclear localization signal (NLS) of the p50 subunit of NF-KBL Any mimetics, derivatives, or homologs of SN50 may be used in the compositions, methods, and kits disclosed herein.
  • the sequence of SN50 is AAV ALLPA VLLALL AP V QRKRQKLMP (SEQ ID NO: 13). Generation and use of SN50 is described in U.S. Pat. Nos. 6,495,518 and 7,553,929.
  • cSN50 is a fragment-designed cyclic peptide combining the hydrophobic region of the Kaposi fibroblast growth factor signal sequence with the nuclear localization signal (NLS) of the p50-NFKBl and inserting a cysteine on each side of the NLS to form an intrachain disulfide bond.
  • the amino acid sequence of cSN50 is
  • AAV ALLPA VLLALLAPCYVQRKRQKLMPC (SEQ ID NO: 1). Any mimetics, derivatives, or homologs of cSN50 may be used in the compositions, methods, and kits disclosed herein. Methods of making and using cSN50 are described, for example, in U.S. Pat. Nos. 8,324,148 and 9,044,433. These patents are incorporated herein by reference in their entireties. 53. cSN50.1 is a cyclized peptide having the sequence of cSN50 with the exception that the tyrosine at position 18 of cSN50, adjacent to the first cysteine, has been removed. Methods of making and using cSN50.1 are described, for example, in U.S. Pat. Nos.
  • AAV ALLPA VLL ALL APC V QRKRQKLMPC (SEQ ID NO: 2).
  • the tyrosine at position 18 was removed from the sequence of cSN50 to increase solubility.
  • cSN50 is soluble at levels of ranging from 2.0 mg/mL to 40 mg/mL depending on the method of synthesis and purification whereas cSN50.1 is soluble at levels of at least 100 mg/ml. Any mimetics, derivatives, or homologs of cSN50.1 may be used in the compositions, methods, and kits disclosed herein.
  • CSN50.1 is also encompassed by SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5.
  • NTMs include fragment-designed and synthesized peptides in which cargo is incorporated as two, rather than one, modules or cargos derived from intracellular proteins other than NF-KB 1. Such additional examples include the sequences of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9.
  • the Nuclear Transport Modifier (NTM) for use in the disclosed methods of treating, inhibiting, reducing, and/or preventing inflammation-mediated diseases such as, for example, allergic, autoimmune, metabolic, microbial, physical, and constitutive inflammation comprising organ-specific or systemic inflammation, such as sepsis as the end stage microbial inflammation
  • inflammation-mediated diseases such as, for example, allergic, autoimmune, metabolic, microbial, physical, and constitutive inflammation comprising organ-specific or systemic inflammation, such as sepsis as the end stage microbial inflammation
  • liver injury such as, for example, alcoholic liver disease
  • the Nuclear Transport Modifier can have the sequence Xaa Xaa Xaa Xaa Xaa Leu Leu Pro Xaa Xaa Leu Leu Ala Leu Leu Ala Pro Cys Xaa Xaa Gin Arg Lys Arg Gin Lys Xaa Xaa Xaa Cys, where Xaa is any amino acid or is absent (SEQ ID NO: 4).
  • the Nuclear Transport Modifier can have the sequence Xaa Xaa Xaa Xaa Xaa Leu Leu Pro Xaa Xaa Leu Leu Ala Leu Leu Ala Pro Cys Xaa Gin Arg Lys Arg Gin Lys Xaa Xaa Xaa Cys, where Xaa is any amino acid or is absent (SEQ ID NO: 5).
  • the Nuclear Transport Modifier is cSN50.1 having the sequence set forth in SEQ ID NO: 2.
  • the NTM has the sequence Xaa Xaa Xaa Xaa Xaa Leu Leu Pro Xaa Xaa Leu Leu Ala Val Leu Ala Pro Xaa Xaa Xaa Gin Arg Lys Arg Gin Lys Xaa Xaa Xaa Xaa, where Xaa is any amino acid or is absent (SEQ ID NO: 6).
  • the NTM has the sequence Ala Ala Val Ala Leu Leu Pro Ala Val Leu Leu Ala Val Leu Ala Pro Cys Val Gin Arg Lys Arg Gin Lys Leu Met Pro Cys (SEQ ID NO: 7).
  • the NTM has the sequence Xaa Xaa Xaa Xaa Xaa Leu Leu Pro Xaa Xaa Leu Leu Ala Leu Leu Ala Pro Xaa Xaa Gin Arg Asp Glu Gin Lys Xaa Xaa Xaa Xaa, where Xaa is any amino acid or is absent (SEQ ID NO: 8).
  • the NTM has the sequence Xaa Xaa Xaa Xaa Xaa Leu Leu Pro Xaa Xaa Leu Leu Ala Leu Leu Ala Pro Cys Xaa Gin Arg Asp Glu Gin Lys Xaa Xaa Xaa Cys (SEQ ID NO: 9).
  • compositions for treating diseases mediated by inflammation (including, but not limited to acute inflammation, subacute inflammation, chronic inflammation, organ-specific inflammation, systemic inflammation, and/or sepsis), said diseases including, but not limited to microbial disease, liver injury (such as, for example, alcoholic liver disease), autoimmune disease, allergies, autoinflammatory disorder, metabolic disorder, neoplastic disorder, inflammatory skin disorder, and/or physical insults include a pharmaceutically acceptable carrier and at least one importin beta-selective and/or at least one importin alpha- selective NTM in an amount effective for modifying (e.g., decreasing) entry into the nucleus of at least one transcription factor that includes but is not limited to NF-KB, AP-1, NFAT, STATl, STAT3, SREBPla, SREBPlc, and SREBP2, and ChREBPs that utilize importins alpha and/or beta for nuclear transport, and treating or preventing the disease.
  • inflammation including, but not limited to acute inflammation, subacute inflammation, chronic inflammation, organ
  • NTMs modulate signaling to the nucleus mediated by transcription factors that include but are not limited to NF-KB, AP-1, NFAT, STATl, STAT3, that utilize importins alpha and beta heterodimer, SREBPla, SREBPlc, and SREBP2, that utilize solely importin beta for nuclear transport whereas ChREBP can utilize both importins alpha and beta for nuclear translocation.
  • the importin beta- selective NTM reduces nuclear translocation of the nuclear forms of SREBPla, SREBPlc, SREBP2, and partially ChREBPs. Any suitable importin beta-selective NTM may be used. Examples of importin beta- selective NTMs include but are not limited to peptide sequences that include an SSHR domain listed in Table 2 below and a cargo listed in Table 1 below.
  • AAV ALLPA VLL ALL AP V QRDEQKLMP (SEQ ID NO: 11) as listed in Table 1 above. Additional examples of peptides designed to inhibit interaction of importin alpha with importin beta necessary for the formation of their heterodimer include AAV ALLPA VLLALLAPRRRRIEVNVELRKAKK (SEQ ID NO: 18) (referred to as SIBB in Table 2), AAV ALLPA VLLALLAPRRRRIEVNVELRKAKKDD (SEQ ID NO: 19) (referred to as SI-1 in Table 2). AAVALLPAVLLALLAPRRQRNEVVVELRKNKRDE (SEQ ID NO: 20) (referred to as SI-3 in Table 2),
  • AAVALLPAVLLALLAPRRHRNEVTVELRKNKRDE SEQ ID NO: 21
  • SI- 4 AAVALLPAVLLALLAPRRRREEEGLQLRKQKREE
  • SI-5 AAVALLPAVLLALLAPRRRREEEGIQLRKQKREQ
  • SI-7 AAV ALLPA VLL ALL APCTEMRRRRIE V C
  • cSIB AAV ALLPA VLL ALL APCTEMRRRRIE V C
  • AAV ALLPA VLLALLAPVELRKAKKDDQMLKRRNVSSF (SEQ ID NO: 25) (referred to as SARI in Table 2)
  • AAV ALLPA VLLALLAPVELRKNKRDEHLLKRRNVPHE (SEQ ID NO: 26) (referred to as SAR3 in Table 2)
  • AAVALLPAVLLALLAPVELRKNKRDEHLLKKRNVPQE (SEQ ID NO: 27) (referred to as SAR4 in Table 2)
  • SAR4 AAV ALLPA VLLALLAPLQLRKQKREEQLFKRRNV AT A
  • SAR5 AAV ALLPA VLLALLAPLQLRKQKREEQLFKRRNV AT A
  • AAVALLPAVLLALLAPIQLRKQKREQQLFKRRNVELI (SEQ ID NO: 29) (referred to as SAR7 in Table 2)
  • AAV ALLPA VLLALLAPCVELRKAKKDDQC (SEQ ID NO: 30) (referred to as cSARl-C in Table 2)
  • AAV ALLPA VLLALLAPCVELRKNKRDEHC (SEQ ID NO: 31) (referred to as cSAR3-C in Table 2)
  • AAV ALLPA VLLALLAPCLQLRKQKREEQC (SEQ ID NO: 32) (referred to as cSAR5-C in Table 2)
  • AAV ALLPA VLLALLAPCIQLRKQKREQQC (SEQ ID NO: 33) (referred to as cSAR7-C in Table 2)
  • a AVALLPA VLLALLAPCQMLKRRNV S SFC (SEQ ID NO: 34) (referred to as cSARl-N in Table 2)
  • AAV ALLPA VLLALLAPCHLLKRRNVPHEC (SEQ ID NO: 35) (referred to as cSAR3-N in Table 2)
  • AAV ALLPA VLL ALL APCHLLKKRNVPQEC (SEQ ID NO: 36) (referred to as cSAR4-N in Table 2)
  • AAV ALLPA VLLALLAPCQLFKRRNV AT AC (SEQ ID NO: 37)
  • an NTM as described herein has the sequence
  • AAV ALLPA VXLAXXAPVELRKNKRDEHLLKRRNVPHE (SEQ ID NO: 39). Additional NTMs include SEQ ID NOs: 1-9, 13, and 16-41. It is to be understood that any derivatives and/or analogues of these sequences are encompassed by the invention.
  • NTM as described herein may be an inhibitor of an importin alpha 3 interaction with importin beta.
  • SI-3 sequence (see Table 2) is designed to block an interaction between importin alpha and importin beta. Hence, this peptide is a cell-penetrating inhibitor of an importin alpha and importin beta interaction. It is to be understood that any derivatives and/or analogues of this sequence is encompassed by the invention.
  • Cargo comprises sequences of functionally active hydrophilic motifs (fragments) listed as linear or cyclized peptides through addition of cysteine at the amino- and carboxy-termini of respective linear peptides. Both linear and cyclized sequences are fused to hydrophobic membrane translocation motif denoted SSHR.
  • diseases mediated by inflammation such as, for example, acute inflammation, subacute inflammation, chronic inflammation, organ-specific inflammation, systemic inflammation, and/or sepsis
  • diseases mediated by inflammation such as, for example, acute inflammation, subacute inflammation, chronic inflammation, organ-specific inflammation, systemic inflammation, and/or sepsis
  • said diseases including, but not limited to liver injury (such as, for example, alcoholic liver disease), metabolic disorder, and/or physical insults, e.g.
  • NTMs including, but not limited to SN50 having the sequence set forth in SEQ ID NO: 1 or cSN50.1 having the sequence set forth in SEQ ID NO: 2, cSN50.1 beta having the sequence set forth in SEQ ID NO: 40, or any of the NTMs disclosed herein having the amino acid sequence set forth in SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19; SEQ ID NO: 20; SEQ ID NO:
  • NTM can be cSN50.1 beta comprising the amino acid sequence
  • cSN50.1 beta is a cyclized peptide having the sequence of cSN50.1 with the exception that the lysine at the position 21 has been replaced by aspartic acid and the arginine residue at the position of 22 has been replaced by glutamic acid.
  • the methods disclosed herein can be used in treating, inhibiting, reducing, and/or preventing diseases mediated by inflammation (such as, for example, acute inflammation, subacute inflammation, chronic inflammation, organ- specific inflammation, systemic inflammation, and/or sepsis) said diseases including, but not limited to microbial disease, liver injury (such as, for example, alcoholic liver disease), autoimmune disease, autoinflammatory disorder, metabolic disorder, neoplastic disorder, inflammatory skin disorder, and/or physical insults.
  • inflammation such as, for example, acute inflammation, subacute inflammation, chronic inflammation, organ- specific inflammation, systemic inflammation, and/or sepsis
  • diseases including, but not limited to microbial disease, liver injury (such as, for example, alcoholic liver disease), autoimmune disease, autoinflammatory disorder, metabolic disorder, neoplastic disorder, inflammatory skin disorder, and/or physical insults.
  • митодти ⁇ и ⁇ и mediated by inflammation such as, for example, acute inflammation, subacute inflammation, chronic inflammation, organ-specific inflammation, systemic inflammation, and/or sepsis
  • diseases including, but not limited to microbial disease, liver injury (such as, for example, alcoholic liver disease), autoimmune disease, autoinflammatory disorder, metabolic disorder, neoplastic disorder, inflammatory skin disorder, and/or physical insults in a subject
  • administering to a subject a therapeutically effective amount of an anti-microbial agent and a composition comprising one or more NTM (such as, for example, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19
  • NTM such as, for example, SEQ ID
  • one of the deleterious effects of inflammation including, for example, allergic, autoimmune, metabolic, microbial, physical, and constitutive inflammation comprising organ-specific or systemic inflammation exemplified by sepsis as the end stage of microbial inflammation
  • liver injury such as, for example, alcoholic liver disease
  • microbial disease autoimmune disease
  • autoinflammatory disorder metabolic disorder
  • neoplastic disorder inflammatory skin disorder, and/or physical insults.
  • inflammation such as, for example, allergic, autoimmune, metabolic, microbial, physical, and constitutive inflammation comprising organ- specific or systemic inflammation exemplified by sepsis as the end stage of microbial inflammation
  • liver injury such as, for example, alcoholic liver disease
  • a disease acute, subacute, and chronic
  • microbial disease liver injury
  • liver injury such as, for example, alcoholic liver disease
  • autoimmune disease autoinflammatory disorder, metabolic disorder, neoplastic disorder, inflammatory skin disorder, and/or physical insults
  • pro- and anti-inflammatory cytokines are produced resulting in the microvascular endothelial injury that evokes activation and deposition of blood platelets thereby resulting in their “consumption” manifested by a decreased platelet count in the blood (thrombocytopenia).
  • fibrinogen can be depleted and both uncontrolled thrombi formation and bleeding may occur while circulating platelets are depleted, a process known as thrombocytopenia without or with Disseminated Intravascular Coagulation.
  • Typical platelet counts for adults is between 150,000 and 400,000/mE, but these numbers can be less 80,000/mE due to microvascular endothelial injury.
  • the thrombocytopenia is ameliorated.
  • thrombocytopenia associated with inflammation including, for example, acute inflammation, subacute inflammation, chronic inflammation, organ-specific inflammation, systemic inflammation, and/or sepsis
  • inflammation including, but not limited to microbial disease, liver injury (such as, for example, alcoholic liver disease), autoimmune disease, autoinflammatory disorder, metabolic disorder, neoplastic disorder, inflammatory skin disorder, and/or physical insults in a subject
  • administering comprising administering to the subject a therapeutically effective amount of a composition comprising one or more NTM (such as, for example, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19; SEQ ID NO: 20; SEQ ID NO: 21
  • NTM such as, for example,
  • SEQ ID NO: 39 SEQ ID NO: 40; and/or SEQ ID NO: 41. It is understood and herein contemplated that the disclosed methods of treating, inhibiting, or reducing thrombocytopenia can further comprise the addition of an anti-microbial agent and/or an anti-inflammatory agent.
  • NTM treatment prevented glycogenolysis that is dependent on the nuclear transport of metabolic transcription factors ChREBPs and leads to the increase of blood glucose (hyperglycemia) and triglycerides (hypertriglyceridemia).
  • compositions for treating an inflammatory disease or disorder e.g., autoimmune, microbial, metabolic, neoplastic, posttraumatic, and autoinflammatory disease
  • the composition includes a pharmaceutically acceptable carrier and at least one (e.g., one, two, three, etc.) importin beta-selective NTM including an SSHR domain and a cargo that does not bind to any importin alpha, or at least one (e.g., one, two, three, etc.) importin alpha-selective NTM, in an amount effective for modifying entry of at least one (e.g., one, two, three, etc.) transcription factor (e.g., NF-KB, AP-1, NFAT, STAT1, SREBPla, SREBPlc, and SREBP2, and ChREBP) into a cell’s (e.g., a mammalian cell's) nucleus and for treating the inflammatory disease or disorder.
  • a pharmaceutically acceptable carrier and at least one (e.g
  • the at least one importin alpha-selective NTM is a peptide or compound that binds to one or more binding pockets of an importin alpha and that modulates nuclear transport of at least one intracellular protein. Modifying entry of at least one transcription factor into a cell's nucleus includes inhibiting entry of the at least one transcription factor into the cell's nucleus.
  • the at least one importin beta-selective NTM can have an amino acid sequence
  • the at least one importin alpha- selective NTM can have, for example, the amino acid sequence AAV ALLPA VXLAXXAPCVQRKRQKLMPC (SEQ ID NO: 41).
  • the composition can be administered with a corticosteroid or a non-steroidal anti-inflammatory agent.
  • the composition can further include a corticosteroid or a non-steroidal anti inflammatory agent.
  • the non-steroidal anti-inflammatory agent can be, for example, acetaminophen or ibuprofen.
  • a method of treating or preventing inflammation in a mammalian subject includes administering a composition including a pharmaceutically acceptable carrier and at least one importin beta-selective NTM including an SSHR domain and a cargo to the mammalian subject in an amount effective for modifying entry of at least one transcription factor (e.g., SREBPla, SREBPlc, and SREBP2, and ChREBP) into a cell's nucleus and for treating or preventing inflammation in the mammalian subject.
  • at least one transcription factor e.g., SREBPla, SREBPlc, and SREBP2, and ChREBP
  • the at least one importin beta-selective NTM binds to and inhibits the activity of at least one importin beta.
  • Modifying entry of at least one transcription factor into a cell's nucleus includes inhibiting entry of the at least one transcription factor into the cell's nucleus.
  • Administration of the composition generally results in inhibition of at least one signaling pathway associated with the inflammation.
  • the at least one importin beta-selective NTM can have an amino acid sequence from the NTM sequences disclosed herein.
  • the composition can be administered by any suitable route, e.g., topically, intranasally, orally, intravenously, or subcutaneously.
  • the method includes administering a composition including a pharmaceutically acceptable carrier and at least one importin alpha- selective NTM to the mammalian subject in an amount effective for modifying entry of at least one transcription factor into a cell's nucleus and for treating or preventing inflammation in the mammalian subject.
  • the at least one important alpha- selective NTM is a peptide or compound that binds to one or more binding pockets of an importin alpha and that modulates nuclear transport of at least one intracellular protein.
  • the at least one importin alpha-selective NTM can be an importin alpha 5-specific NTM or importin alpha 1-specific or importin alpha3- specific NTM or importin alpha 4-specific and an importin alpha 7-specific NTM.
  • the at least one importin alpha- selective NTM can have the amino acid sequence
  • the at least one importin alpha- selective NTM binds to and inhibits the activity of the at least one importin alpha. Modifying entry of at least one transcription factor into a cell's nucleus includes inhibiting entry of the at least one transcription factor into the cell's nucleus.
  • the at least one importin alpha- selective NTM can be specific for importin alpha 5, for example.
  • the at least one importin alpha- selective NTM includes an Importin Alpha Diversity Region 1 or 2 sequence.
  • the composition can be administered with a corticosteroid or a non-steroidal anti-inflammatory agent. In another embodiment, the composition can further include a corticosteroid or a non-steroidal anti-inflammatory agent.
  • the non-steroidal anti-inflammatory agent can be, for example, acetaminophen or ibuprofen.
  • the method includes administering a composition including a pharmaceutically acceptable carrier and at least one agent that inhibits an interaction between at least one importin alpha (e.g., importin alpha 1, importin alpha 3, importin alpha 4, importin alpha 5 and importin alpha 7), and at least one importin beta and that modulates nuclear transport of at least one intracellular protein, to the mammalian subject in an amount effective for modifying entry of at least one transcription factor into a cell's nucleus and for treating or preventing inflammation in the mammalian subject.
  • the at least one agent binds specifically to the at least one importin alpha and is an importin alpha-selective inhibitor.
  • Alcoholic liver disease encompasses fatty liver, alcoholic hepatitis, and cirrhosis. Liver cirrhosis was the 12 th leading cause of death in 2000 (Mann RE, Smart RG, and Govoni R (2003) Alcohol Research& Health). It affects an estimated 2 million persons in the US. Among these cases, the combination of liver cirrhosis and alcoholic hepatitis contributes to an astonishing 65% mortality rate over a 4-year period, a toll greater than many oncologic diseases. The mechanism of ALD progression is vested in inflammation and apoptosis which are tightly linked to innate and adaptive immunity.
  • TLRs Toll-like Receptors
  • phagocytes exemplified by Kupffer cells in the liver, to prospect their microenvironment for LPS, lipoteichoic acids, viral RNA, and/or bacterial DNA, which provide the source for proinflammatory cues.
  • these cues evoke intracellular signaling to the nucleus to reset the genome to proinflammatory and proapoptotic program.
  • TLRs contribute to the development of hepatitis induced by a wide range of microbial agents.
  • alcoholic liver disease now represents the fourth leading cause of death among city dwellers in the US.
  • Inflammatory liver injury is mediated by cytokines and chemokines, which are produced primarily by mononuclear phagocytes such as Kupffer cells in the liver and other macrophages in extrahepatic sites. Moreover, Innate Lymphoid Cells (ILC) and the subsets of T lymphocytes including NK-T cells are contributory. In response to proinflammatory microbial cues sensed by their TLRs and/or cytokine receptors, these cells send intracellular signals to Stress-Responsive Transcription Factor (SRTFs; see Fig. 1).
  • SRTFs Stress-Responsive Transcription Factor
  • SRTFs encompass Nuclear Factor -KB (NF-KB), Activator Protein (API) comprised of c-Fos and c-Jun, Nuclear Factor of Activated T cells (NFAT) and Signal Transduction and Transcription (STAT) 1 and 3.
  • SRTFs are regulated by kinases, phosphatases, and ubiquitinating enzymes.
  • NF-KB is kept in check by IKB inhibitory proteins, which are targeted for phosphorylation, ubiquitination, and proteasome-mediated degradation during an inflammatory response.
  • NFAT is activated via dephosphorylation involving the phosphatase calcineurin.
  • Calcineurin is regulated by Ca++, calmodulin, and cyclophilins, targeted by cyclosporin and other immunosuppressants.
  • TNFoc Proinflammatory cytokines TNFoc and IFNyplay a key role in experimentally produced inflammatory injury of the liver that culminates in massive apoptosis and necrosis. This injury is ablated in mice lacking cognate receptors for these cytokines.
  • TNFoc responds almost immediately to proinflammatory inducers, LPS and superantigen SEB, in two distinct models of inflammatory liver injury mediated by macrophages (Kupffer cells) and T cells, respectively. Its rapid rise at 90 min is followed by a steep decline due to negative regulation of transcript stability through the 3' untranslated region. Ethanol counteracts this destabilizing process. TNFoc interacts with its cognate receptors, TNFR1 and TNFR2.
  • TRADD cytoplasmic adaptor protein
  • JNK cytoplasmic adaptor protein
  • NF-KB cytoplasmic adaptor protein
  • INFy Upon binding to its cognate receptor, INFy induces the activation of Janus Protein Tyrosine Kinases (JAK) 1 and 2.
  • JNK Janus Protein Tyrosine Kinases
  • a phosphotyrosine-docking site is formed for binding of STAT-1 through its SH2 domain.
  • STAT-1 is phosphorylated on a critical tyrosine residue, prompting the formation of a STAT-1 dimer.
  • the nuclear import machinery importin a5/importin b ⁇ complex (that is targeted by NTM peptides) translocates the STAT-1 dimer to the nucleus, where it interacts with regulatory elements in the promoter of multiple IENg responsive genes. These genes encode cytokines, proapoptotic factors, signal transducers (NADPH oxidase complex generating Reactive Oxygen Intermediates), MHC molecules, antiviral and antibacterial inhibitors.
  • IFN gamma signaling through its cognate receptor and STAT1 is very important for raising the level of immune response toward intracellular pathogens, e.g. mycobacteria and some viruses
  • pathogen-derived agonists such as LPS and superantigen SEB
  • apoptosis of hepatocytes can occur in fulminant hepatitis, an inflammatory process that is caused by viral and non-viral agents.
  • the sequence of intracellular signaling events that lead to ultimately fatal liver apoptosis in fulminant liver inflammation remains incompletely understood.
  • NTM Nuclear Transport Modifier
  • a composition comprising a Nuclear Transport Modifier (NTM) such as, for example, an NTM that comprises the sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19; SEQ ID NO: 20; SEQ ID NO: 21; SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27,
  • NTM Nuclear Transport Modifier
  • the inflammatory liver disease is caused by ethanol such as in alcoholic liver disease.
  • the inflammatory liver disease is caused by hyperlipidemia producing liver steatosis, steatohepatitis, and the end stage liver disease, such as liver cirrhosis Development of a fatty liver, termed steatosis, is caused by elevated levels of blood triglycerides.
  • Fatty liver is one of the signs of Metabolic Syndrome afflicting est. 100 million Americans and millions more worldwide. The other signs encompass elevated blood glucose (hyperglycemia) due to insulin resistance, and obesity. Excessive level of lipids and glucose in blood is caused by overfeeding or inborn metabolic errors.
  • the metabolic products evoke importin b ⁇ -mediated signaling to the nucleus of metabolic transcription factors (MTFs) encompassing SREBP1, SREBP2, and ChREBPs among others.
  • MTFs metabolic transcription factors
  • fatty liver evolves into non-alcoholic steatohepatitis leading to liver cirrhosis. Up to 27% of these patients develop liver cancer that has become the predominant indication for liver transplantation in the United States.
  • Treatment with Nuclear Import Pathway-Selective NTMs Improves Metabolic Markers in the Blood and Reduces Nuclear Content of Proinflammatory Transcription Factor, NF-KB RelA, in the Liver (Liu y et al 2021 Sci.Rep)-see attached manuscript.
  • hypertriglyceridemia was precipitously reduced by NTM selectively targeting Importin b ⁇ -mediated nuclear import of SREBPs).
  • This 3-week treatment produced a 2.5-fold decline in blood triglycerides and lowered the elevated blood glucose level.
  • These two abnormal metabolic markers represent a” deadly combination” in individual with Metabolic Syndrome with fatty liver, or worse, with steatohepatitis.
  • NTMs target the nuclear transport shuttles, Imp a5 and Imp b ⁇ , that translocate SRTFs, ChREBPs, and SREBPs to the nucleus and control signal transduction pathways, which culminate in genomic reprogramming.
  • the novel forms of immunotherapy that targets nuclear import as described herein can arrest inflammation-driven organ injury, including damage resulting from metabolic disorders, such as, for example, metabolic syndrome that encompasses fatty liver, hypercholesterolemia, hypertriglyceridemia, diabetes mellitus, and obesity.
  • Gaucher s disease, Phenylketonuria (PKU), Maple syrup urine disease (MSUD), hyperuricemia (gout), calcium pyrophosphate deposition disease (pseudo-gout), hyperthyroidism, hypothyroidism, dyslipidemia, hypolipidemia, and galactosemia).
  • a composition comprising a NTM (such as, for example, an NTM that comprises the sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19; SEQ ID NO: 20; SEQ ID NO: 21; SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29; SEQ ID NO: 30; SEQ ID NO: 31; SEQ ID NO: 32, SEQ ID NO: 33
  • inflammatory liver injury and inflammatory liver disease can result from autoimmune inflammation.
  • the NTMs disclosed herein can target the nuclear transport shuttles, Imp a5 and Imp b ⁇ , that translocate SRTFs to the nucleus and control signal transduction pathways, which culminate in genomic reprogramming.
  • the novel forms of immunotherapy disclosed herein that targets nuclear import as described herein can arrest inflammation-driven destruction associated with diseases including autoimmune diseases.
  • autoimmune disease refers to a set of diseases, disorders, or conditions resulting from an adaptive immune response (T cell and/or B cell response) against the host organism. In such conditions, either by way of mutation or other underlying cause, the host T cells and/or B cells and/or antibodies are no longer able to distinguish host cells from non-self-antigens and attack host cells bearing an antigen for which they are specific.
  • autoimmune diseases include, but are not limited to graft versus host disease, transplant rejection, Achalasia, Acute disseminated encephalomyelitis, Acute motor axonal neuropathy, Addison’s disease, Adiposis dolorosa , Adult Still's disease, Agammaglobulinemia, Alopecia areata, Alzheimer’s disease, Amyloidosis, Ankylosing spondylitis, Anti-GBM/Anti-TBM nephritis, Antiphospholipid syndrome, Aplastic anemia , Autoimmune angioedema, Autoimmune dysautonomia, Autoimmune encephalomyelitis, Autoimmune enteropathy, Autoimmune hemolytic anemia, Autoimmune hepatitis, Autoimmune inner ear disease (AIED), Autoimmune myocarditis, Autoimmune oophoritis, Autoimmune orchitis, Autoimmune pancreatitis, Autoimmune poly
  • a composition comprising a Nuclear Transport Modifier (NTM) such as, for example, an NTM that comprises the sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19; SEQ ID NO: 20; SEQ ID NO: 21; SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ
  • autoimmune inflammation alone is not sufficient to cause inflammatory liver disease or inflammatory liver injury.
  • pharmaceutical treatments such as, for example, salicylates, methotrexate
  • arsenic when combined with pharmaceutical treatments (such as, for example, salicylates, methotrexate) or exposure to arsenic, inflammatory liver injury due to inflammatory liver disease can result.
  • methods of treating, inhibiting, decreasing, reducing, ameliorating, and/or preventing inflammatory liver injury due to an inflammatory liver disease in a subject comprising administering to the subject a therapeutically effective amount of a composition comprising a Nuclear Transport Modifier (NTM); wherein liver injury is caused by autoimmune inflammation and exposure to salicylates, methotrexate, and/or arsenic.
  • NTM Nuclear Transport Modifier
  • compositions comprising NTM disclosed herein (such as, for example, NTM that comprises the sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19; SEQ ID NO: 20; SEQ ID NO: 21; SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29;
  • SEQ ID NO: 30; SEQ ID NO: 31; SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39; SEQ ID NO: 40; and/or SEQ ID NO: 41) are not limited in treatment of inflammation resulting from adaptive immune responses, but are also effective in arresting inflammation-driven destruction associated with the inborn errors of innate immune responses (i.e. Constitutive inflammation that underlies autoinflammatory diseases).
  • autoinflammatory diseases refer to disorders where the innate immune response attacks host cells.
  • autoinflammatory disorders include, Familial Cold Autoinflammatory Syndrome (FCAS), Muckle-Wells Syndrome (MWS), Neonatal-Onset Multisystem Inflammatory Disease (NOMID) (also known as Chronic Infantile Neurological Cutaneous Articular Syndrome (CINCA)), Familial Mediterranean Fever (FMF) and other cryopyrin-associated periodic syndromes (CAPS), Tumor Necrosis Factor (TNF) - Associated Periodic Syndrome (TRAPS), TNFRSF11 A-associated hereditary fever disease (TRAPS 11), Hyperimmunoglobulinemia D with Periodic Fever Syndrome (HIDS), Mevalonate Aciduria (MA), Mevalonate Kinase Deficiencies (MKD), Deficiency of Interleukin- 1b (IL-Ib) Receptor Antagonist (DIRA) (also known as Osteomyelitis, Sterile Multifocal with Periostitis Pustulosis), Majeed Syndrome, Chronic Nonbacterial Osteomyelitis (CNO), Early-On
  • NLRP12- Associated Periodic Fever Syndrome Proteasome- associated Autoinflammatory Syndromes (PRAAS), Spondyloenchondrodysplasia with immune dysregulation (SPENCDI), STING-associated vasculopathy with onset in infancy (SAVI), Aicardi-Goutieres syndrome and other Type 1 Interferonopathies, Acute Febrile Neutrophilic Dermatosis, X-linked familial hemophagocytic lymphohistiocytosis, Lyn kinase-associated Autoinflammatory Disease (LAID), and intestinal and skin inflammatory disorders caused by deletion mutation of the carboxy-terminal segment of the NF-KB essential modulator (NEMO).
  • PRAAS Proteasome- associated Autoinflammatory Syndromes
  • SPENCDI Spondyloenchondrodysplasia with immune dysregulation
  • SAVI STING-associated vasculopathy with onset in infancy
  • Aicardi-Goutieres syndrome and other Type 1 Interferonopathies Acute
  • NTM Nuclear Transport Modifier
  • a composition comprising a Nuclear Transport Modifier (NTM) such as, for example, an NTM that comprises the sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19; SEQ ID NO: 20; SEQ ID NO: 21; SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29; SEQ ID NO: 30; SEQ ID NO:
  • Inflammatory liver injury can also result from physical injuries mediated by inflammation (such as, for example abrasion, puncture, laceration, contusion, including brain trauma, blunt force trauma, ischemia, surgery, transplant, sunburn, chemical bum, high temperature bum, low temperature bum, radiation).
  • inflammation such as, for example abrasion, puncture, laceration, contusion, including brain trauma, blunt force trauma, ischemia, surgery, transplant, sunburn, chemical bum, high temperature bum, low temperature bum, radiation.
  • the NTMs disclosed herein can target the nuclear transport shuttles, Imp a5 and Imp b ⁇ , that translocate SRTFs, ChREBPs, and SREBPs to the nucleus and control signal transduction pathways, which culminate in genomic reprogramming in response to trauma or burns.
  • novel forms of immunotherapy disclosed herein that targets nuclear import as described herein can arrest inflammation-driven organ damage associated with these physical injuries that cause swelling, redness, elevated temperature, pain, and loss of organ function.
  • methods of treating, reducing, inhibiting, decreasing, ameliorating, and/or preventing inflammatory liver injury and/or inflammatory liver disease caused by physical injury comprising administering to a subject with a physical injury a therapeutically effective amount of a composition comprising an NTM (such as, for example, a composition comprising an NTM an NTM that comprises the sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, S
  • the NTM can be administered orally, topically, intravenously, and/or a medicated adhesive bandage, wound dressing, surgical drape, suture, salve, cream, or wound adhesive comprising a therapeutically effective amount of a composition comprising a Nuclear Transport Modifier (NTM).
  • NTM Nuclear Transport Modifier
  • Inflammatory liver injury can also result from exposure to chemical agents, including, but not limited to narcotics and pharmaceutical agents, In some instances the exposure resulting in inflammation can be singular and/or a large dosage. In other instances exposure leading to inflammation can be prolonged.
  • a chemical agent such as, for example, salicylates, methotrexate, arsenic, cocaine, synthetic estrogens, Vitamin A, retinoids, azathioprine or other cytotoxic drugs, and oral contraceptives
  • administering to a subject with a physical injury a therapeutically effective amount of a composition comprising an NTM such as, for example, a composition comprising an NTM an NTM that comprises the sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5
  • Examples of chemical agents that can cause inflammatory liver injury and/or inflammatory liver disease include, but are not limited to salicylates, methotrexate, arsenic, cocaine, synthetic estrogens, Vitamin A, retinoids, azathioprine or other cytotoxic drugs, oral contraceptives, Abemaciclib, Abiraterone Acetate, Abitrexate (Methotrexate), Abraxane (Paclitaxel Albumin- stabilized Nanoparticle Formulation), ABVD, ABVE, ABVE-PC, AC, AC-T, Adcetris (Brentuximab Vedotin), ADE, Ado-Trastuzumab Emtansine, Adriamycin (Doxorubicin Hydrochloride), Afatinib Dimaleate, Afinitor (Everolimus), Akynzeo (Netupitant and Palonosetron Hydrochloride), Aldara (Imiquimod), Aldesleukin
  • Talimogene Laherparepvec Tamoxifen Citrate, Tarabine PFS (Cytarabine), Tarceva (Erlotinib Hydrochloride), Targretin (Bexarotene), Tasigna (Nilotinib), Taxol (Paclitaxel), Taxotere (Docetaxel), Tecentriq , (Atezolizumab), Temodar (Temozolomide),
  • Temozolomide Temsirolimus, Thalidomide, Thalomid (Thalidomide), Thioguanine, Thiotepa, Tisagenlecleucel, Tolak (Fluorouracil— Topical), Topotecan Hydrochloride, Toremifene, Torisel (Temsirolimus), Tositumomab and Iodine 1 131 Tositumomab, Totect (Dexrazoxane Hydrochloride), TPF, Trabectedin, Trametinib, Trastuzumab, Treanda (Bendamustine Hydrochloride), Trifluridine and Tipiracil Hydrochloride, Trisenox (Arsenic Trioxide), Tykerb (Lapatinib Ditosylate), Unituxin (Dinutuximab), Uridine Triacetate, VAC, Vandetanib, VAMP, Varubi (Rolapitant Hydrochloride),
  • the host immune system attempts to eliminate the infecting microbe by employing arms of the innate and adaptive immune systems including the production of cytokines, antibodies, and effector mechanisms of granulocyte, monocyte, macrophage, dendritic cell, innate lymphoid cells, NK cells, NK T cells, T cells, B cells, and plasma cells.
  • a microbe such as, for example, a virus, bacterium, fungus, protozoa, or parasite
  • the host immune system attempts to eliminate the infecting microbe by employing arms of the innate and adaptive immune systems including the production of cytokines, antibodies, and effector mechanisms of granulocyte, monocyte, macrophage, dendritic cell, innate lymphoid cells, NK cells, NK T cells, T cells, B cells, and plasma cells.
  • SRTFs Stress-Responsive Transcription Factor
  • Microbial inflammation refers to a condition associated with its cardinal signs such as redness, swelling, increase in temperature, pain, and impairment of organ function such as disordered respiration as a result of the epithelial injury with adjacent microvascular endothelial injury in the lungs (and other organs) due to a microbial infection such as a vims, bacteria, fungi, or parasite. That is, “Microbial inflammation” is a mechanism of disease caused by infection (“microbial insult”). Microbial inflammation evolves from innate immune response to an infection due to a microbe such as, for example, a vims, bacterium, fungus, or parasite.
  • the microbial injury caused by microbial virulence factors is aggravated by the host-produced inflammatory mediators that impede the clearance of invading microbes and add insult to organ’s injury. It is understood and herein contemplated that the microbial inflammation and its end stage, sepsis can result from any microbial insult elicited by known (or unknown) virulence factors and microbial antigens.
  • a composition comprising an NTM such as, for example, a composition comprising an NTM an NTM that comprises the sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO:
  • SEQ ID NO: 20 SEQ ID NO: 21; SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29; SEQ ID NO: 30; SEQ ID NO: 31; SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38,
  • SEQ ID NO: 39 SEQ ID NO: 40; and/or SEQ ID NO: 41).
  • a “pathogen” is a microbe that causes infection or disease, especially a virus, bacterium, fungus, protozoa, or parasite.
  • the pathogen can be a virus.
  • the pathogen can be selected from the group consisting of Herpes Simplex vims- 1, Herpes Simplex virus-2, Varicella-Zoster vims, Epstein-Barr vims, Cytomegalovirus, Human Herpes virus-6, Variola vims, Vesicular stomatitis vims, Hepatitis A vims, Hepatitis B virus,
  • Hepatitis C virus, Hepatitis D virus, Hepatitis E virus, Rhino virus, Coronavims including, but not limited to avian coronavirus (IBV), porcine coronavirus HKU15 (PorCoV HKU15), Porcine epidemic diarrhea vims (PEDV), HCoV-229E, HCoV-OC43, HCoV-HKUl, HCoV- NL63, SARS-CoV, SARS-CoV-2, or MERS-CoV), Influenza vims A, Influenza vims B, Measles virus, Polyomavirus, Human Papillomavirus, Respiratory syncytial virus, Adenovirus, Coxsackie virus, Chikungunya vims, Dengue vims, Mumps virus, Poliovirus, Rabies virus, Rous sarcoma virus, Reovims, Yellow fever vims, Ebola virus, Marburg virus, Lassa fever virus, Eastern Equine En
  • the pathogen is a bacterium.
  • the pathogen can be selected from the group of bacteria consisting of Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium bovis strain BCG, BCG substrains, Mycobacterium avium, Mycobacterium intracellular, Mycobacterium africanum, Mycobacterium kansasii, Mycobacterium marinum, Mycobacterium ulcerans, Mycobacterium avium subspecies paratuberculosis, Mycobacterium chimaera, Nocardia asteroides, other Nocardia species, Legionella pneumophila, other Legionella species, Acetinobacter baumanii, Salmonella typhi, Salmonella enterica, other Salmonella species, Shigella boydii, Shigella dysenteriae, Shigella sonnei, Shigella flexneri, other Shigella species, Yersinia pestis, Pasteurella haemolytica, Pasteurella
  • the pathogen is a fungus selected from the group of fungi consisting of Candida albicans, Cryptococcus neoformans, Histoplasma capsulatum, Aspergillus fumigatus, Amanita Phalloides, Amanita Verna, Coccidiodes immitis, Paracoccidiodes brasiliensis, Blastomyces dermitidis, Pneumocystis carinii, Penicillium marneffi, and Altemaria alternata.
  • the pathogen is a parasite selected from the group of parasitic organisms consisting of Toxoplasma gondii, Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, other Plasmodium species, Entamoeba histolytica, Naegleria fowleri, Rhinosporidium seeberi, Giardia lamblia, Enterobius vermicularis, Enterobius gregorii, Ascaris lumbricoides, Ancylostoma duodenale, Necator americanus, Cryptosporidium spp., Trypanosoma brucei, Trypanosoma cruzi, Leishmania major, other Leishmania species, Diphyllobothrium latum, Hymenolepis nana, Hymenolepis diminuta, Echinococcus granulosus, Echinococcus multilocularis, Echinococcus vogeli, E
  • the microbial inflammation being treated can be in any tissue, organ, or system in the subject where a microbial infection can take place, including, but not limited to the blood, brain, sinuses, upper respiratory tract, or lungs heart, bone marrow, spleen, liver, kidneys, genito-urinary tract, bladder, aural cavities, stomach, intestines, skin, eyes, teeth, or gingiva, and musculoskeletal system.
  • microbial inflammation at its different stages such as, for example, acute inflammation, subacute inflammation, chronic inflammation, organ-specific inflammation, systemic inflammation, and/or sepsis and septic shock as the end stage of microbial inflammation
  • administering to the subject an anti-microbial agent and a composition comprising one or more NTM, wherein the microbial inflammation is in the blood, brain, sinuses, upper respiratory tract, or lungs, heart, bone marrow, spleen, liver, kidneys, genito-urinary tract, bladder, aural cavities, stomach, intestines, skin, eyes, teeth, or gingiva and musculoskeletal system.
  • the methods of reducing/inhibiting the presence of a pathogenic microbe can further comprise the administration of an anti microbial agent.
  • Anti-microbial agents include but are not limited to antibiotics, antibodies, small molecules, and functional nucleic acids (siRNA, RNAi, anti-sense oligonucleotides), that directly attack the infecting microbe or alter host conditions rendering the host system inhospitable to the microbe.
  • Such agents include, but are not limited to Abacavir, Acyclovir, Adefovir, Amantadine, Amprenavir, Ampligen, Arbidol, Atazanavir, Atripla, Balavir, Beta- D-N4-hydroxycitidine (NHC, EIDD-1931), Cidofovir, Combivir, Dolutegravir, Darunavir, Delavirdine, Didanosine, Docosanol, Edoxudine, Efavirenz, Emtricitabine, Enfuvirtide, Entecavir, Ecoliever, Famciclovir, Fomivirsen, Fosamprenavir, Foscamet, Fosfonet, Ganciclovir, Hydroxy-chloroquine, Ibacitabine, Imunovir, Idoxuridine, Imiquimod, Indinavir, Inosine, Lamivudine, Lopinavir, Loviride, Maraviroc, Moroxydine, Me
  • compositions for treating inflammation disorders (such as, for example, allergic, autoimmune, metabolic, microbial, physical, and constitutive inflammation comprising organ-specific or systemic inflammation exemplified by sepsis as the end stage of microbial inflammation) including, but not limited to microbial disease, liver metabolic disease (such as, for example, alcoholic liver disease), autoimmune disease, autoinflammatory disorder, other metabolic disorder, neoplastic disorder, inflammatory skin disorder, and/or physical insults that are mediated by inflammation in a subject (e.g., a human subject) include a therapeutically effective amount of a Nuclear Transport Modifier (such as cSN50, cSN50.1, cSN50.1 beta, or a NTM as set forth in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and/or SEQ ID NO: 9) sufficient for treating inflammation disorders at their different stages and location
  • inflammation disorders such as, for example, allergic, autoimmune, metabolic,
  • compositions described herein for treating microbial inflammation in a subject include a therapeutically effective amount of a Nuclear Transport Modifier (such as cSN50, cSN50.1, cSN50.1 beta, or a NTM as set forth in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and/or SEQ ID NO: 9) sufficient for reducing nuclear levels of a SRTFs, ChREBPs, and SREBPs in a subject with an inflammation disorder (for example, at its different stages and location (such as, for example, acute inflammation, subacute inflammation, chronic inflammation, organ-specific inflammation, systemic inflammation, and/or sepsis and septic shock as the end stage of microbial inflammation) including, but not limited to microbial disease, liver injury (such as, for example, alcoholic liver disease), autoimmune disease, autoinflammatory disorder, metabolic disorder, neoplastic disorder, inflammatory
  • a Nuclear Transport Modifier such as cSN
  • compositions can also be administered in vivo in a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to a subject, along with the nucleic acid or vector, without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.
  • the carrier would naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art.
  • compositions may be administered orally, parenterally (e.g., intravenously), by intramuscular injection, subcutaneous injection, by intraperitoneal injection, transdermally, extracorporeally, topically or the like, including topical intranasal administration or administration by inhalant.
  • topical intranasal administration means delivery of the compositions onto any dermal or exposed mucosal surface of the nose. Delivery can include creams, lotions, salves, wound adhesives, adhesive bandage, wound dressing, surgical drape, suture, spraying mechanism or droplet mechanism, or through aerosolization. Delivery can also be directly directed to any area of the respiratory system (e.g., lungs) via intubation or inhalation.
  • compositions required will vary from subject to subject, depending on the species, age, weight and general condition of the subject, the severity of the allergic disorder being treated, the particular nucleic acid or vector used, its mode of administration and the like. Thus, an appropriate amount can be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein.
  • compositions for parenteral use may be provided in unit dosage forms (e.g., in single-dose ampoules), or in vials containing several doses and in which a suitable preservative may be added (see below).
  • the composition may be in the form of a solution, a suspension, an emulsion, an infusion device, or a delivery device for implantation, or it may be presented as a dry powder to be reconstituted with water or another suitable vehicle before use.
  • the composition may include suitable parenterally acceptable carriers and/or excipients.
  • the active therapeutic agent(s) may be incorporated into microspheres, microcapsules, nanoparticles, liposomes, or the like for controlled release.
  • the composition may include suspending, solubilizing, stabilizing, pH-adjusting agents, tonicity adjusting agents, and/or dispersing agents.
  • the materials may be in solution, suspension (for example, incorporated into microparticles, liposomes, or cells). These may be targeted to a particular cell type via antibodies, receptors, or receptor ligands.
  • the following references are examples of the use of this technology to target specific proteins in tumor tissue (Senter, et al., Bioconjugate Chem., 2:447-451, (1991); Bagshawe, K.D., Br. J. Cancer, 60:275-281, (1989); Bagshawe, et al., Br. J.
  • Vehicles such as "stealth” and other antibody conjugated liposomes (including lipid mediated drug targeting to colonic carcinoma), lymphocyte directed tumor targeting such as Chimeric Antigen Receptor (CART) therapy, and highly specific therapeutic retroviral targeting of murine glioma cells in vivo.
  • compositions including antibodies, can be used therapeutically in combination with a pharmaceutically acceptable carrier.
  • Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (19th ed.) ed. A.R. Gennaro, Mack Publishing Company, Easton, PA 1995.
  • an appropriate amount of a pharmaceutically-acceptable salt is used in the formulation to render the formulation isotonic.
  • the pharmaceutically- acceptable carrier include, but are not limited to, saline, Ringer's solution and dextrose solution.
  • the pH of the solution is preferably from about 5 to about 8, and more preferably from about 7 to about 7.5.
  • Further carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, liposomes or microparticles. It will be apparent to those persons skilled in the art that certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of composition being administered.
  • compositions can be administered intramuscularly or subcutaneously. Other compounds will be administered according to standard procedures used by those skilled in the art.
  • compositions may include carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the molecule of choice.
  • Pharmaceutical compositions may also include one or more active ingredients such as antimicrobial agents, anti-inflammatory agents, anesthetics, and the like.
  • the pharmaceutical composition may be administered in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated. Administration may be topically (including ophthalmically, vaginally, rectally, intranasally), orally, by inhalation, or parenterally, for example by intravenous drip, subcutaneous, intraperitoneal or intramuscular injection using a two- compartment injector.
  • the disclosed compositions can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, or transdermally.
  • Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.
  • 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.
  • Parenteral vehicles include sodium chloride solution, 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 may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.
  • Materials for use in the preparation of microspheres and/or microcapsules are, e.g., biodegradable/bioerodible polymers such as polygalactin, poly-(isobutyl cyanoacrylate), poly(2-hydroxyethyl-L-glutamine), and poly(lactic acid).
  • Biocompatible carriers that may be used when formulating a controlled release parenteral formulation are carbohydrates (e.g., dextrans), proteins (e.g., albumin), lipoproteins, or antibodies.
  • Materials for use in implants can be non-biodegradable (e.g., poly dimethyl siloxane) or biodegradable (e.g., poly(caprolactone), poly(lactic acid), poly(glycolic acid) or poly(ortho esters) or combinations thereof).
  • biodegradable e.g., poly(caprolactone), poly(lactic acid), poly(glycolic acid) or poly(ortho esters) or combinations thereof.
  • Formulations for oral use include tablets containing the active ingredient(s) (e.g., cSN50, CSN50.1, cSN50.1 alpha, cSN50.1 beta, or a NTM as set forth in SEQ ID NO: 3,
  • SEQ ID NO: 4 SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and/or SEQ ID NO: 9) in a mixture with non-toxic pharmaceutically acceptable excipients.
  • Such formulations are known to the skilled artisan.
  • Excipients may be, for example, inert diluents or fillers (e.g., sucrose, sorbitol, sugar, mannitol, microcrystalline cellulose, starches including potato starch, calcium carbonate, sodium chloride, lactose, calcium phosphate, calcium sulfate, or sodium phosphate); granulating and disintegrating agents (e.g., cellulose derivatives including microcrystalline cellulose, starches including potato starch, croscarmellose sodium, alginates, or alginic acid); binding agents (e.g., sucrose, glucose, sorbitol, acacia, alginic acid, sodium alginate, gelatin, starch, pregelatinized starch, microcrystalline cellulose, magnesium aluminum silicate, carboxymethylcellulose sodium, methylcellulose, hydroxypropyl methylcellulose, ethylcellulose, polyvinylpyrrolidone, or polyethylene glycol); and lubricating agents, glidants, and antiad
  • the tablets may be uncoated or they may be coated by known techniques, optionally to delay disintegration and absorption in the gastrointestinal tract and thereby providing a sustained action over a longer period.
  • the coating may be adapted to release the active drug in a predetermined pattern (e.g., in order to achieve a controlled release formulation) or it may be adapted not to release the active drug until after passage of the stomach (enteric coating).
  • the coating may be a sugar coating, a film coating (e.g., based on hydroxypropyl methylcellulose, methylcellulose, methyl hydroxyethyl cellulose, hydroxypropylcellulose, carboxymethylcellulose, acrylate copolymers, polyethylene glycols and/or polyvinylpyrrolidone), or an enteric coating (e.g., based on methacrylic acid copolymer, cellulose acetate phthalate, hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate succinate, polyvinyl acetate phthalate, shellac, and/or ethylcellulose).
  • a time delay material such as, e.g., glyceryl monostearate or glyceryl distearate may be employed.
  • the solid tablet compositions may include a coating adapted to protect the composition from unwanted chemical changes, (e.g., chemical degradation prior to the release of the active therapeutic substance).
  • the coating may be applied on the solid dosage form in a similar manner as that described in Swarbrick, J. and Boylan, J. C., vide supra.
  • At least two therapeutics e.g., a composition including cSN50, cSN50.1 or any of the NTM as set forth in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 16, as well as any anti-microbial
  • the first active therapeutic is contained on the inside of the tablet, and the second active therapeutic is on the outside, such that a substantial portion of the second active therapeutic is released prior to the release of the first active therapeutic.
  • Formulations for oral use may also be presented as chewable tablets, or as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent (e.g., potato starch, lactose, microcrystalline cellulose, calcium carbonate, calcium phosphate or kaolin), or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example, peanut oil, liquid paraffin, or olive oil.
  • an inert solid diluent e.g., potato starch, lactose, microcrystalline cellulose, calcium carbonate, calcium phosphate or kaolin
  • an oil medium for example, peanut oil, liquid paraffin, or olive oil.
  • Powders and granulates may be prepared using the ingredients mentioned above under tablets and capsules in a conventional manner using, e.g., a mixer, a fluid bed apparatus or a spray drying equipment.
  • Compositions as described herein can also be formulated for inhalation and topical applications.
  • an anti-microbial agent may be
  • compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders may be desirable.
  • compositions may potentially be administered as a pharmaceutically acceptable acid- or base- addition salt, formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with an inorganic base such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, trialkyl and aryl amines and substituted ethanol amines.
  • inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid
  • organic acids such as formic acid, acetic acid, propionic acid,
  • Effective dosages and schedules for administering the compositions may be determined empirically, and making such determinations is within the skill in the art.
  • the dosage ranges for the administration of the compositions are those large enough to produce the desired effect in which the symptoms of the disorder are affected.
  • the dosage should not be so large as to cause adverse side effects, such as unwanted cross -reactions, and the like.
  • the dosage will vary with the age, condition, sex and extent of the disease in the patient, route of administration, or whether other drugs are included in the regimen, and can be determined by one of skill in the art.
  • the dosage can be adjusted by the individual physician in the event of any counterindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products.
  • SEQ ID NO: 2 sets forth a particular sequence of an NTM (cSN50.1). Specifically disclosed are variants of these and other genes and proteins herein disclosed which have at least, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,
  • sequence homology is used interchangeably with sequence identity.
  • Protein variants and derivatives are well understood to those of skill in the art and in can involve amino acid sequence modifications.
  • amino acid sequence modifications typically fall into one or more of three classes: substitutional, insertional or deletional variants.
  • Insertions include amino and/or carboxyl terminal fusions as well as intrasequence insertions of single or multiple amino acid residues. Insertions ordinarily will be smaller insertions than those of amino or carboxyl terminal fusions, for example, on the order of one to four residues.
  • Cell-penetrating fusion protein derivatives are made by fusing a polypeptide sufficiently large to confer intracellular delivery of the targeting sequence by cross-linking in vitro or by recombinant cell culture transformed with DNA encoding the fusion.
  • Deletions are characterized by the removal of one or more amino acid residues from the protein sequence. Typically, no more than about from 2 to 6 residues are deleted at any one site within the protein molecule.
  • These variants ordinarily are prepared by site specific mutagenesis of nucleotides in the DNA encoding the protein, thereby producing DNA encoding the variant, and thereafter expressing the DNA in recombinant cell culture. Techniques for making substitution mutations at predetermined sites in DNA having a known sequence are well known, for example M13 primer mutagenesis and PCR mutagenesis.
  • Amino acid substitutions are typically of single residues, but can occur at a number of different locations at once; insertions usually will be on the order of about from 1 to 10 amino acid residues; and deletions will range about from 1 to 30 residues. Deletions or insertions preferably are made in adjacent pairs, i.e. a deletion of 2 residues or insertion of 2 residues. Substitutions, deletions, insertions or any combination thereof may be combined to arrive at a final construct. The mutations must not place the sequence out of reading frame and preferably will not create complementary regions that could produce secondary mRNA structure. Substitutional variants are those in which at least one residue has been removed and a different residue inserted in its place. Such substitutions generally are made in accordance with the following Tables 3 and 4 and are referred to as conservative substitutions.
  • Substantial changes in function or immunological identity are made by selecting substitutions that are less conservative than those in Table 4, i.e., selecting residues that differ more significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site or (c) the bulk of the side chain.
  • substitutions which in general are expected to produce the greatest changes in the protein properties will be those in which (a) a hydrophilic residue, e.g. seryl or threonyl, is substituted for (or by) a hydrophobic residue, e.g.
  • an electropositive side chain e.g., lysyl, arginyl, or histidyl
  • an electronegative residue e.g., glutamyl or aspartyl
  • substitutions include combinations such as, for example, Gly, Ala; Val, lie, Leu; Asp, Glu; Asn, Gin; Ser, Thr; Lys, Arg; and Phe, Tyr.
  • substitutions include combinations such as, for example, Gly, Ala; Val, lie, Leu; Asp, Glu; Asn, Gin; Ser, Thr; Lys, Arg; and Phe, Tyr.
  • Such conservatively substituted variations of each explicitly disclosed sequence are included within the mosaic polypeptides provided herein.
  • Substitutional or deletional mutagenesis can be employed to insert sites for N- glycosylation (Asn-X-Thr/Ser) or O-glycosylation (Ser or Thr).
  • Deletions of cysteine or other labile residues also may be desirable.
  • Deletions or substitutions of potential proteolysis sites, e.g. Arg is accomplished for example by deleting one of the basic residues or substituting one by glutaminyl or histidyl residues.
  • Certain post-translational derivatizations are the result of the action of recombinant host cells on the expressed polypeptide. Glutaminyl and asparaginyl residues are frequently post-translationally deamidated to the corresponding glutamyl and asparyl residues. Alternatively, these residues are deamidated under mildly acidic conditions. Other post-translational modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the o-amino groups of lysine, arginine, and histidine side chains (T.E. Creighton, Proteins: Structure and Molecular Properties, W. H. Freeman & Co., San Francisco pp 79-86 [1983]), acetylation of the N- terminal amine and, in some instances, amidation of the C-terminal carboxyl.
  • variants and derivatives of the disclosed protein-derived peptides herein is through defining the variants and derivatives in terms of homology /identity to specific known sequences.
  • SEQ ID NO:2 sets forth a particular sequence of cSN50.1. Specifically disclosed are variants of these and other proteins herein disclosed which have at least, 70%, 75%, 80% , 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1,%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity to the stated sequence.
  • the homology can be calculated after aligning the two sequences so that the homology is at its highest level.
  • nucleic acids that can encode those protein-derived peptide sequences are also disclosed. This would include all degenerate sequences related to a specific protein sequence, i.e. all nucleic acids having a sequence that encodes one particular protein sequence as well as all nucleic acids, including degenerate nucleic acids, encoding the disclosed variants and derivatives of the protein sequences. Thus, while each particular nucleic acid sequence may not be written out herein, it is understood that each and every sequence is in fact disclosed and described herein through the disclosed protein sequence.
  • Molecules can be produced that resemble peptides, but which are not connected via a natural peptide linkage.
  • Amino acid analogs and analogs and peptide analogs often have enhanced or desirable properties, such as, more economical production, greater chemical stability, enhanced pharmacological properties (half-life, absorption, potency, efficacy, etc.), altered specificity (e.g., a broad-spectrum of biological activities), reduced antigenicity, and others.
  • D-amino acids can be used to generate more stable peptides, because D amino acids are not recognized by peptidases and such.
  • Systematic substitution of one or more amino acids of a consensus sequence with a D-amino acid of the same type e.g., D-lysine in place of L-lysine
  • Cysteine residues can be used to cyclize or attach two or more peptides together. This can be beneficial to constrain peptides into particular conformations.
  • Stapled alpha-helical sequence of signal-sequence hydrophobic region and/or other fragments used as cargo” can be used to stabilize their conformation in NTM peptides.
  • Tissue injury induced by alcohol abuse constitutes a worldwide health problem.
  • the primary targets of alcohol toxicity are the gastrointestinal system and liver.
  • Alcoholic liver disease (ALD) characterized by infiltrating T cells in regenerating nodules, intralobular zones of inflammation, and central sclerosis, afflicts an estimated 2 million patients in the US.
  • ALD Alcoholic liver disease
  • the combination of liver cirrhosis and alcoholic hepatitis contributes to an astonishing 65% mortality rate over a 4 -year period. This represents a toll greater than many oncologic diseases.
  • Ethanol (EtOH) - induced hepatocellular injury involves not only excessive oxidant stress but also T cell- and Kupffer cell-mediated inflammatory responses.
  • T cell- mediated hepatitis can be recapitulated in mice by injection of a polyclonal T cell activator, plant lectin concanavalin A (Con A), which evokes production of proinflammatory cytokines/chemokines, granulocyte accumulation in liver sinusoids, and apoptosis/necrosis of hepatocytes. It was discovered that these pathologic changes can be significantly attenuated by an innovative cell-penetrating nuclear import inhibitory peptide (cSN50) as the platform in a series of recent studies on inflammatory liver injury. Thus, the role of nuclear import adaptors in the mechanism of EtOH-altered intracellular proinflammatory and proapoptotic signaling to the nucleus can be established. a) Investigating the Mechanism of Ethanol-Enhanced Liver Injury
  • EtOH alters homeostatic balance between these two signal transducers: (ii) that interruption of proinflammatorv signaling in Kupffer cells slow down disease progression in EtOH-enhanced liver injury in the background of excessive oxidant stress and proinflammatorv/proapoptotic signaling: and (iii) that the nuclear import adaptors for Stress-Responsive Transcription Factors (SRTFs) play an essential role in EtOH- associated inflammatory liver injury mediated by macrophages (Kupffer cells) and caused bv endotoxic lipopolvsaccharide (LPS).
  • SRTFs Stress-Responsive Transcription Factors
  • SRTFs regulate the genome response to proinflammatory and proapoptotic cues.
  • the mechanism of action of cell-penetrating peptides SN50 and its cyclized analog cSN50 that inhibit the nuclear import of SRTFs is shown herein.
  • SN50 binds to a nuclear import adaptor protein termed importin a5/karyopherin al (/KPNA1/SRP1) and inhibits its function as a cytoplasmic/nuclear shuttle for SRTFs.
  • nuclear entry is controlled by the complex system of pores that allow free passage to small ( ⁇ 40 kD) karyophilic proteins.
  • Larger proteins such as SRTFs bear a zip code denoted “nuclear localization sequence” (NLS) that is recognized by the "nuclear mail carriers” known as importins/karyopherins.
  • NLS nuclear localization sequence
  • importins/karyopherins these adaptor proteins, also called NLS receptors, deliver SRTFs to their nuclear site of action.
  • nuclear import constitutes the last signaling checkpoint before SRTFs are translocated to the nucleus.
  • Six different human importin/karyopherin alpha adaptor proteins have been reported.
  • importin/karyopherin alpha 5 (SRP1/KPNA1) is the target for a cell-permeant peptide (SN50). Further studies with this new class of cell-penetrating peptide inhibitors of nuclear import allows for the development of inhibitors that target other nuclear import shuttles involved in inflammatory liver injury.
  • Example 9 Ethanol Feeding Enhances T cell-mediated Hepatitis Induced by Concanavalin A (Con A).
  • T cell mediated hepatitis model induced by Con A was modified to include ethanol as a potential liver injury enhancing agent.
  • ethanol a range of Con A concentrations was tested to establish the threshold dose (0.5mg/kg) which induces subclinical liver injury in C57BL/6J mice.
  • EtOH ethanol
  • the ethanol (EtOH) feeding protocol was applied based on an experimental model of LPS-induced liver injury following ethanol exposure. Briefly, 7-week- old female C57BL-6J mice were fed control liquid diets for 3-5 days, then the mice were divided into two groups. Half were fed EtOH-containing (6%) liquid diet and half were fed similar control diet in which EtOH was substituted isocalorically with dextrin maltose. Both diets were purchased from BioServ, Inc.
  • mice were pair-fed (PF) the same volume of an isocaloric of ethanol-free diet daily.
  • PF the same volume of an isocaloric of ethanol-free diet daily.
  • mice received a single injection of Con A (Sigma) via tail vein. Animals were sacrificed at 12- and 24- hours post- Con A. At these time points, fragments of each liver were freeze-clamped in liquid nitrogen and stored at - 80°C until analysis. The remaining liver fragments and other organs were fixed in buffered formalin for subsequent histologic evaluation.
  • EtOH feeding raised the level of the liver enzyme ALT and proinflammatory cytokines IL6 and IFN gamma (Fig. 3).
  • Example 2 Liver Apoptosis and Necrosis Induced by SEB and Mediated by T cells is Suppressed by Nuclear Import Inhibitors.
  • Staphylococcal immunotoxin termed enterotoxin B
  • enterotoxin B and related toxins that target T cells have the capacity to elicit systemic inflammation, liver injury, and death.
  • Cell-penetrating peptides targeting importin/karyopherin alpha 5 /KPNA1, a nuclear import adaptor protein, are delivered to T cells where they inhibit the Staphylococcal enterotoxin B -induced production of inflammatory cytokines ex vivo in cultured primary spleen cells and in vivo.
  • mice The systemic production of tumor necrosis factor alpha, interferon gamma, and interleukin-6 was attenuated in mice either by a cell-penetrating cyclized form of SN50 peptide or by a transgene whose product suppresses the nuclear import of transcription factor nuclear factor /cB in T cells.
  • Example 3 Inflammation-Associated Liver Apoptosis and Necrosis Induced by LPS and Mediated by Toll-Like Receptor 4 (TLR4)-Expressing Macrophages (Kupffer Cells) Requires Nuclear Import of SRTFs.
  • TLR4 Toll-Like Receptor 4
  • mice challenged with LPS displayed very early bursts of the inflammatory cytokines/chemokines TNFoc(l h), IL-6 (2 h), IL-1/3 (2 h), and MCP-1 (2 h).
  • Example 4 Ethanol Feeding Enhances Inflammatory Liver Injury Induced by LPS:
  • cytokine IL10 cell-penetrating nuclear import inhibitory peptide (cSN50) injected i.p. at 30 min before and 30, 90, 150, 210 min and 6, and 12 hours after LPS.
  • the control and treated animals were sacrificed at 12 and 24 hours post-LPS.
  • fragments of each liver were freeze-clamped in liquid nitrogen and stored at -80°C until analysis.
  • the remaining liver tissues and other organs were fixed in buffered formalin for subsequent evaluation of histology.
  • EtOH feeding enhanced substantially production of proinflammatory cytokines and chemokines (TNF alpha, IL6, and MCP1) whereas anti-inflammatory cytokine IL10 remained unchanged.
  • Example 5 The Mechanism of Intracellular Delivery of Cell-Penetrating Peptides that Carry Membrane-Translocating Motif Based on Signal Sequence Hydrophobic Region (SSHR).
  • SSHR Signal Sequence Hydrophobic Region
  • SSHR signal sequence hydrophobic region
  • a nuclear localization sequence comprised of a positively charged cluster of amino acids was rapidly translocated by SSHR enantiomers to the interior of unilamellar phospholipid vesicles.
  • SSHR-based MTM possesses a number of desirable attributes. It is based on the hydrophobic region of a signal sequence that has been conserved through evolution. It translocates freely across a phospholipid bilayer, bypassing a more complex endocytic pathway apparently used by other MTMs such as Antennapedia-based or HIV TAT-based sequences.
  • Example 6 The Mechanism of Inhibition of Nuclear Import of Stress-Responsive Transcription Factors by Cell-Penetrating SN50 Peptide.
  • SRTFs Stress-Responsive Transcription Factors
  • NLS nuclear localization sequence
  • Inducible nuclear import of NF-KB, AP-1, NFAT, and STAT1 in Jurkat T lymphocytes is significantly inhibited by a cell-penetrating peptide carrying the NLS of the NF-KB1 (p50 subunit).
  • NLS peptide-mediated disruption of the nuclear import of these transcription factors results in inhibition of 1/cBa and IL-2 gene expression, processes dependent on NF-KB or the combination of NF-KB, AP-1, and NFAT.
  • inhibitory NLS peptide interacts with a cytoplasmic NLS receptor complex comprised of the SRPl/importin alpha 5 (karyopherin alpha l)/importin beta 1 heterodimer expressed in Jurkat T cells.
  • Example 7 Nuclear Import Inhibitor cSN50 Attenuates Expression of ⁇ 50% LPS- lnducible Genes in the Liver.
  • Endotoxic lipopoly saccharide (LPS, endotoxin) is one of the most potent known pathogen-derived inducers of inflammation ⁇ LPS-evoked inflammatory response is dependent on expression of Toll-like receptor (TLR) 4 expressed in macrophages abundantly present in the liver and known as Kupffer cells.
  • TLR Toll-like receptor
  • mice In response to a high dose of LPS (40 mg/kg without D- galactosamine) mice produce robustly inflammatory cytokines/chemokines and succumb within 72 hours.
  • SRTFs Stress-Responsive Transcription Factor
  • nuclear import adaptor importin/karyopherin alpha 5 (SRP1, KPNA1), which is targeted by cell-penetrating cSN50, is responsible for cytoplasmic/nuclear translocation of SRTFs and metabolic transcription factors that regulate almost half of the genes induced by LPS signaling to the nucleus through TLR4.
  • SRP1, KPNA1 nuclear import adaptor importin/karyopherin alpha 5
  • This hitherto not reported comprehensive analysis of nuclear import-regulated genome response to proinflammatory agonist, LPS is consistent with the demonstration of highly protective effect of cell penetrating nuclear import inhibitor cSN50 peptide in a murine model of LPS-induced lethal inflammation, apoptosis, microvascular thrombosis, and hemorrhagic necrosis.
  • EtOH-induced liver injury is dependent on continuing stimulation of Kupffer cells by endotoxic lipopolysaccharide (LPS) shed from Gram-negative bacteria in the gut and entering the portal circulation through EtOH-altered intestinal mucosa.
  • LPS endotoxic lipopolysaccharide
  • Kupffer cells produce proinflammatory cytokines and chemokines which evoke proapoptotic signaling pathways in hepatocytes that are "sensitized” by oxidant stress induced by EtOH interaction with hepatocytes and other liver cells.
  • IRAK- M a physiologic inhibitor of LPS signaling in macrophages and other phagocytes, can render Kupffer cells "LPS tolerant" upon delivery of cell-penetrating form of this dominant negative inhibitor of LPS signaling.
  • Kupffer cells and other LPS responders become refractory to LPS and unable to produce proinflammatory cytokines/chemokines required for inflammatory and apoptotic injury of hepatocytes.
  • EtOH-induced oxidant stress is significantly enhanced by continuing activation of Kupffer cells.
  • LPS tolerance reduces EtOH-related liver injury in the setting of excessive oxidant stress. This understanding can be supported by rendering SOD-/- mice refractory to LPS. a) Rationale and Results.
  • Kupffer cells can be prevented from generating proinflammatory and proapoptotic cytokines in response to LPS and other bacterial products that gained entry to portal circulation.
  • IRAK-M mediates refractoriness to LPS ("endotoxin tolerance") predominantly in macrophages (e.g. Kupffer cells), dendritic cells, and other phagocytes of myelomonocytic lineage.
  • CP-IRAK-M cell-penetrating (CP) forms of IRAK-M to Kupffer cells
  • LPS-evoked proinflammatory signaling can be ablated in vivo in models of EtOH-induced tissue injury.
  • CP-IRAK-M can be studied not only in EtOH- fed mice challenged with LPS or Con A, but also in Sodl-/- mice which are inherently prone to EtOH-associated liver injury due to oxidative stress. These experiments can define the role of LPS tolerance in EtOH-associated liver injury in wild type and SOD-/- mice that display an excessive oxidative stress.
  • Example 11 Suppressing EtOH-Associated Liver Injury by Targeting Relevant Nuclear Import Adaptors.
  • inflammatory liver injury mediated by macrophages (Kupffer cells) or T cells depends on the nuclear import of SRTFs and metabolic transcription factors SREBPs and ChREBPs. These transcription factors regulate the genome response to proinflammatory, metabolic, and proapoptotic cues.
  • SN50 nuclear import factor
  • ChREBPs metabolic transcription factors
  • Prior studies have established the mechanism of action of SN50 and its congeners, the cell-penetrating peptides that inhibit the nuclear import of SRTFs, SREBPs and ChREBPs.
  • SRP1/KPNA1 nuclear import adaptor protein
  • SARM nuclear import adaptor protein
  • SARM TIR domain expressed in human hepatoma cell line (HepG2) reduced Interleukin 1 beta-induced expression of the NF-KB- regulated reporter gene expression.
  • SRTFs Stress-Responsive Transcription Factor
  • ChREBPs Carbohydrate- Responsive Element-Binding proteins
  • SREBPs Sterol Regulatory Element-Binding Proteins
  • microbial diseases bacteria, viruses, fungi, protozoa
  • microbial diseases bacteria, viruses, fungi, protozoa
  • SRTFs transcription factors
  • ChREBPs the metabolic transcription factors
  • SREBPs the metabolic transcription factors
  • the products of the genes regulated by these transcription factors (cholesterol, triglycerides, glucose) also activate proinflammatory signaling pathway mediated by the Imp a5, thereby producing more severe and difficult to control stage of microbial inflammation.
  • This advanced stage of microbial inflammation comprises inflammatory liver injury, acute respiratory distress syndrome (ARDS), septic cardiomyopathy, microvascular thrombosis (known as Disseminated Intravascular Coagulation), and Acute Kidney Injury.
  • ARDS acute respiratory distress syndrome
  • septic cardiomyopathy a microvascular thrombosis
  • microvascular thrombosis a microvascular thrombosis
  • Acute Kidney Injury a) Rationale and Results.
  • Alcoholic liver disease is associated with the action of endotoxin (lipopolysaccharide, LPS) absorbed from the gut microbiota, and oxidative stress. Both LPS and EtOH-induced metabolic changes, produce oxidative stress that activate a set of intracellular Stress-Responsive Transcription Factor (SRTFs), Carbohydrate-Responsive Element-Binding proteins (ChREBPs), and Sterol Regulatory Element-Binding Proteins (SREBPs). They are imported to the nucleus to reprogram the genome toward continuous expression of proinflammatory, metabolic, and proapoptotic mediators.
  • SRTFs Stress-Responsive Transcription Factor
  • ChREBPs Carbohydrate-Responsive Element-Binding proteins
  • SREBPs Sterol Regulatory Element-Binding Proteins
  • the inflammatory response is mediated by one or more SRTFs, ChREBPs, and SREBPs that are shuttled to the nucleus by a set of adaptor proteins known as importins/karyopherins alpha/beta and possibly a new adaptor SARM.
  • nuclear SRTFs, ChREBPs, and SREBPs activate a myriad of genes that encode inflammatory cytokines/chemokines such as TNFoc, IFNy, and MCP-1, metabolic genes responsible for hyperglycemia, and hypertriglyceridemia.
  • importin a5/karyopherinal KPNA1/SRP1
  • SRTFs Stress-Responsive Transcription Factor
  • Importin a5 was identified as the target of the cell-penetrating peptide SN50 that prevents nuclear import of four SRTFs in human T lymphocytes.
  • Importin b ⁇ was identified as a sole carrier of SREBPs. Both importins, a5 and b ⁇ , are carriers of ChREBPs.
  • Hyperlipidemia the hallmark of Metabolic Syndrome that afflicts millions of people worldwide, exacerbates life-threatening infections.
  • SRTFs stress-responsive transcription factors
  • SREBPs Sterol Regulatory Element- Binding Proteins
  • ChREBPs Carbohydrate-Responsive Element-Binding Proteins
  • Bode, J.G., Nimmesgern, A., Schmitz, J., Schaper, F., Schmitt, M., Frisch, W., Haussinger, D., Heinrich, P.C. and Graeve, L., LPS and TNFalpha induce SOCS3 mRNAand inhibit IL-6- induced activation of STATS in macrophages.
  • Croker, B.A. Krebs, D.L, Zhang, J.G., Wormald, S., Willson, T.A., Stanley, E.G., Robb, L, Greenhalgh, C.J., Forster, I., Clausen, B.E., Nicola, N.A., Metcalf, D., Hilton, D.J., Roberts, A.W. and Alexander, W.S., SOCS3 negatively regulates IL-6 signaling in vivo. Nat. Immunol., 2003. 4(6): p. 540-5.
  • IFN-gamma/STATl acts as a proinflammatory signal in T cell-mediated hepatitis via induction of multiple chemokines and adhesion molecules: a critical role of IRF-1. Am J Physiol Gastrointest Liver Physiol, 2004. 287(5): p. G1044-52.
  • Kobayashi, K., Hernandez, L.D., Galan, J.E., Janeway, C.A., Jr., Medzhitov, R. and Flavell, R.A., IRAK M is a negative regulator of Toll-like receptor signaling. Cell, 2002.110(2): p. 191- 202.
  • CCCH zinc finger proteins with mRNA Binding of tristetraprolin-related zinc finger proteins to Au-rich elements and destabilization of mRNA. J Biol Chem, 2000. 275(23): p. 17827-37.
  • Mink, M., Fogelgren, B., Olszewski, K., Maroy, P. and Csiszar, K. A novel human gene (SARM) at chromosome 17ql 1 encodes a protein with a SAM motif and structural similarity to Armadillo/betacatenin that is conserved in mouse, Drosophila, and Caenorhabditis elegans. Genomics, 2001. 74(2): p. 234-44.
  • Lipopolysaccharide induces in macrophages the synthesis of the suppressor of cytokine signaling 3 and suppresses signal transduction in response to the activating factor IFN-gamma. J. Immunol., 1999.163(5): p. 2640-7.

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Abstract

L'invention concerne des compositions et des méthodes de traitement d'états induits par une inflammation comme la stéatose hépatique, la stéatohépatite, l'apoptose et la cirrhose, la thrombocytopénie, l'hypoglycogénémie, l'hyperglycémie et l'hypertriglycéridémie. Selon un aspect, les compositions et les méthodes selon l'invention peuvent également être utilisées pour améliorer la clairance des microbes des tissus, organes ou systèmes infectés chez un sujet. L'invention concerne également des compositions et des méthodes pour réduire les niveaux des facteurs de transcription sensibles au stress et des facteurs de transcription métaboliques dans une cellule chez un sujet présentant une inflammation du foie microbienne, allergique, auto-immune, métabolique et post-traumatique.
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