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WO2008016707A2 - Utilisation de l'il-6 pour le diagnostic et le traitement de pathologies neuro-inflammatoires - Google Patents

Utilisation de l'il-6 pour le diagnostic et le traitement de pathologies neuro-inflammatoires Download PDF

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WO2008016707A2
WO2008016707A2 PCT/US2007/017376 US2007017376W WO2008016707A2 WO 2008016707 A2 WO2008016707 A2 WO 2008016707A2 US 2007017376 W US2007017376 W US 2007017376W WO 2008016707 A2 WO2008016707 A2 WO 2008016707A2
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subject
level
agent
neuroinflammatory
biological sample
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PCT/US2007/017376
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WO2008016707A3 (fr
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Douglas A. Kerr
Adam I. Kaplin
Chitra Krishnan
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The Johns Hopkins University
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • G01N33/6869—Interleukin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines
    • G01N2333/54—Interleukins [IL]
    • G01N2333/5412—IL-6
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/24—Immunology or allergic disorders
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/285—Demyelinating diseases; Multipel sclerosis

Definitions

  • Neuroinflammatory conditions including transverse myelitis (TM), multiple sclerosis, and neuromyelitis optica, are characterized by abrupt neurologic deficits associated with inflammation, demyelination, and axonal damage. In each of these disorders, neuroinflarnmation damages the myelin sheath that insulates nerve cell fibers in the brain and spinal cord, ultimately causing extensive and often permanent damage to the underlying nerves. Patients suffering from a neuroinflammatory condition experience dramatic and sometimes permanent losses in sensory and motor function. Neuroinflammatory disorders are notoriously difficult to diagnose and treat. Inaccurate diagnoses result in uncertainty for patients and their families. Quick and accurate methods of diagnosing neuroinflammatory conditions are important to ensure that appropriate methods of treatment are implemented to ameliorate neuroinflammatory symptoms and preserve neurological function.
  • the invention features compositions and methods that are useful for the diagnosis and treatment of neuroinflammatory disorders (e.g., transverse myelitis, multiple sclerosis, optic neuritis, neuromyelitis optica).
  • neuroinflammatory disorders e.g., transverse myelitis, multiple sclerosis, optic neuritis, neuromyelitis optica.
  • the invention generally provides methods for diagnosing, treating, or preventing a neuroinflammatory disorder (e.g., transverse myelitis, multiple sclerosis, optic neuritis and neuromyelitis optica); as well as methods for determining a patient prognosis and selecting an appropriate therapy (i.e., an aggressive therapy for a patient having a poor prognosis, and a less aggressive therapy for a patient having a good prognosis).
  • a neuroinflammatory disorder e.g., transverse myelitis, multiple sclerosis, optic neuritis and neuromyelitis optica
  • an appropriate therapy i.e., an aggressive therapy for a patient having a poor prognosis, and a less aggressive therapy for a patient having a good prognosis.
  • the invention provides a method of identifying a subject as having or having a propensity to develop a neuroinflammatory disorder (e.g., transverse myelitis, multiple sclerosis, optic neuritis and neuromyelitis optica), the method involving detecting an increase in the level of IL-6 in a biological sample of the subject relative to a reference level, where the increase in IL-6 indicates that the subject has or has a propensity to develop a neuroinflammatory disorder.
  • a neuroinflammatory disorder e.g., transverse myelitis, multiple sclerosis, optic neuritis and neuromyelitis optica
  • the invention provides a method of identifying a subject as having or having a propensity to develop a neuroinflammatory disorder, the method involving detecting an increase in the level of nitric oxide (NO) in a biological sample of the subject relative to a reference level, where the increase in NO indicates that the subject has or has a propensity to develop a neuroinflammatory disorder.
  • the invention provides a method of selecting a therapy for a subject identified as having a neuroinflammatory disorder, the method involving detecting an increase in the level of IL-6 in a biological sample of a subject relative to a reference level, where the level of IL-6 indicates an appropriate therapy.
  • the invention provides a method of selecting a therapy for a subject identified as having a neuroinflammatory disorder, the method involving detecting an increase in the level of NO in a biological sample of a subject relative to a reference level, where the level of NO indicates an appropriate therapy.
  • the invention provides a method of identifying a subject as having a propensity to develop a neuroinflammatory relapse, the method involving detecting a decrease in levels of NO or IL-6 in response to therapy, where a failure to observe a reduction in NO or IL-6 levels identifies a patient as having a propensity to relapse.
  • levels greater than about 15-20 ⁇ M total nitrate indicates and nitrite levels are indicative of poor prognosis.
  • the method indicates that an aggressive therapy should be selected.
  • the invention features a method of monitoring therapy for a subject identified as having a neuroinflammatory disorder, the method involving detecting an alteration in the level of IL-6 in a biological sample of the subject relative to a reference level, where a reduction in the level of IL-6 indicates therapeutic efficacy.
  • the invention features a method of determining the prognosis of a subject identified as having a neuroinflammaory disorder, the method involving detecting an increase in the level of IL-6 or NO in a biological sample of a subject relative to a reference level, where the level of IL-6 or NO is indicative of a clinical outcome.
  • an amount of IL-6 between about 3.5 pg and 50 pg/ml CSF or NO levels less than about 15 uM indicates a good prognosis. For patients having a good prognosis (i.e., unlikely to experience long term disability or relapse) less aggressive (i.e., steroid therapy) is appropriate.
  • an amount of IL-6 between about 3.5 pg and 50 pg/ml or NO levels less than about 10-15 ⁇ M in CSF indicates that the subject has a good prognosis.
  • the good prognosis identifies the subject as unlikely to experience severe neurological disability or relapse
  • a level of IL-6 in the biological sample of greater than about 50 pg and 5000 pg or NO levels of about 15-20 ⁇ M indicates that the subject has a poor prognosis.
  • the poor prognosis identifies the subject as likely to experience severe neurological disability or relapse. For patients having a poor prognosis, aggressive therapy is indicated.
  • the invention features a pharmaceutical composition for the treatment of neuroinflammation, containing an effective amount of an agent selected from the group consisting of a JAK/STAT signal transduction pathway inhibitor, a PARP inhibitor, an iNOS inhibitor, or analogs thereof.
  • the agent is any one or more of 4- amino-1, 8-napthalimide, the iNOS inhibitor 1400W, GPI-5693, 15427, 16539, 16072, and GPI-21016.
  • the effective amount is 10 mg/kg, 20 mg/kg, preferably 30 mg/kg or 100 mg/kg.
  • the agent is GPI 5693
  • the effective amount is selected from the group consisting of 10 mg/kg, 30 mg/kg, 40 mg/kg, 50 mg/kg, 75 mg/kg, and 100 mg/kg.
  • the invention features a method of treating a neuroinflammatory disorder, the method involving administering to a subject an effective amount of an agent selected from the group consisting of a JAK/STAT signal transduction pathway inhibitor, a PARP inhibitor, an iNOS inhibitor, or analogs thereof.
  • the invention features a method of preventing a neuroinflammatory relapse in a subject at risk thereof, the method involving administering to a subject an effective amount of an agent selected from the group consisting of a JAK/STAT signal transduction pathway inhibitor, a PARP inhibitor, an iNOS inhibitor, a soluble IL-6 receptor or analogs thereof.
  • an agent selected from the group consisting of a JAK/STAT signal transduction pathway inhibitor, a PARP inhibitor, an iNOS inhibitor, a soluble IL-6 receptor or analogs thereof.
  • the invention features a kit for the treatment of a neuroinflammatory disorder, the kit containing a JAK/STAT signal transduction pathway inhibitor, a PARP inhibitor, an iNOS inhibitor, a soluble IL-6 receptor or analogs thereof.
  • the agent is any one or more of GPI-5693, 15427, 16539, 16072, or GPI- 21016.
  • the kit is labeled for the treatment of a neuroinflammatory disorder.
  • the invention features a kit for the diagnosis of a neuroinflammatory disorder, the kit containing an IL-6 detecting agent.
  • the IL-6 detecting agent is an IL-6 antibody.
  • the kit further contains directions for the use of the kit in diagnosing or treating a neuroinflammatory condition or a neuroinflammatory relapse.
  • the kit further comprises an agent for detecting NO.
  • the neuroinflammatory disorder is any one or more of transverse myelitis (TM), multiple sclerosis (MS), optic neuritis and neuromyelitis optica (NMO).
  • the method involves measuring the amount of IL-6 or NO present in a biological fluid selected from the group consisting of: cerebrospinal fluid, serum, urine, and saliva.
  • the reference level is the amount of IL-6 or NO present in a biological sample derived from a control subject.
  • the reference level of IL-6 is between about undetectable 0.5 and 3 pg/ml in cerebrospinal fluid.
  • an increase in IL-6 levels of at least about 2-, 3-, or -4 fold relative to a reference identifies the subject as having a neuroinflammatory disorder.
  • an increase in IL-6 levels between about 5-5,000 (e.g., 5, 10, 20, 50, 100, 200, 300, 500, 750, 1000, 2000, 3000, 5000, 10,000) fold identifies the subject as having a neuroinflammatory condition.
  • an amount of IL-6 between about 3.5 pg and 50 pg indicates that the subject has or has a propensity to develop muscular sclerosis.
  • a level of IL-6 in the biological sample of between about 50 pg and 5000 pg indicates that the subject has or has a propensity to develop transverse myelitis.
  • the method further involves conducting a neurological examination or a diagnostic test.
  • NO is measured indirectly by detecting nitrates, nitrites, or combinations thereof.
  • the level of NO present in a reference is virtually undetectable.
  • the level of NO present in a reference is about 0.5-2 ⁇ M.
  • the level of NO present in a reference is about 0.1 ⁇ M.
  • a level of NO greater than about 5 ⁇ M identifies the subject as having a neuroinflammation.
  • the reference level is the amount of IL-6 or NO present in a biological sample derived from a control subject.
  • the biological sample is cerebrospinal fluid, serum, salive, or urine.
  • the reference level of IL-6 is between about 0.5 and 3 pg/ml in cerebrospinal fluid.
  • an increase in IL-6 or NO level of at least about 2-15 (e.g., 2, 3, 4, 5, 10, 15, 20) fold relative to a reference indicates that steroid therapy is appropriate.
  • an amount of IL-6 between about 3.5 pg and 50 pg indicates that steroid therapy is appropriate.
  • a level of IL-6 in the biological sample of greater than about 50 -5000 pg/ml CSF indicates that the subject has or has a propensity to develop transverse myelitis.
  • a level of IL-6 in the biological sample of greater than about 50-5000 pg/ml CSF indicates that an aggressive
  • the method further involves measuring the amount of NO in the biological sample.
  • total levels of nitrate are detected as a method of measuring NO.
  • an effective amount of any one or more of the following agents are used or are contained in a composition as therapeutics: the agent is 4-amino-l, 8-napthalimide (e.g., administered at about 1 ⁇ M, 5 ⁇ M, 10 ⁇ M, or 20 ⁇ M) an iNOS inhibitor 1400W (e.g., administered at about 50 ⁇ M, 100 ⁇ M, 200 ⁇ M, or 300 ⁇ M), GPI-5693, 15427, 16539, 16072, GPI 15427, or GPI-21016, where an effective amount of GPI 15427 or GPI 21016 is 10 mg/kg, 20 mg/kg, preferably 30 mg/kg or 100 mg/kg; and for GPI 5693, an effective amount is 10 mg/kg, 30 mg/kg, 40 mg/kg, 50 mg/kg, 75 mg/kg, or 100 mg/kg.
  • the method further involves administering a corticosteroid
  • the invention provides diagnostic compositions and methods for measuring IL-6 or NO in a biological sample, which are useful for the identification of a neuroinflammatory condition, as well as improved methods for treating or preventing such a condition.
  • the invention further provides methods for determining a patient prognosis and selecting an appropriate therapy.
  • neuroinflammatory condition is meant a disease associated with inflammation, demyelination, or axonal damage.
  • exemplary neuroinflammatory conditions include transverse myelitis (TM), neuritis optica, multiple sclerosis, and neuromyelitis optica.
  • neuroinflammatory relapse is meant the recurrence of a neuroinflammatory condition or symptom thereof.
  • ameliorate decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease.
  • alteration is meant a change (increase or decrease) in the levels or activity of a gene, polypeptide, or other marker as detected by standard art known methods such as those described herein.
  • an alteration includes a 10% change in levels, preferably a 25% change, more preferably a 40% change, and most preferably a 50% or greater change in levels.
  • antibody is meant any immunoglobulin polypeptide, or fragment thereof, having immunogen binding ability.
  • agent any small molecule chemical compound, antibody, nucleic acid molecule, or polypeptide, or fragments thereof.
  • disease is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ.
  • diseases include bacterial invasion or colonization of a host cell.
  • reference is meant a standard or control condition.
  • subject is meant a mammal, including, but not limited to, a human or non-human mammal, such as a bovine, equine, canine, ovine, or feline.-
  • the terms “treat,” treating,” “treatment,” and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.
  • the terms “prevent,” “preventing,” “prevention,” “prophylactic treatment” and the like refer to reducing the probability of developing a disorder or condition in a subject, who does not have, but is at risk of or susceptible to developing a disorder or condition.
  • marker any protein or polynucleotide having an alteration in expression level or activity that is associated with a disease or disorder.
  • “Microarray” means a collection of nucleic acid molecules or polypeptides from one or more organisms arranged on a solid support (for example, a chip, plate, or bead).
  • Figures IA - IF show that IL-6 is selectively upregulated in the cerebrospinal fluid (CSF) of transverse myselitis (TM) patients and correlates with long-term disability.
  • CSF cerebrospinal fluid
  • TM transverse myselitis
  • a cytokine array was used to profile forty-two inflammatory proteins in the CSF of six TM and eight control patients. The mean value of each cytokine was defined for the control group, and fold induction was calculated for each TM patient.
  • the inset is an immuno fluorescent micrograph showing the results of IL-6 immunohistochemistry performed on the cervical spinal cord of a TM patient who died of respiratory failure. In that section, IL-6 expression colocalized with glial fibrillary acidic protein (GFAP)-positive astrocytes. Magnification, x60.
  • GFAP glial fibrillary acidic protein
  • Figure IB quantitative IL-6 levels in the CSF and serum of control (Con) and TM patients were determined by Enzyme-Linked Immunosorbent Assay (ELISA). Box plots represent the interquartile range, and the outliers shown are outside the fifth and ninety-fifth percentiles. Mean ⁇ SEM for each group is indicated above each box.
  • Figure 1C is a graph showing that among TM patients, acute CSF IL-6 levels strongly correlated with sustained disability (as measured by expanded disability status scale (EDSS).
  • Figure ID is a graph showing that CSF IL-6 levels strongly correlated with total nitric oxide (NO) metabolites during the acute phase of TM.
  • NO nitric oxide
  • Figure IE is a graph showing that total nitric oxide (NO) levels correlated with 14-3-3, a neuronal injury marker in TM patients.
  • Figure IF is a graph showing that levels of 14-3-3 strongly correlated with sustained disability in TM patients. For panels 1C — F, correlation coefficients and statistical significance are shown. Intensity denotes chemiluminescent signal intensity.
  • Figures 2A - 2H show that IL-6 is necessary and sufficient to induce injury of oligodendrocytes and axons in spinal cord organotypic cultures by generating nitric oxide.
  • Figure 2A is a graph that quantitates cell death in spinal cord organotypic cultures.
  • CSF 100 ⁇ l
  • TM or control patient was added to the culture media of spinal cord organotypic cultures, and cellular injury was assessed by ethidium homodimer uptake with Hoechst counterstain (inset).
  • IL-6 was immunodepleted by preincubating TM CSF with an IL-6 antibody and clearing the IL-6 antibody complex with protein A sepharose. Magnification, ⁇ 20.
  • Figure 2B shows the results of SDS-PAGE and immunoblot analysis. Tissue lysates from spinal cord organotypics were generated at various times after the administration of IL- 6 and subjected to SDS-PAGE followed by immunoblot analysis.
  • Figure 2C is a graph showing a quantification of the data shown in Figure 2B by chemiluminescent signal intensity of three independent experiments.
  • Figure 2D shows RT-PCR analysis of RNA derived from spinal cord organotypic cultures at the indicated times after addition of IL-6 at 2,000 pg/ml.
  • Figure 2E is a photomicrograph. Dual-color confocal microscopy was carried out with spinal cord organotypic cultures treated with IL-6 for twenty-four hours.
  • Microglia were identified by incubating live cultures with l,r-dioctadecyl-3,3,3',3'- tetramethylindocarbocyanineperchlorate (DiI) -acetylated (Ac)-low density lipoprotein (LDL), which is endocytosed by phagocytosing cells. After fixation, inducible nitric oxide synthase (iNOS) immunohistochemistry was carried out, revealing the expression of iNOS within microglia. Scale bar: 50 ⁇ m.
  • Figure 2F shows that nitrotyrosine (NT) and iNOS preferentially accumulated within the exterior white matter of spinal cord organotypic cultures. Scale bar: 200 ⁇ m.
  • Figure 2G is a graph showing the effect of adding IL-6 to a final concentration of 2,000 pg/ml to spinal cord organotypic cultures in the presence or absence of the iNOS inhibitor 1400W. *P ⁇ 0.05.
  • WT wild-type
  • KO iNOS knockout
  • Figures 3 A — 3H show cellular participants in IL-6— induced injury.
  • dual-color confocal microscopy was performed using an antibody to nitrotyrosine (NT) as a marker of NO excess, and the cell-specific markers GFAP, RIP, and neurofilament (NF).
  • Figure 3F is a graph showing the results of experiments where dissociated rat spinal neuron cultures were exposed to IL-6 either alone or 2 days after plating 10 4 purified microglial cells in coculture with the dissociated neurons.
  • Figure 3G is a graph showing the results of experiments wherepurified, cultured microglial cells were exposed to either 500 or 2,000 pg/ml of IL-6.
  • FIG. 3H is a graph showing the results of experiments where microglia were plated in a trans well, exposed to IL-6 for 2 hours, and then washed. Microglia were then transferred to 24-well dishes with plated, dissociated neurons, and the neurons were assessed for cellular death up to 24 hours later. *P ⁇ 0.05.
  • Figures 4A - 4G show that IL-6 induced weakness with axonal degeneration and loss of myelin when infused into the spinal subarachnoid space of adult rats.
  • Figure 4A is a graph showing the results of experiments where IL-6 was infused via a subarachnoid spinal catheter into adult rats over a 7-day period. Control animals received saline through the spinal catheter, while another cohort of animals received intrathecal IL-6 and were also given the iNOS inhibitor aminoguanidine (AG) intraperitoneally for the length of the experiment beginning at day 0. *P ⁇ 0.05.
  • AG iNOS inhibitor aminoguanidine
  • Figure 4B is a micrograph showing that pathologic specimens from IL-6— infused rat spinal cords exhibited reduced myelin staining and white matter vacuolation (asterisks). Scale bar: 20 ⁇ m.
  • Figure 4C is a micrograph showing that white matter vacuoles (asterisks) were strongly neurofilament (NF) positive, confirming the presence of axonal degeneration. Scale bar: 20 ⁇ m.
  • Figures 4D and 4E show micrographs of plastic sections (1 ⁇ M) from IL-6-infused rat spinal cords.
  • FIG. 4D shows swollen axons with intact myelin (asterisks), consistent with axonal degeneration
  • panel 4E shows demyelinated axons (arrows).
  • the term "NT” denotes Scale bar: 10 ⁇ m (D and E).
  • Figure 4F is a series of four micrographs showing that these pathologic features could also be seen in autopsy material from a patient with severe TM and high CSF IL-6 levels (1,997 pg/ml).
  • FIG. 4G shows an immunohistochemical analysis of the autopsy material. This revealed axonal dysfunction as defined by disruption of NF staining and accumulation of APP, a marker of disrupted axonal transport.
  • Figures 5 A — 5F show that PARP activation is necessary for IL-6— induced toxicity.
  • Figure 5 A is a graph that shows the results of experiments where PARP activity of IL-6— treated spinal cord (SC) organotypic cultures was assessed in the presence or absence of the PARP inhibitor 4-ANI or the iNOS inhibitor 1400W.
  • Figure 5B is a graph showing the results of experiments where spinal cord organotypic cultures were incubated with IL-6 in the presence or absence of the PARP inhibitor 4-ANI. The amount of nitrotyrosine (NT) accumulation was assessed as a marker of iNOS activity.
  • NT nitrotyrosine
  • Figure 5C is a graph showing the results of experiments where spinal cord (SC) organotypic cultures were incubated with IL-6 in the presence or absence of 4-ANI, and cell death was assessed by propidium iodide uptake.
  • Figure 5D includes two micrographs showing PARP immunoreactivity in the spinal cord of IL-6-infused rats 4 days after initiation of IL-6 infusion. Magnification, ⁇ 20.
  • Figure 5E is a graph showing the results of experiments where PARP activity was assessed in spinal cord tissue lysates generated from IL-6-inrused rats, control rats, or rats both infused with IL-6 and given the PARP inhibitor 4- ANI intraperitoneally. Lysates were generated 4 days after initiation of IL-6 infusion.
  • Figure 5F is a graph showing the results of experiments where IL- 6 was infused into the spinal subarachnoid space of adult rats in the presence or absence of the PARP inhibitors 4- ANI or 3 -AB given systemically. Hind limb grip strength was assessed daily for 7 days as described in Methods. *P ⁇ 0.05.
  • Figures 6A — 6H show that IL-6 induced regionally specific neural injury in the spinal cord.
  • Figure 6A is a graph showing the results of experiments where IL-6 was administered to cortical, hippocampal, and spinal cord organotypic cultures at increasing doses, and cell death was assessed 36 hours later. Data is plotted as the fold induction of death relative to cultures with no IL-6 addition.
  • Figure 6B is a graph showing the results of experiments where adult rats were infused with IL-6 or saline through an intracerebroventricular (IC) cannula at the same rate (0.5 ⁇ l/h for 7 days) and concentration (2,000 pg/ml) as that previously administered via a spinal subarachnoid catheter and assessed for weakness.
  • IC intracerebroventricular
  • Figure 6C is a graph showing the results of experiments where cortical organotypic cultures were treated with IL-6 and assessed for PARP activity up to 20 hours later.
  • Figure 6D shows confocal micrographs of cortex and spinal cord organotypic cultures, performed after the administration of IL-6 to the culture.
  • the term "NT” denotes nitrotyrosine; the term “RIP” denotes an oligodendrocyte marker. Scale bars: 50 ⁇ m.
  • Figure 6E is a gel showing an RT- PCR analysis of iNOS from cortex or spinal cord organotypic cultures performed at various times after the addition of IL-6. GAPDH serves as a PCR control.
  • Figure 6F is a graph showing results of a quantitative immunoblot of IL-6R expression (inset) present in human autopsy tissue lysates.
  • Spinal cord grey and white matter (SCGM and SCWM 5 respectively) and cortex grey and white matter (CoWM and CoGM, respectively) lysates were generated, subjected to SDS-PAGE, and probed for IL-6R immunoreactivity.
  • Figure 6G is a graph showing results of a quantitative immunoblot of sIL-6R from the same lysates (shown in chemiluminescent units).
  • Figure 6H is a graph showing the results of experiments where adult rats were infused with either IL-6 or IL-6 plus sIL-6R at a 1 :1 molar ratio through a spinal subarachnoid catheter as before. Animals were assessed for hind limb grip strength for the 10-day duration of the experiment. *P ⁇ 0.05; **P ⁇ 0.04.
  • Figure 7 is a graph showing that iNOS knockout mice exhibit an early peak of PARP activity following IL-6 administration in spinal cord organotypic cultures. No late peak was observed.
  • Mouse spinal cord organotypic cultures were generated from wild-type (WT) or iNOS knockout (KO) mice and IL-6 was added at 500 pg/ml.
  • Figure 8 is a graph showing that intracerebroventricular infusion of IL-6 (2000 pg/ml) does not induce cognitive/behavioral changes in adult rats.
  • the figure depicts the latencies to eating in the IL-6-treated and control rats. Although the control rats had a trend towards a greater latency to eating, no significant differences between the two groups were seen, P > 0.05
  • the invention features compositions and methods that are useful for the diagnosis and treatment of a neuroinflammatory condition (e.g., transverse myelitis, multiple sclerosis, optic neuritis, neuromyelitis optica).
  • a neuroinflammatory condition e.g., transverse myelitis, multiple sclerosis, optic neuritis, neuromyelitis optica.
  • the invention is based, at least in part, on the observation that IL-6 and nitric oxide levels are significantly increased in biological samples derived from subjects suffering from a neuroinflammatory condition, and that levels of IL-6 correlate with the degree of neurological pathology.
  • the invention provides diagnostic and prognostic methods for detecting increases in the level of IL-6 present in a biological sample derived from a patient, where an increase in IL-6 indicates not only that the subject has a neuroinflammatory condition, but can also indicate whether the patient is likely to have a good or a poor prognosis, as well as whether the patient is likely to relapse. Because the treatment regimen selected for each patient is determined by the patient's prognosis and risk of relapse, the invention further provides for the selection of appropriate prophylactic and/or therapeutic regimens.
  • the invention also provides methods for preventing or treating a neuroinflammatory disorder. Such methods are based, at least in part, on the observation that IL-6 activates the JAK/STAT pathway, resulting in increased iNOS and poly(ADP-ribose) polymerase (PARP) activity, which are associated with neuroinflammatory pathology. Accordingly, the invention further provides compositions and methods for reducing the activity of the JAK/STAT pathway, iNOS, and PARP. Such compositions and methods were found to be useful for the treatment or prevention of neuroinflammat ⁇ ry disease in vivo.
  • IL-6 is a glycoprotein cytokine that mediates signal transduction between immune cells, induces acute-phase protein synthesis, and controls growth and differentiation of cells of the immune and hematopoietic systems (2).
  • IL-6 is likely to be a trophic factor that, under some circumstances, supports neuronal and glial differentiation and survival (2).
  • Introduction of members of the IL-6 superfamily, including IL-6 itself, into some systems has been shown to ameliorate demyelination, perhaps by providing trophic support for oligodendrocytes (3).
  • IL-6 levels in the adult CNS are usually low or undetectable under baseline conditions. Surprisingly, as reported in more detail below, levels of IL-6 were increased dramatically in patients suffering from transverse myelitis, a neuroinflammatory disorder. Similar increases were observed in patients suffering from multiple sclerosis and neuromyelitis optica.
  • IL-6 within the CNS may stimulate iNOS expression, resulting in the production of NO and leading to free radical-induced tissue injury (10).
  • IL-6 produces its effects by binding to IL-6 receptors (IL-6Rs), which form complexes with gpl 30. Once formed, the IL- 6/IL-6R/gpl30 complex stimulates the following two main signal transduction cascades that lead to activation of a number of transcription factors responsible for IL-6-mediated effects: JAK/STAT and Ras/MEK/MAPK (11).
  • IL-6 induced activation of the JAK2/STAT3 signaling pathway in cardiac myocytes results in activation of iNOS with subsequent NO production and decreased cardiac contractility (10).
  • iNOS is normally expressed either minimally or not at all in the CNS, but in pathological conditions, iNOS levels can increase dramatically in glial cells or influxing macrophages in response to injury or inflammation (12).
  • iNOS generates significantly greater, sustained amounts of NO (picomolar vs. micromolar levels, respectively) (13).
  • iNOS peroxynitrite anion
  • ONOO triggers DNA single-strand breakage that activates the DNA-repair enzyme poly(ADP-ribose) polymerase (PARP) (20).
  • PARP poly(ADP-ribose) polymerase
  • TM transverse myelitis
  • neuritis optica e.g., multiple sclerosis
  • neuromyelitis optica e.g., TM
  • TM can exist as part of a multifocal CNS disease (e.g., multiple sclerosis), a multi-system disease (e.g., systemic lupus erythematosus), or as an isolated idiopathic entity.
  • Neuroinflammation causes extensive damage to nerve fibers of the spinal cord.
  • Subjects suffering from TM typically display any one or more of the following symptoms: weakness of the legs and arms, pain, sensory alterations, and/or bowel and bladder dysfunction. These symptoms may develop over the course of days or weeks.
  • TM patients About one-third of patients suffering from TM fail to recover motor function, remaining wheel chair bound or bedridden for the remainder of their lives. In the most severe cases, TM patients lose the ability to breathe on their own, and are dependent on mechanical ventilation. In some patients, the initial TM attack is followed by a recurrence that results in further losses in neurological function.
  • Multiple sclerosis is another neuroinflammatory condition where inflammation damages the myelin sheath of nerves within the spinal cord and CNS. It is a progressive disease where loss of sensory and motor function occurs over months or even years. Symptoms of MS vary because the location and extent of each attack varies. Symptoms may include pain, tingling, muscle weakness, paralysis loss of vision or hearing, incontinence, vertigo, and spasticity.
  • NMO Neuromyelitis optica
  • CNS central nervous system
  • Individuals with NMO develop transverse myelitis and optic neuritis.
  • Optic neuritis is an inflammation or demyelination of the optic nerve that is typically associated with an acute loss or blurring of vision effecting one eye.
  • NMO is associated with spinal cord and CNS demyelination, and patients suffering from NMO often experience unpredictable relapses.
  • IL-6 was elevated in the cerebrospinal fluid (CSF) of transverse myelitis patients at the time of their acute clinical presentation and that levels of IL-6 correlated with the patients' eventual long-term disability.
  • CSF cerebrospinal fluid
  • IL-6 was shown to mediate the kind of spinal cord injury found in patients with TM.
  • nitric oxide (NO) production was necessary to achieve this tissue damage.
  • the targets of IL-6-mediated injury are oligodendrocytes and axons, which result in demyelination and axonal injury.
  • the invention features compositions and methods that are useful for the treatment or prevention of symptoms associated with any one or more of these neuroinflammatory conditions.
  • the present invention provides methods of treating disease and/or disorders or symptoms thereof which comprise administering a therapeutically effective amount of a pharmaceutical composition comprising a therapeutic agent that disrupts or reduces the activity of the JAK/STAT signal transduction pathway or a downstream effector of this pathway, such as PARP or iNOS herein to a subject (e.g., a mammal such as a human).
  • a subject e.g., a mammal such as a human.
  • the method includes the step of administering to the mammal a therapeutic amount of a compound herein (e.g., a compound that reduces the expression or activity of a molecular component of the JAK/STAT pathway or reduces the expression or activity of inducible Nitric Oxide Synthase (iNOS) or PARP, such as 1400W or soluble IL-6 receptor) sufficient to treat the disease or disorder or symptom thereof, under conditions such that the disease or disorder is treated.
  • a compound herein e.g., a compound that reduces the expression or activity of a molecular component of the JAK/STAT pathway or reduces the expression or activity of inducible Nitric Oxide Synthase (iNOS) or PARP, such as 1400W or soluble IL-6 receptor
  • the methods herein include administering to the subject (including a subject identified as in need of such treatment) an effective amount of a compound described herein, or a composition described herein to produce such effect. Identifying a subject in need of such treatment can be in the judgment of a subject or a health care professional and can be subjective (e.g. opinion) or objective (e.g. measurable by a test or diagnostic method).
  • the therapeutic methods of the invention (which include prophylactic treatment) in general comprise administration of a therapeutically effective amount of the compounds herein, such as a compound of the formulae herein to a subject (e.g., animal, human) in need thereof, including a mammal, particularly a human.
  • Such treatment will be suitably administered to subjects, particularly humans, suffering from, having, susceptible to, or at risk for a disease, disorder, or symptom thereof. 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 (e.g., genetic test, enzyme or protein marker, Marker (as defined herein), family history, and the like).
  • a diagnostic test or opinion of a subject or health care provider e.g., genetic test, enzyme or protein marker, Marker (as defined herein), family history, and the like.
  • the compounds herein may be also used in the treatment of any other disorders in which neuroinflammation may be implicated.
  • the invention provides a method of monitoring treatment progress.
  • the method includes the step of determining a level of diagnostic marker (Marker) (e.g., IL-6, NO, 14-3-3, or any target delineated herein modulated by a compound herein, a protein or indicator thereof, etc.) or diagnostic measurement (e.g., screen, assay) in a subject suffering from or susceptible to a disorder or symptoms thereof associated with neuroinflammation, in which the subject has been administered a therapeutic amount of a compound herein sufficient to treat 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 in the subject is determined at a time point later than the determination of the first level, and the two levels are compared to monitor the course of disease or the efficacy of the therapy.
  • a pre- treatment level of Marker in the subject is determined prior to beginning treatment according to this invention; 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.
  • the present invention provides methods for diagnosing a neuroinflammatory condition by assaying the IL-6 and/or NO in a biological sample.
  • the method involves detecting an increase in the level of IL-6 in a sample of cerebrospinal fluid relative to the level present in a sample obtained from a control subject.
  • the method employs an immunoassay, such as a cytokine antibody assay.
  • Patients having a significant increase e.g., about 3-5 fold, 10-100 fold, 100-300 fold, 300-500 fold, 500-1000 fold, or 1000-5000 fold or more
  • an increase of at least about 250, 300, or 350 fold identifies the patient as having transverse myelitis.
  • IL-6 protein levels include, but are not limited to ELISA, ELIspot assays, chemiluminescent enzyme immunoassay (Shikano et al., Nephron. 2000 May;85(l):81-5); radioimmunoassay, and Western blot.
  • Methods of measuring NO include measuring total nitrate levels (e.g., using the Griess method).
  • Other assays for measuring NO are commercially available, for example, from R & D Systems (Minneapolis, MN).
  • Methods of measuring IL-6 or iNOS gene expression are also known in the art and include but are not limited to quantitative PCR, RT-PCR, and Northern blot. Kits for measuring IL-6 are commercially available, for example, from R & D Systems (Minneapolis, MN).
  • the diagnostic methods of the invention maybe combined with conventional methods for diagnosing neuroinflammatory disorders.
  • Such methods involve taking a medical history, carrying out a neurological examination, including characterizing focal neurologic deficits, which may include decreased or abnormal sensation, decreased ability to move a part of the body, speech or vision changes, or other loss of neurologic functions, head MRI scan, spine MRI scan, lumbar puncture (spinal tap), myelography, CSF oligoclonal banding, and/or CSF IgG index.
  • the invention provides compositions and methods for the treatment or prevention of a neuroinflammatory disorder (e.g., transverse myelitis (TM), neuritis optica, multiple sclerosis, and neuromyelitis optica) or neuroinflammatory relapse.
  • a neuroinflammatory disorder e.g., transverse myelitis (TM), neuritis optica, multiple sclerosis, and neuromyelitis optica
  • the invention provides therapeutic and prophylactic compositions that inhibit the expression or activity of a component of the JAK/STAT pathway or a downstream effector of that pathway, such as PARP or iNOS, or that disrupt IL-6 signalling, such as blocking antibodies or soluble IL-6 receptors (seee, for example, Jones et al., 2002, Biochim. Biophys. Acta 1592:251.-263; Kaplanski et al., 2003, Trends Immunol.
  • the activity of a specified compound as an inhibitor of a JAK kinase may be assessed in vitro or in vivo.
  • the activity of a specified compound can be tested in a cellular assay. Suitable assays include assays that determine inhibition of either the phosphorylation activity or ATPase activity of a JAK kinase.
  • an agent is said to inhibit the JAK/STAT pathway if it inhibits the activity of a JAK kinase, if it inhibits phosphorylation of STAT3, for example at Tyr705, or if it inhibits the ATPase activity of a JAK kinase.
  • the invention provides therapeutic and prophylactic compositions comprising an effective amount of 4-amino-l,8-napthalimide (4 ANT) and/or 1400W.
  • the PARP inhibitors are In one preferred embodiment, 10-(4-methyl-piperazin-l-ylmethyl)-2H-7-oxa-l,2-diaza- benzo[de]anthracen-3-one (GPI 15427) and 2-(4-methyl-piperazin-l-yl)-5H-benzo[c][l,5] naphthyridin-6-one (GPI 16539) (Di Paola et al., European Journal of Pharmacology 527 (2005) 163-171; Tentori et al., Clinical Cancer Research Vol. 9, 5370-5379, November 1, 2003).
  • the PARP inhibitor is GPI-5693, 15427, 16539, 16072, or GPI-21016.
  • GPI 5693 bulk drug should be kept at 4°C.
  • 50 mM HEPES buffered saline e.g., to prepare 400 ml of 50 mM Hepes buffered saline solution: dilute 20 ml of IM Hepes stock solution with 380 ml of normal saline solution. Degas by bubbling Argon or nitrogen through the buffer) to solubilize the compounds for efficacy studies) and solubilize concentrations of up to 100 mg/ml GPI 5693 or 10 mg/ml GPI 16072 in HEPES buffered saline (high concentrations may require some sonication). Solutions are somewhat acidic.
  • the pH may be adjusted to 6-7 with NaOH before systemic dosing, (pH should not exceed pH 7.0).
  • pH 4 is fine. Some NaOH addition may be needed to help solubilize higher concentrations. Add this dropwise and vortex between additions.
  • Dosing solutions may be made from dry powder preferably fresh daily or at least every two days. Minimize exposure of the drug to air by capping solutions frequently. Capped solutions should be refrigerated when not in use.
  • JAK/STAT inhibitors which may be useful in the methods of this invention include, but are not limited to: PIAS proteins, which bind and inhibit at the level of the STAT proteins; members of an SH2 containing family of proteins, which are able to bind to JAKs and/or receptors and block signaling; cytokine-inducible Src homology 2-containing (CIS) protein, an inhibitor of STAT signaling; CIS-related proteins, which can inhibit STAT signaling or directly bind to Janus kinases; Suppressor of Cytokine Signaling-I protein (SOCS-I, also referred to as JAB or SSI-I), which appears to associate with all JAKs to block the downstream activation of STAT3; Tyrphostins, which are derivatives of benzylidene mal
  • JAK/STAT inhibitors which may be useful in the methods of this invention include, but are not limited to: PIAS proteins, which bind and inhibit at the level of the STAT proteins; members of an SH2 containing family of proteins, which are able to bind to JAKs and/or receptors and block signaling; cytokine-inducible Src homology 2-containing (CIS) protein, an inhibitor of STAT signaling; CIS-related proteins, which can inhibit STAT signaling or directly bind to Janus kinases; Suppressor of Cytokine Signaling-I protein (SOCS-I, also referred to as JAB or SSI-I), which appears to associate with all JAKs to block the downstream activation of STAT3; Tyrphostins, which are derivatives of benzylidene malononitrile, resembling tyrosine and erbstatin moieties; AG-490, a member of the tyrophostin family of tyrosine kinase
  • the invention provides therapeutic and prophylactic agents that inhibit the expression or activity of iNOS.
  • Methods for assaying the activity or expression of iNOS are known in the art and described herein. Such methods include assaying nitrosylation of tyrosine residues using an immunoassay, assaying iNOS RNA, for example, using RT-PCR, and assaying iNOS protein levels, for example, using immunohistochemistry.
  • Agents that reduce the expression or activity of iNOS are known in the art and described herein.
  • INOS inhibitors include, but are not limited to 1400W.
  • the invention provides therapeutic and prophylactic agents that inhibit the expression or activity of PARP.
  • agents are known in the art and described herein.
  • Agents that inhibit PARP include but are not limited to 4-amino-l,8- napthalimide (4ANI); nicotinamide, 3 aminobenzamide, 6(5H)-Phenanthridinone, 5- Aminoisoquinolinone (5-AIQ), Hydrochloride, 4-Hydroxyquinazoline, 4-Quinazolinol, 1,5- Isoquinolinediol, 5 -Hydroxy- 1 (2H)-isoquinolinone, 3 ,4-Dihydro-5-[4-(l -piperidinyl)butoxy]- l(2H)-isoquinolinone (DPQ); 3-aminobenzamide; 1,5-isoquinolinediol; 6(5H)- phenanthidone; l,3,4,5,-tetrahydrobenzo(c)
  • 10-(4-methyl-piperazin-l-ylmethyl)-2H-7-oxa-l,2- diaza-benzo[dejanthracen-3-one (GPI 15427) and 2-(4-methyl-piperazin-l-yl)-5H- benzo[c][l,5] naphthyridin-6-one (GPI 16539) (Di Paola et al., European Journal of Pharmacology 527 (2005) 163-171; Tentori et al., Clinical Cancer Research Vol. 9, 5370- 5379, November 1, 2003).
  • the PARP inhibitor is GPI-5693, 15427, 16539, 16072, or GPI-21016.
  • the PARP inhibitors, GPI 15427 and GPI 21016 are dissolved in saline and PBS, and the compounds are administered, for example, at 10 mg/kg, 20 mg/kg, preferably 30 mg/kg or 100 mg/kg.
  • the compounds are administered at about 30 mg/kg and 50 mg/kg for GPI 5693.
  • dosing solutions of 3 mg/ml, 5 mg/ml and 10 mg/ml as appropriate.
  • the present invention features pharmaceutical preparations for the treatment or prevention of a neuroinflammatory disorder (e.g., transverse myelitis (TM), neuritis optica, multiple sclerosis, and neuromyelitis optica) or neuroinflammatory relapse comprising an agent that inhibits the expression or activity of a component of the JAK/STAT signal transduction pathway, a PARP inhibitor, an iNOS inhibitor, an agent that blocks IL-6 signalling, such as a blocking antibody or soluble IL-6 receptor, or analogs thereof, together with pharmaceutically acceptable carriers, where the compounds provide for the treatment of virtually any neuroinflammatory condition or neuroinflammatory relapse.
  • the pharmaceutical composition comprises an effective amount of 4-amino-l,8- napthalimide (4 ANI) and/or 1400W.
  • a pharmaceutical composition includes an effective amount of a JAK/STAT signal transduction pathway inhibitor, a PARP inhibitor, or an iNOS inhibitor.
  • the compositions should be sterile and contain a therapeutically effective amount of a JAK/STAT signal transduction pathway inhibitor, a PARP inhibitor, or an iNOS inhibitor in a unit of weight or volume suitable for administration to a subject (e.g., a human patient).
  • the compositions and combinations of the invention can be part of a pharmaceutical pack, where the JAK/STAT signal transduction pathway inhibitor, PARP inhibitor, or iNOS inhibitor is present in individual dosage amounts.
  • compositions of the invention to be used for prophylactic or therapeutic administration should be sterile. Sterility is readily accomplished by filtration through sterile filtration membranes (e.g., 0.2 ⁇ m membranes), by gamma irradiation, or any other suitable means known to those skilled in the art. Therapeutic compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle. These compositions ordinarily will be stored in unit or multi-dose containers, for example, sealed ampoules or vials, as an aqueous solution or as a lyophilized formulation for reconstitution.
  • a JAK/STAT signal transduction pathway inhibitor, a PARP inhibitor, or an iNOS inhibitor may be combined, optionally, with a pharmaceutically acceptable excipient.
  • pharmaceutically-acceptable excipient means one or more compatible solid or liquid filler, diluents or encapsulating substances that are suitable for administration into a human.
  • carrier denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate administration.
  • the components of the pharmaceutical compositions also are capable of being co-mingled with a JAK/STAT signal transduction pathway inhibitor, a PARP inhibitor, or an iNOS inhibitor of the present invention, and with each other, in a manner such that there is no interaction that would substantially impair the desired pharmaceutical efficacy.
  • Compounds of the present invention can be contained in a pharmaceutically acceptable excipient.
  • the excipient preferably contains minor amounts of additives such as substances that enhance isotonicity and chemical stability.
  • Such materials are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, succinate, acetate, lactate, tartrate, and other organic acids or their salts; tris- hydroxymethylaminomethane (TRIS), bicarbonate, carbonate, and other organic bases and their salts; antioxidants, such as ascorbic acid; low molecular weight (for example, less than about ten residues) polypeptides, e.g., polyarginine, polylysine, polyglutamate and polyaspartate; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone (PVP), polypropylene glycols (PPGs), and polyethylene glycols (PEGs);
  • additives such as stabilizers, anti-microbials, inert gases, fluid and nutrient replenishers (i.e., Ringer's dextrose), electrolyte replenishers, and the like, which can be present in conventional amounts.
  • compositions as described above, can be administered in effective amounts.
  • the effective amount will depend upon the mode of administration, the particular condition being treated and the desired outcome. It may also depend upon the stage of the condition, the age and physical condition of the subject, the nature of concurrent therapy, if any, and like factors well known to the medical practitioner. For therapeutic applications, it is that amount sufficient to achieve a medically desirable result.
  • an effective amount is sufficient to prevent, reduce, stabilize, or reverse an alteration associated with neuroinflammation.
  • an effective amount is an amount sufficient to stabilize, slow, or reduce a symptom associated with the neuroinflammatory condition.
  • doses of the compounds of the present invention would be from about 0.01 mg/kg per day to about 1000 mg/kg per day. In one embodiment, 25, 50, 75, 100, 125, 150 or 200 mg of a JAK/STAT signal transduction pathway inhibitor, a PARP inhibitor, or an iNOS inhibitor is administered to a subject.
  • a JAK/STAT signal • transduction pathway inhibitor, a PARP inhibitor, or an iNOS inhibitor is administered.
  • Effective doses range from 0.1 nM to 200 nM, where the bottom of the range is any integer between 1 and 199, and the top of the range is any integer between 2 and 200. It is expected that doses ranging from about 5 to about 2000 mg/kg will be suitable — depending on the specific JAK/STAT signal transduction pathway inhibitor, a PARP inhibitor, or an iNOS inhibitor used. Lower doses will result from certain forms of administration, such as intravenous, subcutaneous, intramuscular, oral, intranasal administration, intraspinal, or arterial release catheter into brain or spinal cord provides local high dose.
  • a composition of the invention is administered orally.
  • Other modes of administration include rectal, topical, intraocular, buccal, intravaginal, intracisternal, intracerebroventricular, intratracheal, nasal, transdermal, within/on implants, or parenteral routes.
  • parenteral includes subcutaneous, intrathecal, intravenous, intramuscular, intraperitoneal, or infusion. Intravenous or intramuscular routes are not particularly suitable for long-term therapy and prophylaxis.
  • compositions comprising a composition of the invention can be added to a physiological fluid, such as cerebrospinal fluid or blood, for example. Oral administration can be preferred for prophylactic treatment because of the convenience to the patient as well as the dosing schedule.
  • compositions of the invention can comprise one or more pH buffering compounds to maintain the pH of the formulation at a predetermined level that reflects physiological pH, such as in the range of about 5.0 to about 8.0.
  • the pH buffering compound used in the aqueous liquid formulation can be an amino acid or mixture of amino acids, such as histidine or a mixture of amino acids such as histidine and glycine.
  • the pH buffering compound is preferably an agent which maintains the pH of the formulation at a predetermined level, such as in the range of about 5.0 to about 8.0, and which does not chelate calcium ions.
  • Illustrative examples of such pH buffering compounds include, but are not limited to, imidazole and acetate ions.
  • the pH buffering compound may be present in any amount suitable to maintain the pH of the formulation at a predetermined level.
  • compositions of the invention can also contain one or more osmotic modulating agents, i.e., a compound that modulates the osmotic properties (e.g, tonicity, osmolality and/or osmotic pressure) of the formulation to a level that is acceptable to the blood stream and blood cells of recipient individuals.
  • the osmotic modulating agent can be an agent that does not chelate calcium ions.
  • the osmotic modulating agent can be any compound known or available to those skilled in the art that modulates the osmotic properties of the formulation. One skilled in the art may empirically determine the suitability of a given osmotic modulating agent for use in the inventive formulation.
  • osmotic modulating agents include, but are not limited to: salts, such as sodium chloride and sodium acetate; sugars, such as sucrose, dextrose, and mannitol; amino acids, such as glycine; and mixtures of one or more of these agents and/or types of agents.
  • the osmotic modulating agent(s) may be present in any concentration sufficient to modulate the osmotic properties of the formulation.
  • compositions comprising a compound of the present invention can contain multivalent metal ions, such as calcium ions, magnesium ions and/or manganese ions. Any multivalent metal ion that helps stabilizes the composition and that will not adversely affect recipient individuals may be used. The skilled artisan, based on these two criteria, can determine suitable metal ions empirically and suitable sources of such metal ions are known, and include inorganic and organic salts.
  • compositions of the invention can also be a non-aqueous liquid formulation.
  • Any suitable non-aqueous liquid may be employed, provided that it provides stability to the active agents (s) contained therein.
  • the non-aqueous liquid is a hydrophilic liquid.
  • non-aqueous liquids include: glycerol; dimethyl sulfoxide (DMSO); polydimethylsiloxane (PMS); ethylene glycols, such as ethylene glycol, diethylene glycol, triethylene glycol, polyethylene glycol ("PEG") 200, PEG 300, and PEG 400; and propylene glycols, such as dipropylene glycol, tripropylene glycol, polypropylene glycol ( 11 PPG") 425, PPG 725, PPG 1000, PPG 2000, PPG 3000 and PPG 4000.
  • Pharmaceutical compositions of the invention can also be a mixed aqueous/non- aqueous liquid formulation.
  • any suitable non-aqueous liquid formulation such as those described above, can be employed along with any aqueous liquid formulation, such as those described above, provided that the mixed aqueous/non-aqueous liquid formulation provides stability to the compound contained therein.
  • the non- aqueous liquid in such a formulation is a hydrophilic liquid.
  • suitable non-aqueous liquids include: glycerol; DMSO; PMS; ethylene glycols, such as PEG 200, PEG 300, and PEG 400; and propylene glycols, such as PPG 425, PPG 725, PPG 1000, PPG 2000, PPG 3000 and PPG 4000.
  • Suitable stable formulations can permit storage of the active agents in a frozen or an unfrozen liquid state.
  • Stable liquid formulations can be stored at a temperature of at least - 70 0 C, but can also be stored at higher temperatures of at least 0 0 C, or between about 0.1 0 C and about 42°C, depending on the properties of the composition. It is generally known to the skilled artisan that proteins and polypeptides are sensitive to changes in pH, temperature, and a multiplicity of other factors that may affect therapeutic efficacy.
  • Other delivery systems can include time-release, delayed release or sustained release delivery systems. Such systems can avoid repeated administrations of compositions of the invention, increasing convenience to the subject and the physician.
  • Many types of release delivery systems are available and known to those of ordinary skill in the art. They include polymer base systems such as polylactides (U.S. Pat. No. 3,773,919; European Patent No. 58,481), poly(lactide-glycolide), copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyric acids, such as poly-D-(-)-3-hydroxybutyric acid (European Patent No.
  • sustained-release compositions include semi-permeable polymer matrices in the form of shaped articles, e.g., films, or microcapsules.
  • Delivery systems also include non-polymer systems that are: lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono- di- and tri-glycerides; hydrogel release systems such as biologically-derived bioresorbable hydrogel (i.e., chitin hydrogels or chitosan hydrogels); sylastic systems; peptide based systems; wax coatings; compressed tablets using conventional binders and excipients; partially fused implants; and the like.
  • lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono- di- and tri-glycerides
  • hydrogel release systems such as biologically-derived bioresorbable hydrogel (i.e., chitin hydrogels or chitosan hydrogels); sylastic
  • colloidal dispersion systems include lipid- based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
  • Liposomes are artificial membrane vessels, which are useful as a delivery vector in vivo or in vitro. Large unilamellar vessels (LUV), which range in size from 0.2 - 4.0 ⁇ m, can encapsulate large macromolecules within the aqueous interior and be delivered to cells in a biologically active form (Fraley, R., and Papahadjopoulos, D., Trends Biochem. Sci. 6: 77- 80).
  • LUV Large unilamellar vessels
  • Liposomes can be targeted to a particular tissue by coupling the liposome to a specific ligand such as a monoclonal antibody, sugar, glycolipid, or protein.
  • a specific ligand such as a monoclonal antibody, sugar, glycolipid, or protein.
  • Liposomes are commercially available from Gibco BRL, for example, as LIPOFECTINTM and LIPOFECTACETM, which are formed of cationic lipids such as N-[I -(2, 3 dioleyloxy)- propyl]-N, N, N-trimethylammonium chloride (DOTMA) and dimethyl dioctadecylammonium bromide (DDAB).
  • DOTMA N-[I -(2, 3 dioleyloxy)- propyl]-N, N, N-trimethylammonium chloride
  • DDAB dimethyl dioctadecylammonium bromide
  • Another type of vehicle is a biocompatible microparticle or implant that is suitable for implantation into a mammalian recipient.
  • exemplary bioerodible implants that are useful in accordance with this method are described in PCT International application no. PCT/US/03307 (Publication No. WO 95/24929, entitled “Polymeric Gene Delivery System”).
  • PCT/US/0307 describes biocompatible, preferably biodegradable polymeric matrices for containing an exogenous gene under the control of an appropriate promoter. The polymeric matrices can be used to achieve sustained release of the exogenous gene or gene product in the subject.
  • the polymeric matrix preferably is in the form of a microparticle such as a microsphere (where an agent is dispersed throughout a solid polymeric matrix) or a microcapsule (wherein an agent is stored in the core of a polymeric shell).
  • a microparticle such as a microsphere (where an agent is dispersed throughout a solid polymeric matrix) or a microcapsule (wherein an agent is stored in the core of a polymeric shell).
  • Microcapsules of the foregoing polymers containing drugs are described in, for example, U.S. Patent 5,075,109.
  • Other forms of the polymeric matrix for containing an agent include films, coatings, gels, implants, and stents.
  • the size and composition of the polymeric matrix device is selected to result in favorable release kinetics in the tissue into which the matrix is introduced.
  • the size of the polymeric matrix further is selected according to the method of delivery that is to be used.
  • Both non-biodegradable and biodegradable polymeric matrices can be used to deliver the compositions of the invention to the subject.
  • Such polymers may be natural or synthetic polymers.
  • the polymer is selected based on the period of time over which release is desired, generally in the order of a few hours to a year or longer. Typically, release over a period ranging from between a few hours and three to twelve months is most desirable.
  • the polymer optionally is in the form of a hydrogel that can absorb up to about 90% of its weight in water and further, optionally is cross-linked with multivalent ions or other polymers.
  • Exemplary synthetic polymers which can be used to form the biodegradable delivery system include: polyamides, polycarbonates, polyalkylenes, polyalkylene glycols, polyalkylene oxides, polyalkylene terepthalates, polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, poly- vinyl halides, polyvinylpyrrolidone, polyglycolides, polysiloxanes, polyurethanes and co-polymers thereof, alkyl cellulose, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitro celluloses, polymers of acrylic and methacrylic esters, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxy-propyl methyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxylethyl cellulose, cellulose triacetate
  • a method of treatment is selected.
  • the level of-IL-6 present in a biological sample such as CSF, is used in selecting a treatment method.
  • less aggressive neuroinflammatory condition have lower IL-6 levels (e.g., less than 50 pg/ml CSF) than neuroinflammatory conditions requiring more aggressive therapies.
  • the level of IL-6 present in a biological sample is correlated with a clinical outcome. Levels of IL-6 greater than 50 pg/ml typically correlate with poor clinical outcomes, and are indicative of a risk for relapse. Accordingly, more aggressive therapeutic interventions are warranted.
  • Aggressive therapeutic interventions include, but are not limited to, combinations of one or more of the following: intravenous steroids (e.g., corticosteroids, methylprednisone, dexamethasone) intravenous IgG therapy, plasmapharesis, cyclophosphamide, thalidomide, cellsept, minocycline, and IL- 6 antibody or other cytokine therapy.
  • intravenous steroids e.g., corticosteroids, methylprednisone, dexamethasone
  • IgG therapy e.g., plasmapharesis, cyclophosphamide, thalidomide, cellsept, minocycline, and IL- 6 antibody or other cytokine therapy.
  • Aggressive therapies are associated with a variety of adverse side-effects. Thus, where less aggressive therapies may be used, such therapies are preferred (e.g., for patients having a good prognosis and low risk of relapse). Levels of IL-6
  • less aggressive therapeutic interventions are appropriate because such patients are less likely to sustain severe losses in neurological function or to experience relapse.
  • Less aggressive therapeutic interventions include treatment with intravenous steroids alone.
  • a clinician will look at whether or not levels of NO or IL-6 are reduced in response to therapy. If no reduction in levels is seen in response to therapy (e.g., aggressive or less aggressive therapy) then the patient is likely to experience a relapse. For example, a patient whose levels of IL-6 fail to drop below about 10 pg/ml CSF and/or whose levels of NO fail to drop below about 5-10 ⁇ M is identified as at risk for a relapse.
  • More aggressive therapy is indicated for patients identified as at risk of neuroinflammatory relapse.
  • the level of NO is measured.
  • Indirect measures for NO include measuring total nitrate and nitrite species, for example, using a commercially available kit from R & D (Minneapolis, MN)- The level of NO present in a reference is virtually undetectable in a standard assay (e.g., Griess assay), for example, less than about l ⁇ M.
  • Levels of NO greater than about 5 ⁇ M are indicative of neuroinflammation; levels greater than about 15-20 ⁇ M total nitrate are indicative of poor prognosis; and indicate that an aggressive therapy should be selected.
  • the diagnostic methods of the invention are also useful for monitoring the course of a neuroinflammatory condition in a patient, for assessing the efficacy of a therapeutic regimen, or for assessing the risk of relapse.
  • the diagnostic methods of the invention are used periodically to monitor the IL-6 or NO levels present in a biological sample of a patient.
  • the neuroinflammatory condition is characterized using a diagnostic assay of the invention prior to administering therapy. This assay provides a baseline that describes the level of IL-6 present in a biological sample of the subject prior to treatment. Additional diagnostic assays are administered during the course of therapy to monitor the efficacy of a selected therapeutic regimen or the likelihood of a neuroinflammatory relapse.
  • a therapy is identified as efficacious when a diagnostic assay of the invention detects a decrease in IL-6 levels, particularly where levels are reduced to less than 35-50 pg/ml, less than 20-35 pg/ml, less than 5-20 pg/ml, and most preferably to about 1-5 pg/ml, or even to 1-2 pg/ml.
  • a therapy that reduces levels of NO to less than about 15 ⁇ M, 10 ⁇ M, or 5 ⁇ M is efficacious. More preferably, levels of NO are reduced to less than about 1-5 ⁇ M.
  • the methods of the invention may be used for microarray-based assays that provide for the high-throughput analysis of IL-6 and other cytokine levels.
  • Useful substrate materials include membranes, composed of paper, nylon or other materials, filters, chips, glass slides, and other solid supports. The ordered arrangement of the array elements allows hybridization patterns and intensities to be interpreted as methylation levels of particular genes.
  • an antibody is fixed to a substrate to allow detection of a bound cytokine antigen.
  • IL-6 and other cytokines are detected in a biological sample using any method known in the art.
  • kits for the treatment or prevention of a neuroinflammatory condition such as transverse myelitis, neuritis optica, multiple sclerosis, and neuromyelitis optica.
  • the kit includes a pharmaceutical pack comprising an effective amount of a JAK/STAT signal transduction pathway inhibitor, a PARP inhibitor, and/or an iNOS inhibitor.
  • the compositions are present in unit dosage form, hi some embodiments, the kit comprises a sterile container which contains a therapeutic or prophylactic composition; such containers can be boxes, ampules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art.
  • Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments.
  • compositions of the invention or combinations thereof are provided together with instructions for administering them to a subject having or at risk of developing a neuroinflammatory condition (e.g., transverse myelitis, neuritis optica, multiple sclerosis, and neuromyelitis optica) or a neuroinflammatory relapse.
  • the instructions will generally include information about the use of the compounds for the treatment or prevention of a neuroinflammatory condition or a neuroinflammatory relapse.
  • the instructions include at least one of the following: description of the compound or combination of compounds; dosage schedule and administration for treatment of a neuroinflammatory condition or symptoms thereof; precautions; warnings; indications; counter-indications; overdosage information; adverse reactions; animal pharmacology; clinical studies; and/or references.
  • the instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container.
  • kits for the monitoring of a level of IL-6 present in a biological sample obtained from a subject includes at least one antibody or other detection agent whose detection of IL-6 determines the level of IL-6 present in a biological sample, together with instructions for using the antibody to identify a neuroinflammatory condition.
  • the kit further comprises a detection agent suitable for measuring the level of NO by measuring nitrates and nitrites (e.g., Griess reaction) in a biological sample.
  • the kit comprises a sterile container which contains the primer or probe; such containers can be boxes, ampules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container form known in the art.
  • Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding nucleic acids.
  • the instructions will generally include information about the use of an IL-6 antibody or other detection agent and their use in diagnosing a neuroinflammatory condition.
  • the kit further comprises any one or more of the reagents described in the diagnostic assays described herein.
  • the instructions include at least one of the following: description of the antibody; methods for using the enclosed materials for the diagnosis of a neuroinflammatory condition; precautions; warnings; indications; clinical or research studies; and/or references.
  • the instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container.
  • compositions of the invention for the treatment of a neuroinflammatory condition or neuroinflammatory relapse are administered in combination with any one or more of the following: azathioprine (Imuran), methotrexate, Rituximab, prednisone, or mycophenolate mofetil (CellCept), immune modulators (e.g., interferon, such as Avonex, Betaseron, or Rebif) or glatiramer acetate (Copaxone); steroids, such as corticosteroid medications (e.g., methylprednisone or dexamethasone, methylprednisolone), lioresal (Baclofen), tizanidine (Zanaflex), or a benzodiazepine to reduce muscle spasticity, cholinergic medications for incontinence, antidepressants for mood or behavior symptoms, amantadine for fatigue.
  • plasma exchange plasma exchange
  • Example 1 IL-6 levels are selectively and dramatically elevated in the CSF of transverse myelitis patients.
  • TM transverse myelitis
  • Two TM patients died of respiratory failure, and immunohistochemical staining of the spinal cord was performed at autopsy in order to define the source(s) of IL-6.
  • CSF IL-6 levels reported here are among the highest reported in any human disease (up to 4,209 pg/ml), related disorders have also been found to have elevated IL-6 within the CNS.
  • Acute disseminated encephalomyelitis, like TM, is a monophasic, inflammatory disorder of the CNS that is often post-infectious (29).
  • IL-6 is involved in the pathogenesis of MS, since patients have elevated IL-6 in the CSF (30) and within plaques (31, 32) and elevated numbers of IL-6- expressing monocytes within the blood and CSF (30).
  • Example 2 IL-6 levels correlate with total NO production and tissue injury in the spinal cord.
  • CSF was added from a TM patient (with IL-6 of 1,997 pg/ml) or a control patient onto spinal cord organotypic culture sections and cell death was evaluated by determining the percentage of cells that took up the red, vital dye propidium iodide. It was found that CSF from the TM patient induced death of spinal cord cells (33% ⁇ 12%), while CSF from a control patient with hydrocephalus did not (4% ⁇ 2.4%, P ⁇ 0.01 ; Figure 2A, black bars and photomicrograph inset).
  • RT- PCR analysis of spinal cord organotypic cultures confirmed a marked upregulation of iNOS RNA at 4 hours after IL-6 administration ( Figure 2D), demonstrating that the upregulation of iNOS occurs at the level of transcription.
  • Immunofluorescent examination of spinal cord organotypic cultures revealed a dramatic upregulation of iNOS within microglial cells (defined by the ability to phagocytose fluorescent acetylated low-density lipoprotein [DiI-Ac- LDL]; Figure 2E) and the accumulation of both iNOS and nitrotyrosine immunoreactivity, especially within the peripheral white matter of the spinal cord ( Figure 2F), in response to IL- 6 administration.
  • Example 4 Targets of IL-6-mediated spinal neural injury.
  • NT a commonly utilized marker of cellular injury due to excess NO (26), with other cellular markers. It was found that nitrotyrosine immunoreactivity was diffusely increased within DL-6— treated tissue ( Figure 3, A, C, and E).
  • astrocytes defined by glial fibrillary acidic protein [GFAP] immunoreactivity
  • GFAP glial fibrillary acidic protein
  • Figure 3A oligodendrocytes (defined by receptor interacting protein [RIP] immunoreactivity; Figure 3, B and C) and axons (defined by neurofilament immunoreactivity; Figure 3,D and 3E) did, and were therefore preferentially susceptible to IL-6-induced injury.
  • Figure 3F When dissociated cultures of pure spinal neurons were generated, it was found that IL-6 did not induce cellular death at any of the studied time points or doses
  • Example 5 Spinal cord from IL-6-infused rats and TM patients exhibit demyelination and axonal degeneration.
  • IL-6 or vehicle was infused through a spinal subarachnoid catheter.
  • IL-6 in response to direct stimulation by proinflammatory cytokines (e.g., TNF- ⁇ and IL- l ⁇ ), viral and bacterial pathogens, and neurotransmitters.
  • proinflammatory cytokines e.g., TNF- ⁇ and IL- l ⁇
  • viral and bacterial pathogens e.g., IL-6
  • neurotransmitters e.g., IL-6, IL-6, neurotransmitters.
  • IL-6-mediated cytotoxicity include oligodendrocytes and axons.
  • the finding of nitrotyrosine accumulation in axons argues for a direct neuropathic effect of IL-6 in mediating neural injury, as opposed to axonal degeneration that is solely the result of demyelination.
  • Example 6 Activation of PARP is necessary for cell death.
  • IL-6 is capable of mediating JAK/STAT signaling pathways other than JAK2/STAT3 and Ras/MEK/MAPK (11), a speculation is that that PARP has a role in signal transduction.
  • Recent evidence from PARP KO mice suggests a role for PARP in enhancing p38/MAPK signaling in response to inflammatory stimulation through lipopolysaccharide (LPS) (41).
  • LPS lipopolysaccharide
  • the early-phase IL- 6-mediated PARP activation described here plays a role in the augmentation of p38/MAPK signaling.
  • Spinal cord iNOS activation is not due to early PARP activation, because it is unaffected by PARP inhibition.
  • Example 7 Regional vulnerability of the spinal cord relative to the brain.
  • IL-6R is similarly expressed within the brain and spinal cord, indicating that IL-6R density does not account for relative susceptibility of spinal cord tissue.
  • IL-6R density does not account for relative susceptibility of spinal cord tissue.
  • the expression of sIL-6R was examined, and it was found to have greater expression in cortical tissue lysates than in spinal cord tissue lysates (Figure 6G).
  • sIL-6R is a critical molecule that dampens the tissue response to IL-6.
  • spatially restricted responses to cytokines including IL-6 may underlie the restricted inflammation seen in a variety of CNS inflammatory disorders, including regionally limited forms of MS, TM, neuromyelitis optica, and optic neuritis.
  • IL-6 family members in preventing cell death as well as potentially playing a causative role in neurodegenerative diseases (2). This discrepancy may result from differences in downstream signaling among IL-6 and its family members, and differing effects in distinct experimental paradigms.
  • the protective or destructive actions of IL-6 may also result from selective dose and regional effects.
  • the results here showed that IL-6 causes preferential cytotoxicity in white matter compared to gray matter in the spinal cord. It was also found that low doses of IL-6 prevented cell death in organotypic cultures of sections from the hippocampus or cortex, whereas higher doses had little effect on cell death. In contrast, no IL-6 dose tested in spinal cord sections was found to be protective, and higher doses were extremely cytotoxic.
  • IL-6 treatment of cortical organotypic cultures did not cause significant PARP activation, iNOS induction, or STAT phosphorylation suggests that the regional selectivity of IL-6 response occurs at the level of IL-6R and not its downstream signaling pathway.
  • the increased abundance of IL-6R in white compared to grey matter which may be due to increased abundance of IL-6R on oligodendrocytes, may account for the preferential white matter injury seen both in vitro and in vivo following IL-6 application.
  • it is the higher concentration of sIL-6R that may explain the increased susceptibility of the spinal cord to IL-6.
  • sIL-6R in preventing cell death
  • IL-6 is bound and isolated by sIL-6R or that the IL-6/sIL-6R complex initiates signaling through alternative pathways in cells without IL-6R (42).
  • Biological markers of regional selectivity within the CNS has been seen previously, such as the selective clearance of alphavirus from spinal cord relative to the cortex (43) and the selective vulnerability of spinal cord neurons to excitotoxic injury when compared to cortical neurons.
  • the results presented herein demonstrate that a single signaling molecule can be a determinant of patient outcome in TM. The implications of these findings are that therapeutic strategies capable of modulating this pathway may improve outcomes in TM patients.
  • the data presented herein provides direct evidence for a signaling cascade involving IL-6, iNOS, and PARP proteins that accounts for the clinicopathologic findings in inflammatory spinal neurodegeneration. Since spinal cord dysfunction is a major determinant of disability in several neurologic disorders including TM and MS, the elucidation of this pathway identifies important therapeutic targets for preventing these and other neurologic disorders or disabilities in the future.
  • Idiopathic TM patients were defined according to previously published criteria (1). Those who did not meet the inflammatory criteria were classified as noninflammatory controls. Spinal fluid was collected, immediately placed on ice, centrifuged at 1,000 g to remove cellular elements, and stored at -80 0 C.
  • Cytokine antibody arrays were purchased (TranSignal RayBio Human Cytokine Antibody Array 3, catalog no. MA6020; Panomics Inc.) and used according to package inserts. For each blot, 1,000 ⁇ l of CSF was used. Signal was analyzed and quantitated by using a Fuji chemiluminescent detection system. Quantitative IL-6 ELISA assay kits and total NO assay kits were purchased from R&D Systems and the LIVE/DEAD Viability/ Cytotoxicity Kit was purchased from Invitrogen Corp.; all were used according to the manufacturers' instructions. All samples were measured in triplicate and average values were determined. Total nitrite concentrations in supernatants collected from in vitro co-culture experiments were determined by using the Total NO Kit (catalog no.
  • Recombinant rat IL-6 (catalog no. 557008; BD Biosciences — Pharmingen) was made as a 2 ⁇ g/ml stock in 1 mg/ml BSA in PBS and used at final concentrations of 500 pg/ml and 2,000 pg/ml.
  • iNOS inhibitor 1400W dihydrochloride (catalog no. ALX-270-073; Alexis Biochemicals) was made at 100 mM stock in water and used at a final concentration of 100 ⁇ M. 4- ANI (catalog no.
  • Antibodies and dilutions used in this study include the following: PhosphoPlus Stat3 (Tyr705) Antibody Kit (1:1,000; Cell Signaling Technology); iNOS/NOS Type II (1:10,000; BD Biosciences); anti-NT, clone 1A6 (1:1,000; Upstate); PARP antibody (1:75; Cell Signaling Technology), biotinylated anti-human EL-6R antibody (1:2,500; R&D Systems), RJP (SMI 91; Sternberger Monoclonals), neurofilament (NF), Heavy Chain (SMI 31/32; Sternberger Monoclonals), and GFAP (1:400, catalog no. MAB360, or 1:2,000, catalog no. AB5804; Chemicon).
  • CSF samples from TM patients were immunodepleted with anti-human IL-6 antibodies (catalog no. ab6672; Novus Biologicals), precipitated with protein A-coated beads, and verified for IL-6 depletion by human IL-6 ELISA assay.
  • Microglial cultures were isolated from adult rats as described by Babas et al. (44), with a few modifications. The cells were derived from the cortical regions of 3 adult Lewis rat brains. Processed tissues were passed through an 18-gauge needle on a 10-CC syringe and filtered over 70- ⁇ m nylon cell strainers (Falcon, catalog no. 352350; BD Biosciences) twice to ensure a single cell suspension. Cells were plated at a density of 4 x 106 cells/well. The next day, cells were washed and changed to fresh growth medium. Cells were maintained for a week before experimentation. Microglial cell purity (at least 90%) was verified by fluorescent staining of microglia with Iba-1 (1:150, catalog no. 01-1974; Wako) and the absence of staining with GFAP.
  • Rats with spinal cannulas were purchased from Zivic Laboratories Inc. These rats had cannulas placed into the cisterna magna and extended caudally through the subarachnoid space with the cannula tip terminating adjacent to the T8 vertebral body. Cannulated rats were anesthetized with avertin, an incision was made behind the head, and the subarachnoid cannula was connected to an Alzet pump (1007D, 0.5 ⁇ l/hr for 7 days). Pumps were filled with 100 ⁇ l IL-6 at 2 ⁇ g/ml or saline. Animals were coded and housed individually.
  • Novelty-induced food intake was utilized as a measure of animal anxiety to determine whether IL-6 induced any change in this measure.
  • the testing apparatus consisted of a Plexiglas box (60 x 60 x 50 cm). Forty-eight hours before testing, all food was removed from the home cage. For testing, 3-4 food pellets were placed in the center of the testing box and an animal was placed into a randomly chosen corner of the testing box and a stopwatch was immediately started. The latency to begin eating was measured and was defined as chewing the food, not simply sniffing or playing with a pellet. If a rat had not eaten within 360 seconds, the test was stopped and the animal was assigned a latency score of 360 seconds and the animal was returned to the home cage. The data for novelty-induced food intake inhibition were analyzed using Student t-test.
  • SPSS software version 12.0; SPSS Inc.
  • Box plots were used to represent the distribution of the data. The outliers shown are outside the fifth and ninety-fifth percentile. Correlations were assessed by Spearman's rank correlation coefficient due to the ordinal nature of the data. Group differences in EDSS and IL-6 levels and between CSF and serum IL-6 levels in the different patient groups were compared using the Mann- Whitney U test due to the non-Gaussian appearance of the data. A P value less than 0.05 was considered significant. Due to the nonparametric nature of the data (as determined by using tests of normality), nonparametric equivalent tests of ANOVA and repeated- measures ANOVA were used to increase the robustness of the results. The Kruskal- Wallis test was performed to analyze differences among groups at each time point, and Friedman's nonparametric repeated measures comparison was used to analyze differences across time within a group. The Mann- Whitney U test was used for the comparison of 2 independent samples.
  • JAK2/STAT3 not ERK1/2, mediates interleukin-6-induced activation of inducible nitric-oxide synthase and decrease in contractility of adult ventricular myocytes. J. Biol. Chem. 278:16304-16309.

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Abstract

La présente invention concerne des compositions et des procédés qui sont utiles pour le diagnostic et le traitement de troubles neuro-inflammatoires (par ex., la myélite transverse, la sclérose en plaques, la neurite optique, la neuromyélite optique).
PCT/US2007/017376 2006-08-02 2007-08-02 Utilisation de l'il-6 pour le diagnostic et le traitement de pathologies neuro-inflammatoires WO2008016707A2 (fr)

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US7956064B2 (en) * 2006-09-01 2011-06-07 Cylene Pharmaceuticals, Inc. Fused tricyclic compounds as serine-threonine protein kinase and PARP modulators
EP3101426A3 (fr) * 2010-12-16 2017-02-08 National Institute of Advanced Industrial Science And Technology Procédé d'enrichissement et de séparation de fluide spinal glycoprotéine, procédé de recherche de marqueur pour les maladies du système nerveux central qui utilise le procédé susmentionné et marqueur pour les maladies du système nerveux central

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Title
MILIKOVIC ET AL.: 'Nitric oxide metabolites and interleukin-6 in cerebrospinal fluid from multiple sclerosis patients' EUR. J. NEUROL. vol. 9, no. 4, July 2002, pages 413 - 418 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7956064B2 (en) * 2006-09-01 2011-06-07 Cylene Pharmaceuticals, Inc. Fused tricyclic compounds as serine-threonine protein kinase and PARP modulators
US9062043B2 (en) 2006-09-01 2015-06-23 Senhwa Biosciences, Inc. Fused tricyclic compounds as serine-threonine protein kinase and PARP modulators
EP3101426A3 (fr) * 2010-12-16 2017-02-08 National Institute of Advanced Industrial Science And Technology Procédé d'enrichissement et de séparation de fluide spinal glycoprotéine, procédé de recherche de marqueur pour les maladies du système nerveux central qui utilise le procédé susmentionné et marqueur pour les maladies du système nerveux central

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