WO2013112834A1 - Compositions et méthodes de traitement d'infections - Google Patents

Compositions et méthodes de traitement d'infections Download PDF

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Publication number
WO2013112834A1
WO2013112834A1 PCT/US2013/023143 US2013023143W WO2013112834A1 WO 2013112834 A1 WO2013112834 A1 WO 2013112834A1 US 2013023143 W US2013023143 W US 2013023143W WO 2013112834 A1 WO2013112834 A1 WO 2013112834A1
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subject
infection
penetrating
cell
myd88
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PCT/US2013/023143
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English (en)
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Jacek J. HAWIGER
Ruth Ann Veach
Jozef ZIENKIEWICZ
Robert D. Collins
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Vanderbilt University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7155—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for interleukins [IL]

Definitions

  • the invention relates generally to the fields of microbiology, bioterrorism, and medicine.
  • Pulmonary anthrax caused by airborne Bacillus anthracis spores is a life-threatening disease often refractory to antimicrobial therapy.
  • PAn Pulmonary anthrax
  • other pulmonary infections caused by ebola viruses, marburgviruses, and SARS coronavirus as well as those caused by Pasteurella tularensis, Staphycoccus aureus, are considered biothreats and emerging infectious diseases for which there are a very few FDA-approved therapeutics.
  • compositions and methods for inhibiting growth of a pathogen e.g., Bacillus anthracis bacteria
  • a pathogen e.g., Bacillus anthracis bacteria
  • the compositions and methods include administration of compositions that include an antimicrobial agent and a nuclear transport modifier (NTM).
  • NTM nuclear transport modifier
  • An 8-day protocol of single-dose ciprofloxacin had no significant effect on mortality (4% survival) of A/J mice lethally infected with B. anthracis Sterne.
  • mice were much more likely to survive infection (52% survival) when treated with ciprofloxacin and a cell-penetrating peptide modifier of host nuclear transport, termed cSN50.
  • cSN50 a muted innate immune response manifested by cytokines, tumor necrosis factor alpha (TNFa), interleukin (IL)-6, and chemokine monocyte chemoattractant protein- 1 (MCP-1) was detected, while the hypoxia biomarker, erythropoietin (EPO), was greatly elevated.
  • cSN50-treated mice receiving ciprofloxacin demonstrated a restored innate immune responsiveness and reduced EPO level. Consistent with this improvement of innate immunity response and suppression of hypoxia biomarker, surviving mice in the combination treatment group displayed minimal histopathologic signs of vascular injury and a marked reduction of anthrax bacilli in the lungs.
  • 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).
  • binding means that one molecule recognizes and adheres to a particular second molecule in a sample or organism, but does not substantially recognize or adhere to other structurally unrelated molecules in the sample.
  • nucleic acid molecule or polypeptide when referring to a nucleic acid molecule or polypeptide, the term “native” refers to a naturally-occurring (e.g., a wild type, WT) nucleic acid or polypeptide.
  • operably linked as used herein may mean a functional linkage between two polynucleotides, for example a first polynucleotide and a second polynucleotide, wherein expression of one polynucleotide affects transcription and/or translation and/or mRNA stability of the other polynucleotide.
  • "Operably linked” may also mean a functional linkage between two or more polypeptides (e.g., a first polypeptide and a second polypeptide).
  • anti-microbial agent is meant to encompass any molecule, chemical entity, composition, drug, therapeutic agent, or biological agent capable of preventing or reducing growth of a microbe (e.g., bacteria, yeast, viruses), or capable of blocking the ability of a microbe to cause disease.
  • a microbe e.g., bacteria, yeast, viruses
  • An example of an antimicrobial agent is an antibiotic.
  • the term includes small molecule compounds, antisense reagents, siRNA reagents, antibodies, enzymes, peptides, organic or inorganic molecules, natural or synthetic compounds and the like.
  • Nuclear Transport Modifier and “NTM” mean a component that is capable of modulating (e.g., decreasing the activity or expression of) at least one nuclear transport adaptor (e.g., importin/karyopherin alpha, importin/karyopherin beta) and thus reducing the transport of factors, such as transcription factors, enzymes, and structural proteins, which are larger than 45 kD into the nucleus.
  • nuclear transport adaptor e.g., importin/karyopherin alpha, importin/karyopherin beta
  • factors such as transcription factors, enzymes, and structural proteins
  • nuclear import adaptor and “nuclear transport adaptor” mean a cell component capable of mediating transport of a protein usually larger than 45 kD (e.g., a transcription factor) into the nucleus.
  • kD e.g., a transcription factor
  • nuclear transport adaptor an importin also known as karyopherin.
  • patient means a mammalian (e.g., human) subject to be treated and/or to obtain a biological sample from.
  • 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.
  • safe and effective amount refers to the quantity of a component which is sufficient to yield a desired therapeutic response without undue adverse side effects (such as toxicity, irritation, or allergic response) commensurate with a reasonable benefit/risk ratio when used in the manner of this invention.
  • therapeutically effective amount is meant an amount of a composition as described herein effective to yield the desired therapeutic response.
  • the specific safe and effective amount or therapeutically effective amount will vary with such factors as the particular condition being treated, the physical condition of the patient, the type of mammal or animal being treated, the duration of the treatment, the nature of concurrent therapy (if any), and the specific formulations employed and the structure of the compounds or its derivatives.
  • the method includes administering to the subject a composition including a pharmaceutically acceptable carrier, an anti-microbial agent, and a nuclear transport modifier (NTM) wherein the composition is in a therapeutically effective amount for treating the infection.
  • the composition can further include a cell penetrating physiologic protein such as cell-penetrating-MAL or recombinant cell-penetrating MyD88.
  • the infection is a B. anthracis infection
  • the anti-microbial agent is ciprofloxacin
  • the NTM is cSN50 or cSN50.1.
  • the subject can be a human suffering from pulmonary anthrax disease and administration of the composition treats the pulmonary anthrax disease. In some embodiments, administration of the composition prevents pulmonary anthrax disease in a subject.
  • the composition can be administered to the subject by any suitable route, e.g., intravenously.
  • the method includes administering to the subject a first composition including a pharmaceutically acceptable carrier and an anti-microbial agent, and a second composition including a pharmaceutically acceptable carrier and a NTM, wherein administration (e.g., intravenously) of the first and second compositions results in eradication of the infection.
  • the first and second compositions can be administered to the subject simultaneously, or can be administered to the subject at two different time points.
  • the composition can further include a cell penetrating physiologic protein such as cell- penetrating-MAL and recombinant cell-penetrating MyD88.
  • the infection is a B.
  • the anti-microbial agent is ciprofloxacin
  • the NTM is cSN50 or cSN50.1.
  • the subject can be a human suffering from pulmonary anthrax disease and administration of the first and second compositions treats the pulmonary anthrax disease. In some embodiments, administration of the first and second compositions prevents pulmonary anthrax disease in the subject.
  • the pharmaceutical composition for treating an infection in a subject (e.g., human, animal).
  • the pharmaceutical composition includes a pharmaceutically acceptable carrier, an anti-microbial agent, and a NTM.
  • the pharmaceutical composition can further include a cell penetrating physiologic protein such as cell-penetrating-MAL and recombinant cell-penetrating MyD88.
  • the infection is a B. anthracis infection
  • the anti-microbial agent is ciprofloxacin
  • the NTM is cSN50 or cSN50.1.
  • the anti-microbial agent and the NTM are in therapeutically effective amounts for treating the infection, and when treating a subject suffering from pulmonary anthrax disease, the antimicrobial agent and the NTM are in therapeutically effective amounts for treating pulmonary anthrax disease in the subject.
  • kits for treating an infection in a subject include a composition including a pharmaceutically acceptable carrier, an anti-microbial agent and at least a first NTM; packaging; and instructions for use.
  • the composition is in a therapeutically effective amount for treating the infection in a subject.
  • the composition may be contained within a syringe.
  • the infection is a B. anthracis infection
  • the anti-microbial agent is ciprofloxacin
  • the at least one NTM is cSN50 or cSN50.1.
  • the composition can further include a second NTM and/or a cell penetrating physiologic protein such as cell-penetrating-MAL and recombinant cell-penetrating MyD88.
  • compositions, kits and methods similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable compositions, kits and methods are described below. All publications, patent applications, and patents mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control. The particular embodiments discussed below are illustrative only and not intended to be limiting.
  • FIG. 1 is a graph showing survival in inhalational anthrax was increased by a combination of cSN50 with Ciprofloxacin.
  • Female A/J mice were infected intranasally (IN) with 10 7 B. anthracis spores and treated with 15 injections of cSN50 during the first 2 days and daily ciprofloxacin (triangles) or saline and ciprofloxacin (squares) or saline without ciprofloxacin (circles).
  • the p value represents the significance of the difference in survival between the two ciprofloxacin-treated groups (with and without cSN50 peptide).
  • FIG. 2 is a series of graphs showing cSN50 restored depressed cytokine/chemokine responses to B. anthracis infection and attenuated a rise in erythropoietin, a hypoxia biomarker.
  • Blood serum levels of TNFa, IL-6, and MCP-1 were measured in female A/J mice infected with 10 7 B. anthracis spores IN and treated with cSN50 and daily ciprofloxacin (solid squares) or saline and ciprofloxacin (solid triangles) or saline without ciprofloxacin (open triangles) as in Fig. l .
  • FIG. 3 is a series of micrographs showing lung injury in mice challenged intranasally with B. anthracis was reduced by cSN50 treatment.
  • E-F Mice treated with saline + ciprofloxacin.
  • FIG. 4 is a schematic illustration depicting a potential site of action of cell- penetrating peptides that counteractanthrax toxins acting within the cell.
  • anti-microbial compositions and kits for inhibiting growth of a pathogen and treating infection by the pathogen in the subject, and treating an associated pulmonary disease in the subject, and methods of production and use thereof. It has been demonstrated, for the first time, that regulating nuclear transport with a cell- penetrating modifier (NTM) provides a cytoprotective effect, which enables the host's immune system to reduce its susceptibility to lethal B. anthracis infection.
  • NTM cell- penetrating modifier
  • a nuclear transport modifier with anti-microbial therapy (e.g., an antibiotic or anti-viral therapeutic that inhibits viral entry or replication)
  • anti-microbial therapy e.g., an antibiotic or anti-viral therapeutic that inhibits viral entry or replication
  • a novel adjunctive measure to control florid pulmonary disease acused by anthrax or other biothreats or emerging infections is offered.
  • the NTM e.g., cSN50
  • one or more other cell penetrating physiologic proteins e.g. cell-penetrating MyD88 that are potentially depleted by intracellularly deployed toxins or viruses
  • the primary mode of infection for anthrax afflicting humans is by spores that are inhaled, ingested through the GI tract, or absorbed through a break in the skin.
  • spores are phagocytized by alveolar macrophages and dendritic cells. These spore-containing phagocytes function as "Trojan horses" that gain entry into regional lymph nodes.
  • anthrax spores germinate rapidly (1 h) into bacillary vegetative forms endowed with an antiphagocytic capsule and three additional virulence factors: protective antigen (PA), lethal factor (LF), and edema factor (EF) - known cumulatively as anthrax toxin.
  • PA protective antigen
  • LF lethal factor
  • EF edema factor
  • Pulmonary anthrax is characterized by perihilar interstitial pneumonia, acute bronchopneumonia with intra-alveolar and interstitial exudates. Alveolar spaces are filled with fluid containing fibrin, and show signs of hemorrhage.
  • Anthrax lethal toxin is a heterodimer of two proteins produced by anthrax vegetative forms: a pore- forming Protective Antigen (PA) and a Zn-dependent metalloprotease known as Lethal Factor (LF).
  • PA is a pH-dependent protein translocator that ferries LF into the cytoplasm.
  • the PA-LF complex is internalized and translocated from the endocytic vesicle to the cytoplasm. Multiple structural changes are required from receptor-bound pore-forming PA and from its catalytic partners LF and EF to reach the cell's interior. This entry process is catalyzed by a phenylalanine clamp.
  • LF docks with at least eight nitrogen-activated protein (MAP) kinases and cleaves their FL terminal segments. Since these segments are required for binding of MAP kinases to their downstream signal transducers, the signaling pathways involved in anti-anthrax innate (non-specific) and adaptive (specific) immune responses are disrupted.
  • MAP nitrogen-activated protein
  • the MKK3 dual specificity kinase that activates p 38 MAP kinase is inactivated in macrophages.
  • inactivation of MKK3 triggered by LF results in the inability of macrophages to produce TNFa and nitric oxide (NO), two important effectors of innate immunity.
  • NO nitric oxide
  • Dendritic cells the most effective antigen-presenting cells, are also targets for this "disarming" function of LF.
  • the reported pattern of mediators activated in response to LF includes increases in inflammatory cytokine Interleukin (IL)-6, and chemokine KC, which are dependent on nuclear import of NF-KB and other stress-responsive transcription factors.
  • IL-6 cytokine Interleukin-6
  • chemokine KC which are dependent on nuclear import of NF-KB and other stress-responsive transcription factors.
  • This pattern of Nf-KB-dependent cytokine/chemokine responses provides a framework for beneficial intervention by modulators of nuclear import as described herein.
  • Nuclear Transport Modifiers also referred to herein as "cell- penetrating peptide modifiers of nuclear transport" target nuclear transport adaptors.
  • Nuclear Transport Modifiers suppress signaling to the nucleus mediated by transcription factors that include but are not limited to NFKB, AP-1, NFAT, STATl, SREBPla, SREBPlc, and SREBP2, and ChREBP that utilize importins alpha and beta for nuclear transport.
  • Nuclear Transport Modifiers include but are not limited to cSN50, cSN50.1, cSN50.1 beta, and SN50.
  • Any cell-penetrating peptide or peptidomimetic that is capable of modulating a nuclear transport adaptors and changing their ability to facilitate or enable entry of a transcription factor, an enzyme, or structural protein into the nucleus may be a Nuclear Transport Modifier.
  • 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 NFKB I . Any mimetics, derivatives, or homologs of SN50 may be used in the compositions, methods, and kits disclosed herein.
  • the sequence of SN50 is AAVALLPAVLLALLAPVQRKRQKLMP (SEQ ID NO: 1). Generation and use of SN50 is described in U.S. Patent No. 7,553,929.
  • cSN50 is a cyclized peptide combining the hydrophobic domain of the K-FGF signal sequence with the NLS of the p50 subunit of NFKB I and inserting a cysteine on each side of the NLS to form an intrachain disulfide bond.
  • the amino acid sequence of cSN50 is AAVALLPAVLLALLAPCYVQRKRQKLMPC (SEQ ID NO: 2). Any mimetics, derivatives, or homologs of cSN50 may be used in the compositions, methods, and kits disclosed herein.
  • 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 are described, for example, in U.S. Patent Nos. 7,553,929 and 6,495,518.
  • the amino acid sequence of cSN50.1 is AAVALLPAVLLALLAPCVQRKRQKLMPC (SEQ ID NO:3).
  • 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 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 amino acid sequence of cSN50.1 Beta is AAVALLPAVLLALLAPCVQRDEQKLMPC (SEQ ID NO:4).
  • NTMs cell-penetrating (CP) MyD88 and its "brother” CP-MAL (also known as “TIRAP”). Both act in concert to transduce signals recognized by Toll-like receptors (TLR) 2 and 4.
  • TLR Toll-like receptors
  • MyD88 and MAL share a TIR domain and are potentially susceptible to anthrax Lethal Toxin which acts as a potent metalloproteinase inside immune cells. Restoring depleted levels of MyD88 and MAL with their CP homologs offers an additional method of treatment when anthrax toxins acting inside immune cells cause havoc in physiologic signaling pathways based on MyD88 and MAL. These "disarmed" signaling pathways cannot support innate and adaptive immunity.
  • compositions for inhibiting growth of B. anthracis bacteria and treating and preventing PAn disease in a subject are described herein.
  • a typical pharmaceutical composition includes a pharmaceutically acceptable carrier, an antibiotic, and a cell-penetrating peptide modifier of nuclear transport.
  • the antibiotic and the cell-penetrating peptide modifier of nuclear transport are in therapeutically effective amounts for treating the B. anthracis infection.
  • the antibiotic and the cell-penetrating peptide modifier of nuclear transport are in therapeutically effective amounts for treating pulmonary anthrax disease in the subject.
  • the antibiotic is ciprofloxacin and the cell-penetrating peptide modifier of nuclear transport is cSN50.
  • the pharmaceutical compositions can be administered to any mammalian subject.
  • mammalian subjects include humans, non-human primates, bovines, canines, equines, etc.
  • the amount of the pharmaceutical composition (therapeutic agent(s)) to be administered varies depending upon the manner of administration, the age and body weight of the patient, and with the pathology of the disease.
  • a composition as described herein is typically administered at a dosage that promotes an immune response against B. anthracis and inhibits growth of B. anthracis in the subject.
  • the therapeutic methods of the invention include administration of a therapeutically effective amount of a composition described herein to a subject (e.g., animal, human) in need thereof, including a mammal, particularly a human.
  • a subject e.g., animal, human
  • Such treatment will be suitably administered to subjects, particularly humans, suffering from, having, susceptible to, or at risk for a disease associated with and/or caused by B. anthracis, or symptom thereof.
  • compositions described herein can be administered to a subject by any suitable delivery vehicle and route.
  • the administration of a pharmaceutical composition including therapeutically effective amounts of an antimicrobial agent and a cell-penetrating nuclear transport modifier (NTM) may be by any suitable means that promotes an immune response against the pathogen of interest (e.g., B. anthracis) and results in effective inhibition of growth and spread of that pathogen (e.g., B. anthracis) in a subject.
  • An anti-microbial (e.g., antibiotic) and a NTM as described herein may be contained in any appropriate amount in any suitable carrier substance, and are generally present in amounts of 1-95% by weight of the total weigh of the composition.
  • cSN50.1 peptide is dissolved at about 10% (100 mg per mL).
  • the composition may be provided in a dosage form that is suitable for local or systemic administration (e.g., orally, parenterally, subcutaneously, intravenously, intramuscularly, and by inhalation).
  • the pharmaceutical compositions may be formulated according to conventional pharmaceutical practice (see, e.g., Remington: The Science and Practice of Pharmacy (20th ed.), ed. A. R. Gennaro, Lippincott Williams & Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York).
  • compositions for Inhibiting Growth of a Pathogen and Treating are provided.
  • compositions described herein can be used to inhibit the growth, spread, and host cell injury caused by any bacteria, fungi, and viruses.
  • additional microorganisms whose growth can be inhibited using the kits, compositions and methods described herein include but are not limited to Escherichia coli, Salmonella enterica serovar typhi, Vibrio cholerae, Burkholderia pseudomallei, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumanii, and Yersinia enterocolitica, ebola viruses, Marburg viruses, SARS coronavirus, Pasteurella tularensis, Staphycoccus aureus, Staphylococcus aureus (including MRSA), Streptococcus pneumoniae, Streptococcus pyogenes, Pseudomonas aeruginosa, Pasteurella
  • a method of inhibiting growth of B. anthracis bacteria in a subject includes administering to the subject a composition including a pharmaceutically acceptable carrier, an antibiotic, and an NTM, wherein the composition is in a therapeutically effective amount for treating the B. anthracis infection.
  • the antibiotic can be any suitable antibiotic, e.g., ciprofloxacin, and any suitable cell-penetrating peptide modifier of nuclear transport, e.g., cSN50, can be used.
  • the subject may be a human suffering from pulmonary anthrax disease and administration of the composition treats the pulmonary anthrax disease.
  • administration of the composition prevents pulmonary anthrax disease in a subject infected with B.
  • composition can be administered to the subject by any suitable route, but in a typical embodiment, is administered parenterally using subcutaneous, intramuscular or intravenous route.
  • the subject is typically mammalian, e.g., a human.
  • a method of inhibiting growth of B. anthracis bacteria in a subject includes administering to the subject a first composition including a pharmaceutically acceptable carrier and an antibiotic, and a second composition including a pharmaceutically acceptable carrier and a cell-penetrating peptide modifier of nuclear transport, wherein administration of the first and second compositions results in eradication of the B. anthracis infection.
  • the first and second compositions can be administered to the subject simultaneously, or at two different time points.
  • the invention provides a method of monitoring treatment progress.
  • the method includes the step of determining a level of diagnostic marker such as plasma level of cytokines, chemokines, and erythropoietin.
  • a level of diagnostic marker such as plasma level of cytokines, chemokines, and erythropoietin.
  • blood levels of bacteria, fungi and viruse can be monitored.
  • Diagnostic imaging of lungs and other organs invaded by anthrax bacilli and other microbial agents can be employed to monitor the treatment outcome or diagnostic measurement (e.g., screen, assay) in a subject suffering from or susceptible to a disorder or symptoms thereof associated with, for example, PAn, in which the subject has been administered a therapeutic amount of a composition as described herein for treating the disease or symptoms thereof.
  • the level of marker determined in the method can be compared to known levels of marker in either healthy normal controls or in other afflicted patients to establish the subject's disease status.
  • a second level of marker e.g., specific antibody response to the invading microorganism
  • a pre-treatment level of marker in the subject is determined prior to beginning treatment according to the methods described herein; this pre-treatment level of marker can then be compared to the level of marker in the subject after the treatment commences, to determine the efficacy of the treatment.
  • diagnostic and theranostic methods useful to determine whether the subject is susceptible to the treatment methods of the invention.
  • the term "theranostics” generally refers to therapy-specific diagnostics, which is the use of diagnostic testing to diagnose the disease, choose the correct treatment regime for that disease, and monitor the patient response to therapy.
  • Theranostic tests can be used to predict and assess drug response in individual patients, and are designed to improve drug efficacy by selecting patients for treatments that are particularly likely to benefit from the treatments.
  • Theranostic tests are also designed to improve drug safety by identifying patients that may suffer adverse side effects from the treatment.
  • the methods described herein can be used to inhibit the growth, spread, and host cell injury caused by any bacteria, fungi, and viruses.
  • additional microorganisms whose growth can be inhibited using the methods described herein include but are not limited to Escherichia coli, Salmonella enterica serovar typhi, Vibrio cholerae, Burkholderia pseudomallei, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumanii, and Yersinia enterocolitica, ebola viruses, Marburg viruses, SARS coronavirus, Pasteurella tularensis, Staphycoccus aureus, Staphylococcus aureus (including MRSA), Streptococcus pneumoniae, Streptococcus pyogenes, Pseudomonas aeruginosa, Pasteurella tularensis
  • kits for treating an infection, and diseases or disorders associated the infection can be used to treat an anthrax infection, and any disease or disorder associated with an anthrax infection (e.g. Pan) in a subject.
  • a typical kit includes: a composition including a pharmaceutically acceptable carrier, an anti-microbial agent (e.g., antibiotic), and a NTM, the anti-microbial agent and NTM in amounts effective for treating the infection (e.g., inhibiting B. anthracis growth, promoting an immune response against B. anthracis, preventing or alleviating PAn), as well as packaging, and instructions for use.
  • an additional therapeutic agent e.g., a secod NTM and/or second antibiotic and/or cell-penetrating- MAL and recombinant cell-penetrating MyD88
  • the NTM may be, for example, cSN50 or cSN50.1.
  • the anti-microbial agent can be, for example, ciprofloxacin.
  • the kit includes one or more syringes each containing a composition as described herein. Early and aggressive therapeutic intervention in pulmonary (inhalational) anthtrax, as well as in post- airborne exposure to other biothreats and emerging pathogens, requires rapid delivery methods.
  • kits may also contain one or more of the following: containers which include positive controls, containers which include negative controls, photographs or images of representative examples of positive results and photographs or images of representative examples of negative results.
  • compositions including an antibiotic and an NTM in amounts effective for treating a B. anthracis infection may be by any suitable means that results in a concentration of the therapeutic that is effective in treating the B. anthracis infection.
  • an additional therapeutic agent such as a second NTM and/or a second antibiotic may be administered. Additional examples of such a therapeutic include a recombinant cell-penetrating MyD88 adaptor protein and/or a recombinant cell-penetrating MAL adaptor protein.
  • the composition may be provided in a dosage form that is suitable for oral, local or systemic administration (e.g., parenteral, subcutaneous ly, intravenously, intramuscularly, or intraperitoneally).
  • the pharmaceutical compositions may be formulated according to conventional pharmaceutical practice (see, e.g., (Gennaro, A. R. ed. (2000) Remington: The Science and Practice of Pharmacy (20th ed.), Lippincott Williams & Wilkins, Baltimore, MD; Swarbrick, J. and Boylan, J. C. eds. (1988-1999) Encyclopedia of Pharmaceutical Technology, Marcel Dekker, New York).
  • compositions as described herein may be administered parenterally by injection, infusion or implantation (subcutaneous, intravenous, intramuscular, intraperitoneal, or the like) in dosage forms, formulations, or via suitable delivery devices or implants containing conventional, non-toxic pharmaceutically acceptable carriers and adjuvants.
  • a composition as described herein is administered via osmotic pump.
  • the composition may be administered orally in sublingual form or with a coating protecting the composition from gastrointestinal peptidases.
  • the formulation and preparation of such compositions are well known to those skilled in the art of pharmaceutical formulation. Formulations can be found in Gennaro supra.
  • 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.
  • 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 pharmaceutical compositions described herein may be in a form suitable for sterile injection.
  • the suitable active therapeutic(s) are dissolved or suspended in a parenterally acceptable liquid vehicle.
  • acceptable vehicles and solvents that may be employed are water, water adjusted to a suitable pH by addition of an appropriate amount of hydrochloric acid, sodium hydroxide or a suitable buffer, 1,3-butanediol, Ringer's solution, and isotonic sodium chloride solution and dextrose solution.
  • the aqueous formulation may also contain one or more preservatives (e.g., methyl, ethyl or n-propyl p-hydroxybenzoate).
  • a dissolution enhancing or solubilizing agent can be added, or the solvent may include 10-60% w/w of propylene glycol or the like.
  • Materials for use in the preparation of microspheres and/or microcapsules are, e.g., biodegradable/bioerodible polymers such as polygalactia 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., polydimethyl 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.
  • 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 hydroxyethylcellulose, 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. supra.
  • At least two therapeutic agents e.g., an antibiotic and an NTM
  • 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.
  • three or more therapeutic agents may be included in a single composition.
  • 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.
  • compositions as described herein may be administered in combination with any standard PNa or anthrax therapy; such methods are known to the skilled artisan and described in Gennaro, A. R. ed. (1990) Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. Combinations are expected to be advantageously synergistic. Therapeutic combinations that increase an immune response against B. anthracis and inhibit B. anthracis growth, for example, are identified as useful in the compositions, methods, and kits described herein.
  • the therapeutic methods described herein in general include administration of a therapeutically effective amount of a composition described herein to a subject (e.g., animal) in need thereof, including a mammal, particularly a human.
  • a subject e.g., animal
  • Such treatment will be suitably administered to subjects, particularly humans, suffering from, having, susceptible to, or at risk for anthrax infection. Determination of those subjects "at risk” can be made by any objective or subjective determination by a diagnostic test or opinion of a subject or health care provider.
  • compositions (pharmaceutical compositions) described herein are preferably administered to an animal (e.g., mammalian (such as human, ovine, bovine, canine, porcine, equine, etc.), reptilian, piscine, avian, etc.) in an effective amount, that is, an amount capable of producing a desirable result in a treated animal (e.g., increasing an immune response against B. anthracis, inhibiting B. anthracis growth, preventing or alleviating PAn).
  • an effective amount that is, an amount capable of producing a desirable result in a treated animal (e.g., increasing an immune response against B. anthracis, inhibiting B. anthracis growth, preventing or alleviating PAn).
  • an effective amount e.g., an amount capable of producing a desirable result in a treated animal (e.g., increasing an immune response against B. anthracis, inhibiting B. anthracis growth, preventing or alleviating PAn).
  • dosage for any one subject depends on many factors, including the subject's size, body surface area, age, the particular composition to be administered, time and route of administration, general health, and other drugs being administered concurrently.
  • Ciprofloxacin 500-mg oral tablet every 12 h as directed
  • doxycline 100-mg oral tablet every 12 h as directed
  • first-line oral antimicrobials for postexposure prophylaxis.
  • intravenous ciprofloxacin is recommended unless there are contraindications to its use.
  • Pulmonary anthrax caused by inhaling B. anthracis spores represents a serious threat in biowarfare and bioterrorism. This threat is underscored by the accidental Sverdlovsk airborne outbreak in the former Soviet Union and more recent attempts to deliberately spread B. anthracis spores via the U.S. Postal Service in 2001. Inhaled B. anthracis spores are disseminated throughout the body causing bacteremia, which is refractory to treatment with antibiotics and leads to extensive lung injury and death. The cardinal features of lung injury involve hemorrhage in the mediastinum and pleural cavity, necrosis of mediastinal lymph nodes, and pulmonary edema with hyaline membrane formation.
  • TLRs Toll-Like Receptors
  • Edema factor acts as a calcium- and calmodulin (CaM)-dependent adenylate cyclase that is 1 ,000 fold more active than mammalian CaM- activated adenylate cyclase. It causes prominent edema at the site of infection, the inhibition of neutrophil function, and suppression of the production of TNFa and IL-6 by monocytes.
  • CaM calcium- and calmodulin
  • EF-generated cAMP activates cAMP-dependent protein kinase A (PKA), which in turn phosphorylates cAMP response element binding protein (CREB) leading to suppression of mitogen-activated protein kinase (MAPK) kinase p38 and inhibition of multiple transcription factors involved in cytokine production such as nuclear factor kappa B ( FKB) and nuclear factor of activated T cells (NFAT).
  • PKA cAMP-dependent protein kinase A
  • CREB cAMP response element binding protein
  • MAPK mitogen-activated protein kinase
  • FKB nuclear factor kappa B
  • NFAT nuclear factor of activated T cells
  • Lethal factor a zinc metalloprotease, suppresses production of two effectors of innate immunity in macrophages, TNFa, and nitric oxide (NO), and reduces expression of other cytokine gene transcripts.
  • LF also inactivates MAPK kinase, leading to aberrant intracellular signaling and contributing to the death of cultured macrophages.
  • anthrax toxins greatly affect the signaling to the nucleus essential for genome reprogramming in macrophages and dendritic cells.
  • a treatment protocol was selected in which pulmonary anthrax was caused by a lethal dose of inhaled B. anthracis spores refractory to an 8-day treatment with the antibiotic ciprofloxacin.
  • the cSN50 peptide employed in this model contains a cyclized form of the NLS from the P50/NFKB1 subunit of NFKB.
  • the NLS was fused to the signal sequence-derived hydrophobic region from fibroblast growth factor 4.
  • This hydrophobic segment serves as a membrane-translocating motif (MTM), which enables peptide or protein cargo to penetrate the plasma membrane of multiple cell types in various organs through a receptor/transporter and endocytosis-independent mechanism.
  • MTM membrane-translocating motif
  • NLS as a cargo, competitively targets the adaptor importin alpha 5/karyopherin alpha 1, among other importins/karyopherins (Zienkiewicz J., Armitage A., and Hawiger J. Biochemistry 2013 under review), thereby modulating its nuclear shuttling function. It was surmised that regulating nuclear transport of the overexpressed repressors of innate immunity generated by the high levels of cAMP induced by LF would help restore normal immune function.
  • a combination therapy was designed by adding ciprofloxacin to treatment with the nuclear transport modifier.
  • a dosing schedule of ciprofloxacin was chosen that would partially control the rapid replication of anthrax bacilli in infected animals but not prevent the lethal outcome.
  • Using a ciprofloxacin protocol that favored the lethal outcome provided a sufficient system for evaluating the impact of cSN50 peptide treatment on the course of infection while controlling rapid replication of bacilli with antibiotic.
  • mice Untreated control mice were given IP saline injections, but no ciprofloxacin. These mice died between 2 to 4 days post-infection (Fig. l), while 50-70% of the infectious spore dose was recovered from the lungs of representative animals sacrificed one hour post-infection. Prior to death, mice developed labored respirations, most likely due to lung and pleural injury noted at autopsy. Mice receiving ciprofloxacin and saline lived longer, but ultimately all but one succumbed to infection within 9 days (4% survival). In contrast, a significant number of mice receiving a combination of nuclear transport modifier, cSN50, and ciprofloxacin survived (Fig. l). Survival at the ninth day in three independent experiments was 52% (p ⁇ 0.001).
  • anthracis spore challenge showed depressed levels of TNFa, IL-6, and MCP-1.
  • treatment with cSN50 and ciprofloxacin restored induction of these mediators of innate immunity in B. anthracis- infected animals (Fig.2).
  • Other pro- and anti-inflammatory cytokines, IL-12p70, interferon gamma (IFNy), and IL-10 remained unchanged.
  • Increased hypoxia manifested by an elevated level of its biomarker, EPO, has been previously reported in mice challenged with anthrax lethal toxin (Moayeri et al, J Clin Invest 112: 670-682, 2003). Consistent with these prior findings, EPO was elevated at 48 h after B.
  • hypoxia marker was attenuated in all mice receiving treatment with cSN50 and ciprofloxacin (Fig. 2).
  • nuclear transport modifier suppressed hypoxia associated with the lethal outcome of pulmonary anthrax.
  • mice treated with cSN50 peptide and ciprofloxacin showed minimal pulmonary edema at the ninth day of observation (Fig. 3G) and lungs were essentially normal in mice euthanized at 21 days (Fig. 3H).
  • PAS-stained sections in mice from these groups of survivors were negative for B. anthracis vegetative forms in all organs examined, and very few spores were detected in lung sections stained with a modified Ziehl's carbol fuchsin dye for spores in tissues. In mice that succumbed to infection, there were fewer stained spores seen in lungs of cSN50-treated animals compared to those treated with ciprofloxacin alone.
  • cSN50 peptide in the lungs correlated with the survival of mice challenged with 10 7 B. anthracis spores and receiving ciprofloxacin also.
  • beneficial effect of cSN50 peptide added to ciprofloxacin facilitated lung clearance of B. anthracis bacilli and prevented their spread to other organs (the heart, spleen, liver, and kidneys).
  • MTM-containing peptide cSN50 was synthesized, purified, filter-sterilized, and analyzed as described previously (Liu et al, J Biol Chem 275: 16774-16778, 2000; Torgerson et al, J Immunol 161 : 6084-6092, 1998).
  • Bacillus anthracis culture and spore preparation Bacillus anthracis culture and spore preparation: Bacillus anthracis Sterne strain was grown in 2xSG medium (nutrient broth supplemented with 2mM Magnesium Sulfate, 27mM Potassium Chloride, ImM Calcium Nitrate, ⁇ Manganese Chloride, and 700nM Ferrous Sulfate) at 37°C with constant shaking (300 rpm) until sporulation (5- 7 days). The culture was centrifuged for 7 min at 8000g at 4°C, resuspended in sterile water, heated at 65°C for lh to kill vegetative bacilli and germinated spores then washed 3 times with sterile water.
  • 2xSG medium nutrient broth supplemented with 2mM Magnesium Sulfate, 27mM Potassium Chloride, ImM Calcium Nitrate, ⁇ Manganese Chloride, and 700nM Ferrous Sulfate
  • the number of colony forming units (cfu) was determined by plating serial dilutions of spores on Luria broth (LB) agar and counting the B. anthracis colonies. Spores were aliquoted in 20% glycerol and stored at -80°C. The number of cfu was reconfirmed before each use.
  • mice Female A/J mice (7-8 weeks) were purchased from the Jackson Laboratories (Bar Harbor, ME), and randomly assigned to study groups. For B. anthracis Sterne spore instillation, mice were anesthetized by intraperitoneal (IP) injection of 50mg/kg Nembutal, and lxlO 7 spores in 50 ⁇ saline were administered to each mouse intranasally (IN). The number of spores reaching the lungs was determined in each experiment. Briefly, 2-3 mice were sacrificed lh post-infection and lungs were removed under sterile conditions, homogenized, and serial dilutions of homogenates plated on LB agar.
  • IP intraperitoneal
  • mice that received doses of 10 5 or 10 6 spores IN were observed for 9 days and all were asymptomatic and survived. In contrast, all mice that received 10 7 spores died within 4 days with signs of overt infection.
  • mice Female A/J mice were injected IP with 200 ⁇ 1 saline or cSN50 peptide (3.5mg/ml) at 30 minutes before and 30 minutes, 1.5h, 2.5h, 3.5h, 6h, 9h, 12h, 15h, 18h, 21h, 24h, 30h, 36h, and 42h after IN infection with 10 7 spores of B. anthracis Sterne strain.
  • Treatment with ciprofloxacin, 50mg/kg was administered subcutaneously (s.c), beginning 24h after spore challenge and continuing once daily for 8 days. Blood was collected from the saphenous vein before infection, at 12, 24, 36 and 48h after spore challenge, and at death.
  • Serum was separated from clotted blood and stored at - 20°C. All animals were observed for morbidity and mortality for up to 9 days and some cSN50 peptide-treated mice were monitored for up to 21 days after spore challenge. Moribund animals were humanely euthanized by IP injection of pentobarbital.
  • Cytokines, Chemokine, and Erythropoietin Assays in Serum IL-6, TNFa, IFNy, IL-10, IL-12, and MCP-1 were measured in serum by a Cytometric Bead Array according to the manufacturer's instructions (BD Biosciences).
  • Murine EPO was measured by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's instructions (R and D Systems). Results are expressed as the mean + S.E.
  • SRTFs NF- ⁇ and other stress-responsive transcription factors
  • cSN50 cell-penetrating nuclear import modulator
  • Nuclear import constitutes a key checkpoint in signaling to the nucleus.
  • a new class of nuclear import modulators has been designed that enable a short peptide sequence representing the nuclear localization motif of NF-KB1 (p50) to cross the plasma membrane by attaching it to the hydrophobic region of the signal peptide sequence.
  • This physiologic and non- immunogenic sequence is utilized by nascent polypeptide chains for translocation across the endoplasmic reticulum membrane in the secretory pathway.
  • MTM membrane translocating motif
  • NLS nuclear localization sequence
  • hypoxia-induced factor 1 responsible for expression of erythropoietin and other biomarkers of tissue hypoxia during the late stages of pulmonary anthrax.
  • HIF-1 hypoxia-induced factor 1
  • a cyclized form of this peptide modulator suppressed the in vivo proinflammatory cytokine cascade induced in mice by a high concentration of LPS. Strikingly, mortality was reduced from 100% to 10% (4).
  • mice deficient in TNFa and IFNy receptors Three inflammatory cytokines (TNFa, IL-6 and IL- ⁇ ⁇ ) are significantly elevated, reaching peaks at 1, 2, and 6 hours respectively, while the chemokine MCP-1 peaks at 2 hours. Strikingly, all inflammatory cytokines and chemokines studied are significantly suppressed in mice treated with nuclear import modulator cSN50. These beneficial effects correlate with the outcome of acute systemic inflammatory response: mortality.
  • mice Whereas only 5% of the mice survived in a control group, 75% survived 3 days after LPS challenge (a subset of 3 mice observed for 10 days showed no signs of disease). Histological examination of cSN50-treated mice showed normal tissue architecture with no apoptotic or hemorrhagic liver injury in contrast to untreated (diluent) controls. The fulminant course of the inflammatory response (90% of the animals died within 12 hours) produced dramatic changes in the liver, reflected by massive apoptosis and hemorrhagic necrosis. The hemorrhagic necrosis of the liver observed in diluent-receiving animals provides dramatic illustration of endothelial injury.
  • Staphylococcal Enterotoxin B (SEB), a Class B biothreat agent, induces intracellular signaling to the nucleus that is blocked by a nuclear import modulator. Described herein is a new approach to the study of nuclear signaling in response to SEB. This alternative approach to antibody-mediated neutralization of SEB has effectively blocked its intracellular signaling to the nucleus required for inflammatory cytokine gene expression.
  • the genes that encode inflammatory cytokines are under the control of stress- responsive transcription factors (SRTFs), including nuclear factor ⁇ ( FKB), activator protein 1 (AP-1), nuclear factor of activated T cells (NFAT), and signal transducer and activator of transcription 1 (STAT1).
  • SRTFs stress- responsive transcription factors
  • FKB nuclear factor ⁇
  • AP-1 activator protein 1
  • NFAT nuclear factor of activated T cells
  • STAT1 signal transducer and activator of transcription 1
  • mice treated with SEB and D-Gal were also explored. At death, all mice exhibited severe liver injury characterized by extensive apoptosis and hemorrhagic necrosis. No systemic toxicity was detected upon the administration of SEB or D-Gal alone. In contrast, the administration of cSN50 before exposure to SEB and D-Gal, and thereafter in six doses over 12 hours, produced a pronounced protective effect. Fourteen of 15 mice recovered fully from SEB challenge and survived at least 72 h. Thus, the cell-penetrating cSN50 peptide reduced SEB-induced lethality by 87%.
  • mice received the first dose of cSN50 30 minutes after SEB and D-Gal. Despite omitting the first dose of cSN50, given previously 30 minutes before SEB and D-gal challenge, significant suppression of inflammatory cytokines TNF-a (p 0.0001), IFN- ⁇ (p 0.0001), and IL-6 (p ⁇ 0.001) was observed using a two-way repeated measure analysis of variance concomitant with 60% survival (p ⁇ 0.02).
  • PAn-induced death following ciprofloxacin treatment is prevented by nuclear import modulator.
  • the nucleus-orchestrated innate immune response is profoundly deranged by the toxins secreted by Bacillus anthracis vegetative forms.
  • Production of both pro- and antiinflammatory cytokines and chemokines is abrogated due to disruption of the mitogen-activated protein (MAP) kinase network.
  • MAP mitogen-activated protein
  • other studies reported systemic expression of proinflammatory cytokines and chemokines upon infection with live anthrax spores. The functional intactness of MyD88 is required for resistance to anthrax spore-induced infection.
  • mice developed labored respiration prior to death, most likely due to excessive pleural exudate noted at autopsy. Histological analysis of the untreated mice that died displayed extreme edema in their lungs, early influx of lung- infiltrating leukocytes, and massive growth of bacilli. In contrast, there were no abnormalities or signs of infection, spores or vegetative bacteria in the sinusoidal system or brain.
  • cSN50 peptide a cell-penetrating nuclear import modulator, cSN50 peptide, was tested in combination with post-exposure ciprofloxacin treatment, for its effect on survival of spore-challenged mice.
  • Mice were infected with approximately 10 7 spores IN and treated with multiple intraperitoneal (i.p.) injections of either cSN50 or saline, and a daily dose of ciprofloxacin (50 mg/kg s.c), begun 24 hours post-exposure and continued for 10 days.
  • the control group mice were given i.p. saline injections instead of cSN50 peptide.
  • mice receiving saline and ciprofloxacin There was a delay in the death of mice receiving saline and ciprofloxacin, but ultimately all succumbed to infection. However, an even greater delay in death was seen in the mice receiving cSN50 and ciprofloxacin, and 52 % survived for up to 10 days.
  • a comparison of the two cipro floxacin-treated groups (without and with cSN50 administration) showed a significant statistical difference (p ⁇ 0.001). Some surviving mice were observed for up to 21 days.
  • the Toll-Like Receptor (TLR) superfamily includes a group of cytoplasmic adapter proteins exemplified by MyD88 and Trif. These proteins transduce signals from a multitude of TLRs and IL-l/IL-18 receptors to the nucleus via NF- ⁇ , AP-1, and type I INF-inducing pathways. Because of its central position in the integration of signals from IL- ⁇ receptor and TLRs, structural requirements were defined for MyD 88 -mediated signaling induced by IL- ⁇ . Homotypic oligomerization of MyD88, due to its forced expression, resulted in robust activation of NF- ⁇ reporter gene activity observed in the absence of ILip stimulation.
  • mutations of Box 3 residues 285-286 reversed the dominant negative effect of MyD88 TIR domain on ILi p-induced and NF-KB-dependent reporter gene activity and IL6 production. Moreover, mutations of residues 171 in helixaA, 195-197 in Box 2, and 275 in the ⁇ strand had similar functional effects. Strikingly, only mutations of residues 195-197 eliminated the TIR-TIR interaction of MyD88 and ILlRAcP while substitution of neighboring canonical Proline 200 by Histidine was without effect. Based on this structure-function analysis, a three-dimensional docking model of TIR-TIR interaction between MyD88 and ILlRAcP was developed.
  • This modeling was based on geometry optimization by energy minimization followed by molecular dynamic computation using program SANDER (AMBER software package).
  • the ribbon structure was developed with the Deep View program while the molecular surface of associated proteins was determined by PSSHOW (SYBIL-Tripos software package).
  • PSSHOW SYBIL-Tripos software package.
  • the separation surface indicates that there is no crossing of molecular surfaces and distance between them is within the range of 0.4 - 4.7 A. Their topology is diverse and contains several deep pockets.
  • an interacting site in Box 2 comprised of residues D195/R196/D 197 potentially has an additional function that may encroach on the ability of MyD88 to interact with ILlRAcP.
  • heterotypic interaction of ILlRAcP TIR domain with MyD88 TIR domain antecedes oligomerization of MyD88.
  • the latter depends on homotypic binding mediated by its TIR domain. Therefore, the interactive site in Box 3 is not likely involved in binding of MyD88 to ILlRAcP. Rather, this site participates in homotypic MyD88 oligomerization and possibly other transactions involving downstream signal transducers.
  • SOCS3 is a modular, S3 ⁇ 4- containing, 225 amino acid protein. SOCS3 was enabled to cross the plasma membrane of cultured cells and subsequently blood cells and other cells in a range of organs, by attaching a membrane translocating motif (MTM). Recombinant SOCS3 proteins were constructed without and with the MTM by amplifying SOCS sequences.
  • MTM membrane translocating motif
  • the MTM composed of 12 amino acids from a hydrophobic signal sequence from fibroblast growth factor 4.
  • the MTM was attached to either the NH 2 -terminus (HMS3) or COOH-terminus (HS3M) to mediate uptake into cells.
  • HMS3 NH 2 -terminus
  • H3M COOH-terminus
  • His-SOCS3 H263
  • H3M COOH-terminus
  • All three contained a full-length SOCS3 sequence generated by PCR and flanked at the NH 2 -terminus by a polyhistidine tag.
  • the PCR product was cleaved with Ndel and cloned into the Ndel site of the expression vector (pET-28a(+)).
  • the resulting plasmids were used to express SOCS3 protein under the control of the lacl promoter in E.
  • coli strain BL21-CodonPlus DE3
  • the recombinant fusion proteins were purified under denaturing conditions by chromatography on nickel-nitrilotriacetic acid (Ni-NTA) meta-affinity beads as directed by the supplier (Qiagen) then stepwise refolded in Dulbecco Minimal Essential Medium (DMEM). Purity and yields of all three recombinant SOCS3 proteins were comparable.
  • Ni-NTA nickel-nitrilotriacetic acid
  • DMEM Dulbecco Minimal Essential Medium
  • the mouse macrophage RAW 264.7 cell line was used to test the intracellular delivery and function of recombinant SOCS3 proteins that contain or lack the membrane translocating motif (MTM).
  • MTM membrane translocating motif
  • FITC Fluorescein isothiocyanate
  • One of the two CP-SOCS3 proteins, HMS3 showed a stronger intracellular signal in blood leukocytes and lymphocytes, prompting us to analyze its time- dependent kinetics in blood and spleen leukocytes and lymphocytes.
  • FITC- labeled HMS3 was detectable, albeit in reduced amounts, at 8 hours and even 24 hours after intraperitoneal injection.
  • CP-SOCS3 was also detectable in the lung, spleen and heart. This direct fluorescence detection of CP-SOCS3 was confirmed with indirect immunodetection using SOCS3 -specific antibody. Mice treated with diluent or FITC alone showed no fluorescence in any of these organs.
  • CP-SOCS3 Histologic analysis documented the protective effect of CP-SOCS3 (HSM3) on SEB-induced massive liver apoptosis and hemorrhagic necrosis. Mutated CP-SOCS3 (HS3NSM) did not display the protective effect. Thus, CP-SOCS3 was effective in the model of acute inflammation caused by SEB.
  • IPT Intracellular Protein/Peptide Therapy
  • the first class of Intracellular Anthrax Signaling Modulators blocks cell signaling to the nucleus in response to the insults of anthrax toxin.
  • IASM Intracellular Anthrax Signaling Modulators
  • cell- penetrating cSN50 peptide blocks the nuclear import of STRFs, hypoxia-induced factor (HIF)-l, and possibly several other karyophilic proteins.
  • This cell-penetrating nuclear import modulator is a "lead compound” designed and tested for in vivo studies (Liu et al, J. Biol. Chem. 279: 19239-46, 2004).
  • the second example of IASM is a cell-penetrating recombinant MyD88 that is expected to render experimental animals resistant to PAn.
  • MyD88 Deficiency of MyD88 renders mice susceptible to PAn (Hughes MA et al Infect Immun 2005;73:7535-7540; Okugawa S et al Infect Immun 201 1; 79: 118-124).
  • a cell-penetrating form of MyD88 is engineered and tested for its protective effect in bone marrow-derived macrophages from MyD88 -/- mice, and then in vivo in the PAn model described herein.
  • the results will provide crucial insight into the poorly understood mechanism of deranged intracellular signaling that underlies death in PAn.
  • the proposed research plan will ultimately provide preclinical evaluation of innovative intracellular protein/peptide therapy designed to restore (or normalize) the innate immune response to spore-induced PAn.
  • the positive outcome of this project may transcend its anthrax-oriented objectives by paving the way for similar therapies of life-threatening infections caused by other class A agents such as tularemia.
  • cSN50 peptide is a cyclized form of SN50 peptide that was shown to target the nuclear import adaptor protein denoted importin/karyopherin alpha5 (/KPNA5).
  • the cSN50 peptide was administered intraperitoneally 30 min before challenge with anthrax spores and at 1-2-6 h intervals after challenge for 42 h.
  • mice treated with cSN50 peptide and ciprofloxaciin died at a much slower rate, reaching 52% survival at 9 d. It is postulated that this new form of treatment for PAn can be optimized to increase survival to 80-90%, as achieved in prior studies with the class B agent, staphylococcal enterotoxin B.
  • importin alpha 1 (PI-1) recognizes the unconventional nuclear localization signal (NLS) identified on the influenza A virus nucleoprotein. Cell-penetrating peptides containing these NLSs will be designed and tested alone or in combination with cSN50 peptide to achieve optimal inhibition of anthrax- induced noxious signaling.
  • NLS unconventional nuclear localization signal
  • Anthrax spores are produced using the toxigenic but nonencapsulated Sterne strain of Bacillus anthracis. The spores are produced, isolated, counted, and prepared for intranasal instillation according to known methods.
  • mice Female A/J mice (7-8 weeks) purchased from the Jackson Laboratory are randomly assigned to study groups. All animal handling and experimental procedures are performed in accordance with the American Association of Accreditation of Laboratory Animal Care guidelines and approved by the Institutional Animal Care and Use Committee.
  • mice are anesthetized by intraperitoneal (IP) injection of 50mg/kg Nembutal, and lxlO 7 spores in 50 ⁇ 1 saline are instilled to each mouse intranasally (IN). To determine the number of spores reaching the lungs, 2-3 mice from each experiment are sacrificed at I hour post-infection.
  • IP intraperitoneal
  • Lungs are removed under sterile conditions, homogenized, and serial dilutions of homogenates plated on LB agar. Usually, 50-70% of the instilled dose of anthrax spores is recovered from the lungs of infected mice.
  • mice are treated with ciprofloxacin (50mg/kg, administered subcutaneously). These injections are continued once daily for 10 days.
  • the treatment with cSN50 peptide is initiated 30 min before the spore challenge and continued at 30 minutes, 1.5 h, 2.5 h, 3.5 h, 6 h, 9 h, 12 h, 15 h, 18 h, 21 h, 24 h, 30 h, 36 h, and 42 h after infection.
  • the cSN50 peptide (200 ⁇ 1, 3.5mg/ml) is injected intraperitoneally (IP).
  • IP intraperitoneally
  • the control group receives 200 ⁇ 1 saline IP at the same time intervals.
  • Blood is collected from the saphenous vein before anthrax spore challenge and at 12, 24, 36 and 48 hours afterwards or at death. Blood is allowed to clot then serum separated and stored at -20°C for later analysis. Animals are observed for morbidity/mortality for 21 days after spore challenge. Moribund animals are humanely euthanized by IP injection of pentobarbital. Surviving animals are euthanized in the same manner 21 days after spore challenge. Organs and heads of mice (with skin removed) are collected at death and immersed in 10% formalin for histological analysis. A log rank test is used for statistical analysis of survival data.
  • IL-6, TNFa, IFNy, IL-10, IL-12 and MCP-1 are measured in serum by a Cytometric Bead Array (CBA) according to the manufacturer's instructions (BD Biosciences).
  • CBA Cytometric Bead Array
  • mEpo Murine erythropoietin
  • IL- ⁇ are measured by Enzyme-Linked Immunosorbent Assay (ELISA) according to the manufacturer's instructions (R and D Systems). Results are expressed as the mean + S.E. and a one-way analysis of variance, a two-way repeated measure analysis of variance and the student's t test were used to determine significance of the difference.
  • Organ samples (lungs, liver spleen, heart and kidney) and heads (skin removed) are collected at death from mice who succumb to anthrax infection, and from surviving mice euthanized after 9 or 21 days.
  • Formalin-fixed, paraffin-embedded sections are stained with hematoxylin and eosin (HE) or periodic acid-Schiff and hematoxylin (PAS) to assess tissue injury due to infection and the presence of bacilli.
  • HE hematoxylin and eosin
  • PAS hematoxylin
  • the initial protocol is based on administration of cSN50 peptide 30 min before anthrax spore challenge.
  • a modified treatment schedule will be tested using delayed administration of cSN50 peptide to coincide with post-exposure ciprofloxacin therapy.
  • This post-exposure therapy is instituted 24 h after anthrax spore challenge. It will be determined whether a delay in treatment with cSN50 for 24h will still result in increased survival of mice exposed to anthrax spores.
  • the cSN50 peptide was initially designed to carry the nuclear localization signal (NLS) derived from transcription factor NF-KB1. This sequence comprises a cluster of basic amino acids that are categorized as the classic NLS motif. Subsequently, it was established that this sequence is recognized by importin alpha 5/karyopherin alpha 1 , also denoted KPNA1. Although cSN50 peptide effectively modulates proinflammatory signaling to the nucleus in in vivo models of staphylococcal enterotoxin B (SEB) and LPS toxicity, its effect in pulmonary anthrax is different in terms of survival and markers of an overstimulated innate immune response (cytokines and chemokines).
  • SEB staphylococcal enterotoxin B
  • LPS LPS toxicity
  • this cell-penetrating peptide will be labeled with FITC according to a known protocol and examined for its intracellular delivery in the mouse macrophage cell line RAW 264.7. Its effect on nuclear import of NFKB and other stress- responsive transcription factors will be determined and compared with cSN50. Subsequently, this new nuclear import modulator denoted SNPIA will be tested in the pulmonary anthrax model according to the protocol delineated above. If the SNPIA peptide demonstrates inhibitory action, a combination of SNPIA and cSN50 will be tested in the PAn model to determine whether these two distinct nuclear import modulators will display an additive, synergistic, or antagonistic effect.
  • peptides are synthesized using FMOC chemistry.
  • the amino acid composition and mass of the synthesized peptides are routinely determined by amino acid analysis and matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS).
  • MALDI-MS matrix-assisted laser desorption ionization mass spectrometry
  • Mutant peptides carrying a mutated functional domain are synthesized and purified for control experiments.
  • the SM peptide is cell-penetrating but functionally inactive.
  • MyD88-deficient mice succumb to PAn cell-penetrating MyD88 (CP- MyD88) isengineered to test its protective role in MyD88-/- mice.
  • CP- MyD88 cell-penetrating MyD88
  • MyD88 function depends on its well- defined sequences in the TIR domain (see Example 2 above). This domain inhibits signaling to the nucleus from multiple TLRs, while full-length MyD88 overexpressed in cultured cells triggers nuclear signaling mediated by NFKB, responsible for activation of a myriad of genes that regulate inflammation, immunity, and apoptosis.
  • NFKB nuclear signaling mediated by NFKB
  • a goal is to develop CP-MyD88 or its CP-modular fragment(s), test their intracellular activity and stability in terms of their half-life, and determine their effect on spore-infected BMDM obtained from MyD88- deficient and MyD88-sufficient (wild type) mice followed by in vivo analysis in PAn developed in MyD88-deficient mice and in A/J mice.
  • CP-MyD88 Constructs encoding mouse MyD88 in its wild-type and cell-penetrating form (CP-MyD88) are prepared as His-tagged proteins according to the established protocols. As discussed in Example 2 above, MyD88 protein containing AU1 and Myc tags was constructed and used for biochemical analysis of its binding activity. Therefore, problems expressing MyD88 when appended to the membrane translocating motif (MTM) are not anticipated.
  • MTM membrane translocating motif
  • the purification yield of CP -recombinant proteins such as CP-SOCS3 and CP-SOCS 1 currently produced is usually >10 mg soluble protein per liter of bacterial culture with 20% recovery. Contaminating LPS (0.2-0 ⁇ g per mg of purified protein) has not been shown to adversely interfere with the action of CP proteins.
  • Recombinant proteins are purified under denaturing conditions by chromatography on nickel- nitrilotriacetic acid (Ni-NTA) meta-affinity beads (Qiagen). At least seven recombinant murine MyD88 proteins will be produced. The control MyD88 protein lacks the MTM and will not penetrate cells. The remaining recombinant CP-MyD88 proteins contain MTM at the COOH-terminus and NH2-terminus, respectively. The four mutated CP-MyD88 proteins comprise potential "loss of function" forms of MyD88 containing two alanine replacements: at position 56 (phenylalanine) and at position 200 (proline).
  • the mutated MyD88 sequences will be generated via site-directed PCR mutagenesis as reported in prior studies (Li C et al J Biol Chem 2005 ). Recombinant MyD88 proteins will be tested for potential cytotoxicity by performing a cell viability assay. Their half-life (Tm) in the MyD88-deficient cells will be determined by immunoblotting using anti-mouse MyD88 antibody using Direct Infrared Fluorescent Detection in the Odyssey Imaging System.
  • CP-MyD88 intracellular replacement in MyD88- deficient mice may depend on its delivery across the plasma membrane and into the cytoplasm where endogenous MyD88 functions as an adaptor protein integrating signals flowing from multiple Toll-like receptors. Recombinant CP-MyD88 entry into cells will be compared with recombinant MyD88 without an MTM. Full-length CP-MyD88 proteins will be labeled with fluorescein isothiocyanate (FITC) according to a previously published protocol. Localization of CP-MyD88 will be analyzed by flow cytometry.
  • FITC fluorescein isothiocyanate
  • FITC-labeled proteins will be introduced into cultures of bone marrow-derived macrophages (BMDM) from MyD88-deficient mice. Proteinase K will then be added to degrade proteins adsorbed on the cell surface while internalized CP-MyD88 proteins are protected from degradation (Protease Accessibility Assay). It is anticipated that CP-MyD88 proteins will localize within cells and function as modulators of intracellular signaling induced by anthrax spores as compared to LPS.
  • BMDM bone marrow-derived macrophages
  • the intracellular function of MyD88 will be analyzed for its effect on (i) ⁇ alpha degradation induced by LPS; (ii) induction of NFKB nuclear translocation in response to LPS; and (iii) cytokine/chemokine production (TNFa, IL-6, MCP-1) in response to anthrax spores or LPS.
  • cytokine/chemokine production TNFa, IL-6, MCP-1
  • a preclinical test of CP-MyD88's potential for therapeutic efficacy will be the outcome of in vivo studies to inhibit Pulmonary Anthrax in MyD88-deficient and A/J mice.
  • Purified CP-MyD88 and its translocation-negative forms at a concentration of 2 mg/Kg (body weight) will be administered IP to mice 30 minutes prior to spore challenge, followed by a post-challenge schedule of administration similar to the cSN50 peptide.
  • the temporal relationship between the anthrax spore challenge and the CP-MyD88 action will be analyzed.
  • CP-MyD88 mutants and modules will be screened for their intracellular function in murine bone marrow-derived macrophages (BMDM) obtained from anthrax- permissive MyD88-/-mice and anthrax non-permissive C57BL6J mice.
  • BMDM murine bone marrow-derived macrophages
  • HKBa heat-killed B. anthracis Sterne strain
  • significant induction of TNFa is observed after 6h.
  • BMDM from MyD88-/- mice do not produce this mediator of innate immunity.
  • MyD88 correlates inversely with anthrax bacilli-permissive phenotypes, a significant discovery confirmed by the same investigators in a pulmonary anthrax model following challenge with aerosolized B. anthracis Sterne spores.
  • This test system will be utilized for screening CP-MyD88 mutants and modules for their reversal of the anthrax-permissive phenotype of MyD88-/-BMDM into non- permissive.
  • CP-MyD88 modules CP-DD and CP-TIR
  • CP-DD and CP-TIR potential dominant-negative inhibitors of intracellular MyD88 interactions responsible for the reversal of the anthrax non-permissive phenotype of MyD88+/+ BMDM, derived from C57BL/6J mice, into anthrax permissive.
  • Figure 4 is a conceptual figure depicting a potential site of action of cell- penetrating peptides that disarm anthrax toxins acting within the cell.

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Abstract

La présente invention concerne des compositions, des kits et des méthodes permettant d'inhiber la croissance d'un agent pathogène et traiter une infection par l'agent pathogène chez un sujet, qui comprend l'administration d'une composition qui contient un agent antimicrobien et un modificateur de transport nucléaire (NTM). L'agent antimicrobien et le NTM sont présents en des quantités thérapeutiquement efficaces pour le traitement de l'infection. Selon un mode de réalisation dans lequel le sujet souffre de maladies pulmonaires bactériennes, virales ou fongiques, l'agent antimicrobien et le NTM sont présents en des quantités thérapeutiquement efficaces pour le traitement d'une maladie pulmonaire chez le sujet. Selon un mode de réalisation, les compositions, les kits et les méthodes sont utilisés de façon à inhiber la croissance des bactéries Bacillus anthracis, traiter une infection à B. anthracis et traiter la maladie du charbon pulmonaire chez un sujet. Selon un mode de réalisation dans lequel le sujet souffre d'une maladie du charbon pulmonaire, l'agent antimicrobien et le NTM sont présents en quantités thérapeutiquement efficaces pour traiter la maladie du charbon pulmonaire chez le sujet.
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018232383A1 (fr) 2017-06-16 2018-12-20 Vanderbilt University Méthodes et compositions pour le traitement d'une inflammation microbienne
CN113543798A (zh) * 2018-09-14 2021-10-22 范德比尔特大学 用于治疗皮肤病的方法和组合物
US11571455B2 (en) 2013-04-11 2023-02-07 Vanderbilt University Methods and compositions for treating alcoholic liver disease

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US20040235075A1 (en) * 2003-03-28 2004-11-25 Cullum Malford E. Rapid immunoassay of anthrax protective antigen in vaccine cultures and bodily fluids by fluorescence polarization
US20040235746A1 (en) * 1994-06-13 2004-11-25 Hawiger Jack J. Cell permeable peptides for inhibition of inflammatory reactions and methods of use
US20080064643A1 (en) * 2004-12-20 2008-03-13 Paolo Carminati Myd88 Homodimerization Inhibitors
US20110105383A1 (en) * 2008-09-10 2011-05-05 Magnus Hook Methods and compositions for stimulation of mammalian innate immune resistance to pathogens

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US20040235746A1 (en) * 1994-06-13 2004-11-25 Hawiger Jack J. Cell permeable peptides for inhibition of inflammatory reactions and methods of use
US20040235075A1 (en) * 2003-03-28 2004-11-25 Cullum Malford E. Rapid immunoassay of anthrax protective antigen in vaccine cultures and bodily fluids by fluorescence polarization
US20080064643A1 (en) * 2004-12-20 2008-03-13 Paolo Carminati Myd88 Homodimerization Inhibitors
US20110105383A1 (en) * 2008-09-10 2011-05-05 Magnus Hook Methods and compositions for stimulation of mammalian innate immune resistance to pathogens

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11571455B2 (en) 2013-04-11 2023-02-07 Vanderbilt University Methods and compositions for treating alcoholic liver disease
WO2018232383A1 (fr) 2017-06-16 2018-12-20 Vanderbilt University Méthodes et compositions pour le traitement d'une inflammation microbienne
JP2020524144A (ja) * 2017-06-16 2020-08-13 ヴァンダービルト ユニヴァーシティ 微生物性炎症を処置するための方法および組成物
US20210145928A1 (en) * 2017-06-16 2021-05-20 Vanderbilt University Methods and compositions for treating microbial inflammation
US11771739B2 (en) 2017-06-16 2023-10-03 Vanderbilt University Methods and compositions for treating microbial inflammation
CN113543798A (zh) * 2018-09-14 2021-10-22 范德比尔特大学 用于治疗皮肤病的方法和组合物
EP3849591A4 (fr) * 2018-09-14 2022-10-12 Vanderbilt University Méthodes et compositions pour traiter des maladies de la peau

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