WO2015051337A2 - Méthodes de traitement de troubles gastro-intestinaux - Google Patents

Méthodes de traitement de troubles gastro-intestinaux Download PDF

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WO2015051337A2
WO2015051337A2 PCT/US2014/059190 US2014059190W WO2015051337A2 WO 2015051337 A2 WO2015051337 A2 WO 2015051337A2 US 2014059190 W US2014059190 W US 2014059190W WO 2015051337 A2 WO2015051337 A2 WO 2015051337A2
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tnf
inhibitor
npy
levels
selective
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PCT/US2014/059190
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WO2015051337A3 (fr
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Shanthi Srinivasan
Bindu CHANDRASEKHARAN
Malu Tansey
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Emory 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
    • G01N33/6863—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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/191—Tumor necrosis factors [TNF], e.g. lymphotoxin [LT], i.e. TNF-beta
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/02—Drugs for disorders of the nervous system for peripheral neuropathies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/06—Gastro-intestinal diseases
    • G01N2800/065—Bowel diseases, e.g. Crohn, ulcerative colitis, IBS

Definitions

  • the present invention relates generally to the administration of a TNF-a inhibitor, preferably a dominant negative TNF-a protein for the treatment of a gastrointestinal disorder associated with reduced neuronal survival and decreased colonic motility.
  • Gastrointestinal (GI) disorders can be grouped into those characterized by inflammation and those that are not. Inflammatory GI disorders include Crohn's disease and inflammatory bowel disease. GI disorders not typically associated with inflammation include irritable bowel syndrome (IBS) and similar disorders. Irritable bowel syndrome is a common disorder that affects the large intestine ⁇ colon) and commonly causes cramping, abdominal pain, bloating gas, diarrhea and constipation, yet does not appear to cause permanent damage to the colon.
  • IBS irritable bowel syndrome
  • TBS is a problematic and uncomfortable GI disorder not caused by inflammation.
  • the present disclosure provides for the first time a treatment of gastrointestinal disorders characterized by increased neuronal survival and colonic cell motility. Accordingly, the disclosure provides a method of improving enteric nerve cell survival in a patient in need thereof exhibiting symptoms of colitis, said method comprising administering to a patient in need thereof a TNF-a inhibitor that reduces neuropeptide -Y (NPY), whereby said patient exhibits improved symptoms of colitis.
  • a TNF-a inhibitor that reduces neuropeptide -Y (NPY), whereby said patient exhibits improved symptoms of colitis.
  • the TNF-a inhibitor is XProl595.
  • following the administration the patient exhibits improved colonic cell motility.
  • the patient prior to the administering, the patient exhibits symptoms associated with irritable bowel syndrome.
  • the symptoms are selected from the group consisting of dyspepsia, bloating, abdominal pain, diarrhea and constipation.
  • the patient following the administration, the patient exhibits reduced claudin-2 expression relative to claudin-2 levels prior to said administration.
  • the disclosure provides a method comprising detecting levels of NPY in a patient suffering from intestinal disorder and administering to the patient a TNF-a inhibitor if levels of NPY are elevated relative to NPY levels in an unafflicted control subject.
  • the disclosure also provides a method of decreasing tight junction permeability comprising administering a TNF-a inhibitor to a patient in need thereof.
  • the method further comprises detecting levels of NPY in a patient suffering from a gastrointestinal disorder and administering to the patient a TNF-a inhibitor if levels of NPY are elevated relative to NPY levels in an unafflicted control subject.
  • the tight junctions are blood brain barrier or GI tight junctions.
  • the disclosure provides a method of diagnosing a disorder associated with GI motility comprising detecting levels of CRP, TNF-a, NPY, claudin-2 or any combinations thereof in a subject, wherein elevated levels of CRP, TNF-a, NPY, claudin-2 or any combinations thereof relative to normal controls indicates the presence of a disorder associated with GI motility in the subject.
  • the disorder associated with GI motility is selected from the group consisting of irritable bowel syndrome and Parkinson's disease.
  • the disclosure also provides a method of treating a subject in need thereof comprising detecting levels of CRP, TNF-a, NPY, claudin-2 or any combinations thereof in a subject, wherein elevated levels of CRP, TNF-a, NPY, claudin-2 or any combinations thereof relative to normal controls indicates the presence of a disorder associated with GI motility in said subject; and administering to said subject a TNF-a inhibitor.
  • the selective inhibitor of TNF-a reduces detectable levels of CRP, TNF-a, claudin-2 or any combinations thereof more than a non-selective TNF-a inhibitor.
  • the selective inhibitor of TNF-a reduces NPY expression more than a non-selective TNF-a inhibitor.
  • the TNF-a inhibitor is a selective inhibitor that inhibits soluble TNF-a but not transmembrane TNF-a.
  • the dominant negative TNF-a inhibitor is a dominant negative TNF-a polypeptide, which more preferably comprises a variant sequence relative to wild-type TNF-a, and which may be PEGylated.
  • Such variant sequence may comprise the amino acid substitutions A145R/I97T or
  • the dominant negative TNF-a polypeptide is XProl595.
  • TNF-a inhibitors for use in reducing neuropeptide -Y (NPY), improving enteric nerve cell survival, and improving symptoms of colitis.
  • the TNF-a inhibitor is for use in improving colonic cell motility.
  • the TNF-a inhibitor is for use in improving symptoms associated with irritable bowel syndrome.
  • the symptoms are selected from the group consisting of dyspepsia, bloating, abdominal pain, diarrhea and constipation.
  • the TNF-a inhibitor is for use in reducing claudin-2 expression.
  • the TNF-a inhibitor is a selective inhibitor that inhibits soluble but not transmembrane TNF-a.
  • the selective inhibitor reduces NPY expression more than a non-selective TNF-a inhibitor.
  • the selective inhibitor of TNF-a comprises VIM, R31C, C69V, Y87H, CIOIA, and A145R mutations relative to wild-type TNF-a.
  • the selective inhibitor of TNF-a is PEGylated.
  • the selective inhibitor of TNF-a is XProl595.
  • TNF-a inhibitors for use in treating a gastrointestinal disorder in a patient having levels of NPY elevated relative to NPY levels in an unafflicted control subject. Also provided are TNF-a inhibitors for use in decreasing tight junction permeability. In some embodiments, the TNF-a inhibitor is for use in decreasing tight junction permeability in a patient having levels of NPY elevated relative to NPY levels in an unafflicted control subject. In some embodiments, the tight junctions are blood brain barrier or GI tight junctions.
  • TNF-a inhibitors for use in diagnosing a disorder associated with GI motility, in a patient having elevated levels of CRP, TNF-a, NPY, claudin-2 or any combinations thereof relative to normal controls.
  • the disorder associated with GI motility is selected from the group consisting of irritable bowel syndrome and Parkinson's disease.
  • TNF-a inhibitors for use in treating a disorder associated with GI motility, in a patient having elevated levels of CRP, TNF-a, NPY, claudin-2 or any combinations thereof relative to normal controls.
  • the TNF-a inhibitor is a selective inhibitor that inhibits soluble but not transmembrane TNF-a.
  • the selective inhibitor of TNF-a reduces detectable levels of CRP, TNF-a, claudin-2 or any combinations thereof more than a non-selective TNF-a inhibitor.
  • the selective inhibitor of TNF-a comprises VIM, R31C, C69V, Y87H, CIOIA, and A145R mutations relative to wild-type TNF-a. In some embodiments, the selective inhibitor of TNF-a is PEGylated. In some
  • the selective inhibitor of TNF-a is XProl595.
  • TNF-a inhibitors in the manufacture of a medicament for improving enteric nerve cell survival, reduces neuropeptide -Y (NPY), and improving symptoms of colitis.
  • the medicament is for improving colonic cell motility.
  • the medicament is for improving symptoms associated with irritable bowel syndrome.
  • the symptoms are selected from the group consisting of dyspepsia, bloating, abdominal pain, diarrhea and constipation.
  • the medicament is for reducing claudin-2 expression.
  • the TNF-a inhibitor is a selective inhibitor that inhibits soluble but not transmembrane TNF-a.
  • the selective inhibitor of TNF-a reduces NPY expression more than a non-selective TNF-a inhibitor.
  • the selective inhibitor of TNF-a comprises VIM, R31C, C69V, Y87H, CIOIA, and A145R mutations relative to wild-type TNF-a.
  • the selective inhibitor of TNF-a is PEGylated.
  • selective inhibitor of TNF-a is XProl595.
  • TNF-a inhibitors in the manufacture of a medicament for treating a gastrointestinal disorder in a patient having levels of NPY elevated relative to NPY levels in an unafflicted control subject. Also provided are uses of TNF-a inhibitors in the manufacture of a medicament for decreasing tight junction permeability. In some embodiments, the medicament is for decreasing tight junction permeability in a patient having levels of NPY elevated relative to NPY levels in an unafflicted control subject. In some embodiments, the tight junctions are blood brain barrier or GI tight junctions.
  • TNF-a inhibitors in the manufacture of a medicament for diagnosing a disorder associated with GI motility, in a patient having elevated levels of CRP, TNF- a, NPY, claudin-2 or any combinations thereof relative to normal controls.
  • the disorder associated with GI motility is selected from the group consisting of irritable bowel syndrome and Parkinson's disease.
  • TNF-a inhibitors in the manufacture of a medicament for treating a disorder associated with GI motility, in a patient having elevated levels of CRP, TNF-a, NPY, claudin-2 or any combinations thereof relative to normal controls.
  • the TNF- ⁇ inhibitor is a selective inhibitor that inhibits soluble but not transmembrane TNF-a.
  • the selective inhibitor of TNF-a reduces detectable levels of CRP, TNF-a, claudin-2 or any combinations thereof more than a non-selective TNF-a inhibitor.
  • the selective inhibitor of TNF-a comprises VIM, R31C, C69V, Y87H, CIOIA, and A145R mutations relative to wild-type TNF-a.
  • the selective inhibitor of TNF-a is PEGylated.
  • the selective inhibitor of TNF-a is XProl595.
  • FIG. 1A shows the nucleic acid sequence of human TNF-a (SEQ ID NO:l). An additional six histidine codons, located between the start codon and the first amino acid, are underlined.
  • FIG. IB shows the amino acid sequence of human TNF-a (SEQ ID NO:2) with an additional 6 histidines (underlined) between the start codon and the first amino acid. Amino acids changed in exemplary TNF-a variants are shown in bold.
  • FIG. 2 shows the positions and amino acid changes in certain TNF-a variants.
  • FIG. 3- Shows the amino acid sequence of human TNF-a (SEQ ID NO:3).
  • FIG. 4- NPY knockdown reduces TNF production from enteric neurons, and TNF activates NPY promoter.
  • Primary enteric neuronal cultures from two-day-old WT and NPT mice pups were cultured for 10 days at 37°C.
  • Colonic explants from eight- week-old WT and NPT mice were cultured in RPMI for 24 h.
  • the supematants from primary cultures and colon explants were assayed for TNF release by ELISA.
  • Enteric neuronal cell lines were stimulated with TNF (25 ng) in presence or absence of TNF inhibitors (25 ng).
  • RNA was extracted after 6 h and amplified for NPY expression by real-time PCR.
  • Enteric neuronal cell line was transfected with various NPY deletion promoter constructs and assayed for luciferase activity in presence and absence of TNF.
  • the chromatin from enteric neuronal cells treated with/ without TNF was immunoprecipitated with phospho-c-jun antibody.
  • Fold change in c-jun binding on NPY promoter was assessed by real-time PCR.
  • the site directed mutagenesis of c-jun binding site was performed using a kit as detailed in Methods, and TNF-induced promoter activity was monitored by luciferase assay in two mutant clones (Mut #1 & #2).
  • A Primary enteric neurons in culture under light microscope (20x)
  • B TNF levels in WT and NPY enteric neurons
  • C TNF levels in colon explants cultures from WT and NPY ' ' ' mice
  • D NPY expression in enteric neuronal cells treated with TNF and TNF inhibitors
  • E luciferase assay depicting activation of NPY promoter constructs by TNF
  • F Fold change in phospho-c-jun expression with TNF
  • FIG. 5- NPY increases permeability of colonic epithelial monolayer via pore-forming claudin-2.
  • WT and NPY ' ' ' mice were gavaged with FITC Dextran (FD4, 4 kD) and amount of FD4 released into the serum (ng / gm serum protein) was measured after 4 h in a spectrofluorimeter (excitation ⁇ 450 nm, emission ⁇ 510 nm).
  • FIG. 6- TNF inhibitors attenuate enteric neuronal NPY expression and inflammation in murine experimental colitis.
  • Mice were given DSS in drinking water for seven days, with three intraperitoneal injections of either etanercept or XProl595 at 10 mg/ kg body weight starting from day 1 as outlined in (A). On day 8, mice were euthanized and colon was collected. The paraffin- embedded colon sections were assessed for (B) NPY expression by immunostaining using peripherin as general neuronal marker, and the staining intensity arbitrarily quantified by
  • Metamorph is graphically represented in (C).
  • the colonic ganglia were isolated by LCM from cryo sections, and NPY expression was assessed by real-time PCR in (D).
  • FIGS. 7-1 and 7-2- TNF inhibitors reduce enteric neuronal apoptosis and enhance neuronal survival via attenuation of oxidative stress.
  • Mice were administered DSS in drinking water for seven days; they also received three intraperitoneal injections (every third day) of either etanercept or XProl595 at 10 mg/ kg body weight.
  • Oxidative stress was evaluated from inducible nitric oxide synthase (iNOS) and antioxidant catalase expression in mouse colonic mucosa by real time PCR. Paraffin-embedded colonic sections were immunostained for cleaved caspase-3, and peripherin was used as a general neuronal marker.
  • iNOS inducible nitric oxide synthase
  • FIG. 8- Administration of TNF inhibitors improves colonic motility in mice with colitis.
  • Mice were given DSS in drinking water for seven days, with three intraperitoneal injections of either etanercept or XProl595 at 10 mg/ kg body weight starting from day 1.
  • mice were euthanized and the longitudinal muscle strips peeled from the colon were subjected to isometric muscle recording (as detailed in Methods) with electrical field stimulation for assessment of neuronally-mediated colonic relaxation and contraction.
  • these muscle strips were stimulated with carbachol to assess smooth muscle contraction, and with sodium nitroprusside (SNP) to evaluate smooth muscle relaxation responses.
  • SNP sodium nitroprusside
  • TNF-a e.g., dominant negative TNFa proteins
  • TNF-a e.g., dominant negative TNFa proteins
  • the present invention addresses many of problems of prior art related to treatment of gastrointestinal disorders not associated with inflammation.
  • the present disclosure provides a method of treating gastrointestinal disorders not associated with inflammation, such as irritable bowel syndrome.
  • Preferred inhibitors of TNFa may be dominant negative TNFa proteins, referred to herein as "DNTNF-a,” “DN-TNF-a proteins,” “TNFa variants,” “TNFa variant proteins,” “variant TNF- a,” “variant TNF-a,” and the like.
  • DNTNF-a dominant negative TNFa proteins
  • a variant of human TNF-a is compared to SEQ ID NO: 1 (nucleic acid including codons for 6 histidines), SEQ ID NO:2 (amino acid including 6 N-terminal histidines) or SEQ ID NO:3 (amino acid without 6 N-terminal histidines).
  • DN-TNF-a proteins are disclosed in detail in U.S. Patent No. 7,446, 174, which is incorporated herein in its entirety by reference.
  • variant TNF-a or TNF-a proteins include TNF-a monomers, dimers or trimers. Included within the definition of "variant TNF-a" are competitive inhibitor TNF-a variants. While certain variants as described herein, one of skill in the art will understand that other variants may be made while retaining the function of inhibiting soluble but not transmembrane TNF-a.
  • the proteins of the invention are antagonists of wild type TNF-a.
  • antagonists of wild type TNF-a is meant that the variant TNF-a protein inhibits or significantly decreases at least one biological activity of wild-type TNF-a.
  • the variant is an antagonist of soluble TNF-a, but does not significantly antagonize transmembrane TNF-a, e.g. , DN-TNF-a protein as disclosed herein inhibits signaling by soluble TNF-a, but not transmembrane TNF-a.
  • inhibits the activity of TNF-a and grammatical equivalents is meant at least a 10% reduction in wild-type, soluble TNF-a activity, more preferably at least a 50% reduction in wild-type, soluble TNF-a activity, and even more preferably, at least 90% reduction in wild-type, soluble TNF-a activity.
  • transmembrane TNF-a In a preferred embodiment, the activity of soluble TNF-a is inhibited while the activity of transmembrane TNF-a is substantially and preferably completely maintained.
  • the TNF proteins of the invention have modulated activity as compared to wild type proteins.
  • variant TNF-a proteins exhibit decreased biological activity (e.g. antagonism) as compared to wild type TNF-a, including but not limited to, decreased binding to a receptor (p55, p75 or both), decreased activation and/or ultimately a loss of cytotoxic activity.
  • cytotoxic activity herein refers to the ability of a TNF-a variant to selectively kill or inhibit cells.
  • variant TNF-a proteins that exhibit less than 50% biological activity as compared to wild type are preferred. More preferred are variant TNF-a proteins that exhibit less than 25%, even more preferred are variant proteins that exhibit less than 15%, and most preferred are variant TNF-a proteins that exhibit less than 10% of a biological activity of wild-type TNF-a.
  • Suitable assays include, but are not limited to, caspase assays, TNF-a cytotoxicity assays, DNA binding assays, transcription assays (using reporter constructs, size exclusion chromatography assays and radiolabeling/immuno-precipitation), and stability assays (including the use of circular dichroism (CD) assays and equilibrium studies), according to methods know in the art.
  • At least one property critical for binding affinity of the variant TNF-a proteins is altered when compared to the same property of wild type TNF-a and in particular, variant TNF-a proteins with altered receptor affinity are preferred. Particularly preferred are variant TNF-a proteins with altered affinity toward oligomerization to wild type TNF-a.
  • the invention provides variant TNF-a proteins with altered binding affinities such that the variant TNF- ⁇ proteins will preferentially oligomerize with wild type TNF-a, but do not substantially interact with wild type TNF receptors, i.e., p55, p75.
  • “Preferentially” in this case means that given equal amounts of variant TNF-a monomers and wild type TNF-a monomers, at least 25% of the resulting trimers are mixed trimers of variant and wild type TNF-a, with at least about 50% being preferred, and at least about 80-90%) being particularly preferred.
  • the variant TNF-a proteins of the invention have greater affinity for wild type TNF-a protein as compared to wild type TNF-a proteins.
  • do not substantially interact with TNF receptors is meant that the variant TNF-a proteins will not be able to associate with either the p55 or p75 receptors to significantly activate the receptor and initiate the TNF signaling pathway(s).
  • at least a 50%> decrease in receptor activation is seen, with greater than 50%, 76%, 80-90% being preferred.
  • the variants of the invention are antagonists of both soluble and transmembrane TNF-a.
  • preferred variant TNF-a proteins are antagonists of the activity of soluble TNF-a but do not substantially affect the activity of transmembrane TNF-a.
  • a reduction of activity of the heterotrimers for soluble TNF-a is as outlined above, with reductions in biological activity of at least 10%, 25, 50, 75, 80, 90, 95, 99 or 100% all being preferred.
  • some of the variants outlined herein comprise selective inhibition; that is, they inhibit soluble TNF-a activity but do not substantially inhibit
  • transmembrane TNF-a it is preferred that at least 80%, 85, 90, 95, 98, 99 or 100%) of the transmembrane TNF-a activity is maintained. This may also be expressed as a ratio; that is, selective inhibition can include a ratio of inhibition of soluble to transmembrane TNF-a. For example, variants that result in at least a 10:1 selective inhibition of soluble to transmembrane TNF-a activity are preferred, with 50:1, 100:1, 200:1, 500:1, 1000:1 or higher find particular use in the invention.
  • one embodiment utilizes variants, such as double mutants at positions 87/145 as outlined herein, that substantially inhibit or eliminate soluble TNF-a activity (for example by exchanging with homotrimeric wild-type to form heterotrimers that do not bind to TNF-a receptors or that bind but do not activate receptor signaling) but do not significantly affect (and preferably do not alter at all) transmembrane TNF-a activity.
  • the variants exhibiting such differential inhibition allow the decrease of inflammation without a corresponding loss in immune response, or when in the context of the appropriate cell, without a corresponding demyelination of neurons.
  • the affected biological activity of the variants is the activation of receptor signaling by wild type TNF-a proteins.
  • the variant TNF-a protein interacts with the wild type TNF-a protein such that the complex comprising the variant TNF-a and wild type TNF-a has reduced capacity to activate (as outlined above for "substantial inhibition"), and in preferred embodiments is incapable of activating, one or both of the TNF receptors, i.e. p55 TNF-R or p75 TNF-R.
  • the variant TNF-a protein is a variant TNF-a protein that functions as an antagonist of wild type TNF-a.
  • the variant TNF-a protein preferentially interacts with wild type TNF-a to form mixed trimers with the wild type protein such that receptor binding does not significantly occur and/or TNF-a signaling is not initiated.
  • mixed trimers is meant that monomers of wild type and variant TNF-a proteins interact to form heterotrimeric TNF-a.
  • Mixed trimers may comprise 1 variant TNF-a protein:2 wild type TNF-a proteins, or 2 variant TNF-a proteins :1 wild type TNF-a protein.
  • trimers may be formed comprising only variant TNF-a proteins.
  • variant TNF-a antagonist proteins of the invention are highly specific for TNF-a antagonism relative to TNF-beta antagonism. Additional characteristics include improved stability, pharmacokinetics, and high affinity for wild type TNF-a. Variants with higher affinity toward wild type TNF-a may be generated from variants exhibiting TNF-a antagonism as outlined above.
  • variant TNF-a proteins for example are experimentally tested and validated in in vivo and in in vitro assays. Suitable assays include, but are not limited to, activity assays and binding assays.
  • TNF-a activity assays such as detecting apoptosis via caspase activity can be used to screen for TNF-a variants that are antagonists of wild type TNF-a.
  • Other assays include using the Sytox green nucleic acid stain to detect TNF-induced cell permeability in an Actinomycin-D sensitized cell line. As this stain is excluded from live cells, but penetrates dying cells, this assay also can be used to detect TNF-a variants that are agonists of wild-type TNF-a.
  • agonists of "wild type TNF-a” is meant that the variant TNF-a protein enhances the activation of receptor signaling by wild type TNF-a proteins.
  • variant TNF-a proteins that function as agonists of wild type TNF-a are not preferred. However, in some embodiments, variant TNF-a proteins that function as agonists of wild type TNF-a protein are preferred.
  • An example of an NF kappaB assay is presented in Example 7 of U.S. Patent 7,446,174, which is expressly incorporated herein by reference.
  • binding affinities of variant TNF-a proteins as compared to wild type TNF-a proteins for naturally occurring TNF-a and TNF receptor proteins such as p55 and p75 are determined.
  • Suitable assays include, but are not limited to, e.g., quantitative comparisons comparing kinetic and equilibrium binding constants, as are known in the art. Examples of binding assays are described in Example 6 of U.S. Patent 7,446,174, which is expressly incorporated herein by reference.
  • the variant TNF-a protein has an amino acid sequence that differs from a wild type TNF-a sequence by at least 1 amino acid, with from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 amino acids all contemplated, or higher.
  • the variant TNF-a proteins of the invention preferably are greater than 90% identical to wild-type, with greater than 95, 97, 98 and 99% all being contemplated. Stated differently, based on the human TNF-a sequence of FIG.
  • variant TNF-a proteins have at least about 1 residue that differs from the human TNF-a sequence, with at least about 2, 3, 4, 5, 6, 7 or 8 different residues.
  • Preferred variant TNF-a proteins have 3 to 8 different residues.
  • a % amino acid sequence identity value is determined by the number of matching identical residues divided by the total number of residues of the "longer" sequence in the aligned region.
  • the "longer" sequence is the one having the most actual residues in the aligned region (gaps introduced by WU-Blast-2 to maximize the alignment score are ignored).
  • percent (%) nucleic acid sequence identity with respect to the coding sequence of the polypeptides identified is defined as the percentage of nucleotide residues in a candidate sequence that are identical with the nucleotide residues in the coding sequence of the cell cycle protein.
  • a preferred method utilizes the BLASTN module of WU-BLAST-2 set to the default parameters, with overlap span and overlap fraction set to 1 and 0.125, respectively.
  • TNF-a proteins may be fused, for example, to other therapeutic proteins or to other proteins such as Fc or serum albumin for therapeutic or pharmacokinetic purposes.
  • a TNF-a protein of the present invention is operably linked to a fusion partner.
  • the fusion partner may be any moiety that provides an intended therapeutic or pharmacokinetic effect. Examples of fusion partners include but are not limited to Human Serum Albumin, a therapeutic agent, a cytotoxic or cytostatic molecule, radionucleotide, and an Fc, etc.
  • an "Fc fusion” is synonymous with the terms “immunoadhesin”, “Ig fusion”, “Ig chimera”, and “receptor globulin” as used in the prior art (Chamow et al, 1996, Trends Biotechnol 14:52-60; Ashkenazi et al, 1997, Curr Opin Immunol 9:195-200, both incorporated by reference).
  • An Fc fusion combines the Fc region of an immunoglobulin with the target-binding region of a TNF-a protein, for example. See for example U.S. Pat. Nos. 5,766,883 and 5,876,969, both of which are incorporated by reference.
  • the variant TNF-a proteins comprise variant residues selected from the following positions 21, 23, 30, 31, 32, 33, 34, 35, 57, 65, 66, 67, 69, 75, 84, 86, 87, 91, 97, 101, 111, 112, 115, 140, 143, 144, 145, 146, and 147.
  • Preferred amino acids for each position, including the human TNF-a residues, are shown in FIG. 3.
  • preferred amino acids are Glu, Asn, Gin, Ser, Arg, and Lys; etc.
  • Preferred changes include: VIM, Q21C, Q21 R, E23C, R31C, N34E, V91E, Q21R, N30D, R31C, R31I, R31D, R31E, R32D, R32E, R32S, A33E, N34E, N34V, A35S, D45C, L57F, L57W, L57Y, K65D, K65E, K651, K65M, K65N, K65Q, K65T, K65S, K65V, K65W, G66K, G66Q, Q67D, Q67K, Q67R, Q67S, Q67W, Q67Y, C69V, L75E, L75K, L75Q, A84V, S86Q, S86R, Y87H, Y87R, V91E, I97R, I97T, CIOIA, Al 11R, Al 1 IE, Kl 12D, Kl 12E, Yl 15D, Yl
  • the invention provides TNF-a variants selected from the group consisting of XENP268 XENP344, XENP345, XENP346, XENP550, XENP551, XENP557, XENP1593, XENP1594, and XENP1595 as outlined in Example 3 of U.S. Patent 7,662,367, which is incorporated herein by reference.
  • the invention provides methods of forming a TNF-a heterotrimer in vivo in a mammal comprising administering to the mammal a variant TNF-a molecule as compared to the corresponding wild-type mammalian TNF-a, wherein said TNF-a variant is substantially free of agonistic activity.
  • the invention provides methods of screening for selective inhibitors comprising contacting a candidate agent with a soluble TNF-a protein and assaying for TNF-a biological activity; contacting a candidate agent with a transmembrane TNF-a protein and assaying for TNF-a biological activity, and determining whether the agent is a selective inhibitor.
  • the agent may be a protein (including peptides and antibodies, as described herein) or small molecules.
  • the invention provides variant TNF-a proteins that interact with the wild type TNF-a to form mixed trimers incapable of activating receptor signaling.
  • variant TNF-a proteins with 1, 2, 3, 4, 5, 6 and 7 amino acid changes are used as compared to wild type TNF-a protein. In a preferred embodiment, these changes are selected from positions 1, 21, 23, 30, 31, 32, 33, 34, 35, 57, 65, 66, 67, 69, 75, 84, 86, 87, 91, 97, 101, 111, 112, 115, 140, 143, 144, 145, 146 and 147.
  • the non-naturally occurring variant TNF-a proteins have substitutions selected from the group of substitutions consisting of VIM, Q21C, Q21R, E23C, N34E, V91E, Q21R, N30D, R31C, R311, R31D, R31E, R32D, R32E, R32S, A33E, N34E, N34V, A35S, D45C, L57F, L57W, L57Y, K65D, K65E, K651, K65M, K65N, K65Q, K65T, K65S, K65V, K65W, G66K, G66Q, Q67D, Q67K, Q67R, Q67S, Q67W, Q67Y, C69V, L75E, L75K, L75Q, A84V, S86Q, S86R, Y87H, Y87R, V91E, I97R, I97T, CIOIA, Al 11R, Al 1 IE,
  • substitutions may be made either individually or in combination, with any combination being possible.
  • Preferred embodiments utilize at least one, and preferably more, positions in each variant TNF-a protein. For example, substitutions at positions 31, 57, 69, 75, 86, 87, 97, 101, 115, 143, 145, and 146 may be combined to form double variants. In addition triple, quadruple, quintuple and the like, point variants may be generated.
  • the invention provides TNF-a variants comprising the amino acid substitutions A145R/I97T.
  • the invention provides TNF-a variants comprising the amino acid substitutions VIM, R31C, C69V, Y87H, CIOIA, and A145R. In a preferred embodiment, this variant is PEGylated.
  • the variant is XProl595, a PEGylated protein comprising VIM, R31C, C69V, Y87H, CIOIA, and A145R mutations relative to the wild type human sequence.
  • the areas of the wild type or naturally occurring TNF-a molecule to be modified are selected from the group consisting of the Large Domain (also known as II), Small Domain (also known as I), the DE loop, and the trimer interface.
  • the Large Domain, the Small Domain and the DE loop are the receptor interaction domains.
  • the Large Domain preferred positions to be varied include: 21, 30, 31, 32, 33, 35, 65, 66, 67, 111, 112, 115, 140, 143, 144, 145, 146 and/or 147.
  • the preferred positions to be modified are 75 and/or 97.
  • the preferred position modifications are 84, 86, 87 and/or 91.
  • the Trimer Interface has preferred double variants including positions 34 and 91 as well as at position 57.
  • substitutions at multiple receptor interaction and/or trimerization domains may be combined. Examples include, but are not limited to, simultaneous substitution of amino acids at the large and small domains (e.g.
  • A145R and I97T large domain and DE loop
  • A145R and Y87H large domain and trimerization domain
  • Additional examples include any and all combinations, e.g., I97T and Y87H (small domain and DE loop).
  • theses variants may be in the form of single point variants, for example Kl 12D, Yl 15K, Yl 151, Yl 15T, A145E or A145R. These single point variants may be combined, for example, Yl 151 and A145E, or Yl 151 and A145R, or Yl 15T and A145R or Yl 151 and A145E; or any other combination.
  • Preferred double point variant positions include 57, 75, 86, 87, 97, 115, 143, 145, and 146; in any combination.
  • double point variants may be generated including L57F and one of Yl 151, Yl 15Q, Yl 15T, D143K, D143R, D143E, A145E, A145R, E146K or E146R.
  • triple point variants may be generated. Preferred positions include 34, 75, 87, 91, 115, 143, 145 and 146. Examples of triple point variants include V91E, N34E and one of Y115I, Yl 15T, D143K, D143R, A145R, A145E E146K, and E146R. Other triple point variants include L75E and Y87H and at least one of Yl 15Q, A145R, Also, L75K, Y87H and Yl 15Q. More preferred are the triple point variants V91E, N34E and either A145R or A145E.
  • variant TNF-a proteins may also be identified as being encoded by variant TNF-a nucleic acids.
  • nucleic acid the overall homology of the nucleic acid sequence is commensurate with amino acid homology but takes into account the degeneracy in the genetic code and codon bias of different organisms. Accordingly, the nucleic acid sequence homology may be either lower or higher than that of the protein sequence, with lower homology being preferred.
  • a variant TNF-a nucleic acid encodes a variant TNF-a protein.
  • nucleic acids may be made, all of which encode the variant TNF-a proteins of the present invention.
  • those skilled in the art could make any number of different nucleic acids, by simply modifying the sequence of one or more codons in a way which does not change the amino acid sequence of the variant TNF-a.
  • the nucleic acid homology is determined through hybridization studies.
  • nucleic acids which hybridize under high stringency to the nucleic acid sequence shown in FIG. 1A (SEQ ID NO:l) or its complement and encode a variant TNF-a protein is considered a variant TNF-a gene.
  • High stringency conditions are known in the art; see for example Maniatis et al, Molecular Cloning: A Laboratory Manual, 2d Edition, 1989, and Short Protocols in Molecular Biology, ed. Ausubel, et al, both of which are hereby incorporated by reference. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures.
  • Tm thermal melting point
  • Stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C. for short probes (e.g. 10 to 50 nucleotides) and at least about 60°C. for long probes (e.g. greater than 50 nucleotides).
  • Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.
  • less stringent hybridization conditions are used; for example, moderate or low stringency conditions may be used, as are known in the art; see Maniatis and Ausubel, supra, and Tijssen, supra.
  • nucleic acid variants encode TNF-a protein variants comprising the amino acid substitutions described herein.
  • the TNF-a variant encodes a polypeptide variant comprising the amino acid substitutions A145R/I97T.
  • nucleic acid variant encodes a polypeptide comprising the amino acid substitutions VIM, R31C, C69V, Y87H, CIOIA, and A145R, or any 1, 2, 3, 4 or 5 of these variant amino acids.
  • nucleic acid may refer to either DNA or RNA, or molecules which contain both deoxy- and ribonucleotides.
  • the nucleic acids include genomic DNA, cDNA and oligonucleotides including sense and anti-sense nucleic acids.
  • Such nucleic acids may also contain modifications in the ribose-phosphate backbone to increase stability and half-life of such molecules in physiological environments.
  • the nucleic acid may be double stranded, single stranded, or contain portions of both double stranded or single stranded sequence.
  • the depiction of a single strand also defines the sequence of the other strand (“Crick”); thus the sequence depicted in FIG. 1A (SEQ ID NO: l) also includes the complement of the sequence.
  • recombinant nucleic acid is meant nucleic acid, originally formed in vitro, in general, by the manipulation of nucleic acid by endonucleases, in a form not normally found in nature.
  • an isolated variant TNF-a nucleic acid, in a linear form, or an expression vector formed in vitro by ligating DNA molecules that are not normally joined are both considered recombinant for the purposes of this invention.
  • vector any genetic element, such as a plasmid, phage, transposon, cosmid, chromosome, virus, virion, etc. , which is capable of replication when associated with the proper control elements and which can transfer gene sequences between cells.
  • vector includes cloning and expression vehicles, as well as viral vectors.
  • nucleic acid once a recombinant nucleic acid is made and reintroduced into a host cell or organism, it will replicate non-recombinantly, i.e. using the in vivo cellular machinery of the host cell rather than in vitro manipulations; however, such nucleic acids, once produced recombinantly, although subsequently replicated non-recombinantly, are still considered recombinant for the purposes of the invention.
  • a "recombinant protein” is a protein made using recombinant techniques, i.e. through the expression of a recombinant nucleic acid as depicted above.
  • a recombinant protein is distinguished from naturally occurring protein by at least one or more characteristics.
  • the protein may be isolated or purified away from some or all of the proteins and compounds with which it is normally associated in its wild-type host, and thus may be substantially pure.
  • an isolated protein is unaccompanied by at least some of the material with which it is normally associated in its natural state, preferably constituting at least about 0.5%, more preferably at least about 5% by weight of the total protein in a given sample.
  • a substantially pure protein comprises at least about 75% by weight of the total protein, with at least about 80% being preferred, and at least about 90% being particularly preferred.
  • the definition includes the production of a variant TNF-a protein from one organism in a different organism or host cell. Alternatively, the protein may be made at a significantly higher concentration than is normally seen, through the use of a inducible promoter or high expression promoter, such that the protein is made at increased concentration levels.
  • all of the variant TNF-a proteins outlined herein are in a form not normally found in nature, as they contain amino acid substitutions, insertions and deletions, with substitutions being preferred, as discussed below.
  • variant TNF-a proteins of the present invention are amino acid sequence variants of the variant TNF-a sequences outlined herein and shown in the Figures. That is, the variant TNF-a proteins may contain additional variable positions as compared to human TNF-a. These variants fall into one or more of three classes: substitutional, insertional or deletional variants.
  • Amino acid substitutions are typically of single residues; insertions usually will be on the order of from about 1 to 20 amino acids, although considerably larger insertions may be tolerated. Deletions range from about 1 to about 20 residues, although in some cases deletions may be much larger.
  • the expression vectors may be either self-replicating extrachromosomal vectors or vectors which integrate into a host genome. Generally, these expression vectors include transcriptional and translational regulatory nucleic acid operably linked to the nucleic acid encoding the variant TNF-a protein.
  • control sequences refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism.
  • the control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.
  • a nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence.
  • DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide;
  • a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or
  • a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.
  • a replacement of the naturally occurring secretory leader sequence is desired.
  • an unrelated secretory leader sequence is operably linked to a variant TNF-a encoding nucleic acid leading to increased protein secretion.
  • any secretory leader sequence resulting in enhanced secretion of the variant TNF-a protein when compared to the secretion of TNF-a and its secretory sequence, is desired.
  • Suitable secretory leader sequences that lead to the secretion of a protein are known in the art.
  • a secretory leader sequence of a naturally occurring protein or a protein is removed by techniques known in the art and subsequent expression results in intracellular accumulation of the recombinant protein.
  • operably linked means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading frame. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.
  • the transcriptional and translational regulatory nucleic acid will generally be appropriate to the host cell used to express the fusion protein; for example, transcriptional and translational regulatory nucleic acid sequences from Bacillus are preferably used to express the fusion protein in Bacillus. Numerous types of appropriate expression vectors, and suitable regulatory sequences are known in the art for a variety of host cells.
  • the transcriptional and translational regulatory sequences may include, but are not limited to, promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, and enhancer or activator sequences.
  • the regulatory sequences include a promoter and transcriptional start and stop sequences.
  • Promoter sequences encode either constitutive or inducible promoters.
  • the promoters may be either naturally occurring promoters or hybrid promoters. Hybrid promoters, which combine elements of more than one promoter, are also known in the art, and are useful in the present invention.
  • the promoters are strong promoters, allowing high expression in cells, particularly mammalian cells, such as the CMV promoter, particularly in combination with a Tet regulatory element.
  • the expression vector may comprise additional elements.
  • the expression vector may have two replication systems, thus allowing it to be maintained in two organisms, for example in mammalian or insect cells for expression and in a prokaryotic host for cloning and amplification.
  • the expression vector contains at least one sequence homologous to the host cell genome, and preferably two homologous sequences that flank the expression construct.
  • the integrating vector may be directed to a specific locus in the host cell by selecting the appropriate homologous sequence for inclusion in the vector. Constructs for integrating vectors are well known in the art.
  • the expression vector contains a selectable marker gene to allow the selection of transformed host cells. Selection genes are well known in the art and will vary with the host cell used.
  • a preferred expression vector system is a retroviral vector system such as is generally described in PCT/US97/01019 and PCT/US97/01048, both of which are hereby incorporated by reference.
  • the expression vector comprises the components described above and a gene encoding a variant TNF-a protein.
  • vector composition the combination of components, comprised by one or more vectors, which may be retroviral or not, is referred to herein as a "vector composition".
  • a number of viral based vectors have been used for gene delivery. See for example U.S. Pat. No. 5,576,201, which is expressly incorporated herein by reference.
  • retroviral systems are known and generally employ packaging lines which have an integrated defective provirus (the "helper") that expresses all of the genes of the virus but cannot package its own genome due to a deletion of the packaging signal, known as the psi sequence.
  • the cell line produces empty viral shells.
  • Producer lines can be derived from the packaging lines which, in addition to the helper, contain a viral vector, which includes sequences required in cis for replication and packaging of the virus, known as the long terminal repeats (LTRs).
  • LTRs long terminal repeats
  • retroviral vectors include but are not limited to vectors such as the LHL, N2, LNSAL, LSHL and LHL2 vectors described in e.g., U.S. Pat. No. 5,219,740, incorporated herein by reference in its entirety, as well as derivatives of these vectors.
  • Retroviral vectors can be constructed using techniques well known in the art. See, e.g., U.S. Pat. No.
  • Adenovirus based systems have been developed for gene delivery and are suitable for delivery according to the methods described herein.
  • Human adenoviruses are double-stranded DNA viruses that enter cells by receptor-mediated endocytosis. These viruses are particularly well suited for gene transfer because they are easy to grow and manipulate and they exhibit a broad host range in vivo and in vitro.
  • Adenoviruses infect quiescent as well as replicating target cells. Unlike retroviruses which integrate into the host genome, adenoviruses persist extrachromosomally thus minimizing the risks associated with insertional mutagenesis. The virus is easily produced at high titers and is stable so that it can be purified and stored. Even in the replication-competent form, adenoviruses cause only low level morbidity and are not associated with human malignancies. Accordingly, adenovirus vectors have been developed which make use of these advantages. For a description of adenovirus vectors and their uses see, e.g., Haj-Ahmad and Graham (1986) J. Virol. 57:267-274; Bett et al.
  • the viral vectors used in the subject methods are AAV vectors.
  • AAV vector is meant a vector derived from an adeno-associated virus serotype, including without limitation, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAVX7, etc.
  • Typical AAV vectors can have one or more of the AAV wild-type genes deleted in whole or part, preferably the rep and/or cap genes, but retain functional flanking inverted terminal repeat (ITR) sequences.
  • ITR inverted terminal repeat
  • An AAV vector includes at least those sequences required in cis for replication and packaging ⁇ e.g., functional ITRs) of the virus.
  • the ITRs need not be the wild-type nucleotide sequences, and may be altered, e.g. , by the insertion, deletion or substitution of nucleotides, so long as the sequences provide for functional rescue, replication and packaging.
  • AAV serotypes see for example Cearley et al, Molecular Therapy, 16: 1710-1718, 2008, which is expressly incorporated herein in its entirety by reference.
  • AAV expression vectors may be constructed using known techniques to provide as operatively linked components in the direction of transcription, control elements including a transcriptional initiation region, the DNA of interest and a transcriptional termination region.
  • the control elements are selected to be functional in a thalamic and/or cortical neuron. Additional control elements may be included.
  • the resulting construct, which contains the operatively linked components is bounded (5' and 3') with functional AAV ITR sequences.
  • AAV ITRs adeno-associated virus inverted terminal repeats
  • AAV ITRs the art- recognized regions found at each end of the AAV genome which function together in cis as origins of DNA replication and as packaging signals for the virus.
  • AAV ITRs, together with the AAV rep coding region, provide for the efficient excision and rescue from, and integration of a nucleotide sequence interposed between two flanking ITRs into a mammalian cell genome.
  • AAV ITR regions The nucleotide sequences of AAV ITR regions are known. See, e.g., Kotin, R. M. (1994) Human Gene Therapy 5:793-801; Berns, K. I. "Parvoviridae and their Replication" in Fundamental Virology, 2nd Edition, (B. N. Fields and D. M. Knipe, eds.) for the AAV-2 sequence. As used herein, an "AAV ITR" need not have the wild-type nucleotide sequence depicted, but may be altered, e.g. , by the insertion, deletion or substitution of nucleotides.
  • the AAV ITR may be derived from any of several AAV serotypes, including without limitation, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAVX7, etc.
  • 5' and 3' ITRs which flank a selected nucleotide sequence in an AAV vector need not necessarily be identical or derived from the same AAV serotype or isolate, so long as they function as intended, i.e., to allow for excision and rescue of the sequence of interest from a host cell genome or vector, and to allow integration of the heterologous sequence into the recipient cell genome when AAV Rep gene products are present in the cell.
  • Suitable DNA molecules for use in AAV vectors will include, for example, a gene that encodes a protein that is defective or missing from a recipient subject or a gene that encodes a protein having a desired biological or therapeutic effect ⁇ e.g., an enzyme, or a neurotrophic factor).
  • a desired biological or therapeutic effect e.g., an enzyme, or a neurotrophic factor.
  • the artisan of reasonable skill will be able to determine which factor is appropriate based on the neurological disorder being treated.
  • the selected nucleotide sequence is operably linked to control elements that direct the transcription or expression thereof in the subject in vivo.
  • control elements can comprise control sequences normally associated with the selected gene.
  • heterologous control sequences can be employed.
  • Useful heterologous control sequences generally include those derived from sequences encoding mammalian or viral genes.
  • Examples include, but are not limited to, the SV40 early promoter, mouse mammary tumor virus LTR promoter; adenovirus major late promoter (Ad MLP); a herpes simplex virus (HSV) promoter, a cytomegalovirus (CMV) promoter such as the CMV immediate early promoter region (CMVIE), a rous sarcoma virus (RSV) promoter, synthetic promoters, hybrid promoters, and the like.
  • HSV herpes simplex virus
  • CMV cytomegalovirus
  • CMVIE CMV immediate early promoter region
  • RSV rous sarcoma virus
  • synthetic promoters hybrid promoters, and the like.
  • sequences derived from nonviral genes such as the murine metallothionein gene, will also find use herein.
  • Such promoter sequences are commercially available from, e.g., Stratagene (San Diego, Calif).
  • the TNF-a protein may be covalently modified.
  • a preferred type of covalent modification of variant TNF-a comprises linking the variant TNF-a polypeptide to one of a variety of nonproteinaceous polymers, e.g. , polyethylene glycol (“PEG”), polypropylene glycol, or polyoxyalkylenes, in the manner set forth in U.S. Pat. No. 4,640,835; 4,496,689;
  • cysteines are designed into variant or wild type TNF-a in order to incorporate (a) labeling sites for characterization and (b) incorporate PEGylation sites.
  • labels that may be used are well known in the art and include but are not limited to biotin, tag and fluorescent labels ⁇ e.g. fluorescein). These labels may be used in various assays as are also well known in the art to achieve characterization.
  • a variety of coupling chemistries may be used to achieve PEGylation, as is well known in the art. Examples include but are not limited to, the technologies of Shearwater and Enzon, which allow modification at primary amines, including but not limited to, lysine groups and the N-terminus. See, Kinstler et al, Advanced Drug Deliveries Reviews, 54, 477-485 (2002) and M J Roberts et al, Advanced Drug Delivery Reviews, 54, 459-476 (2002), both hereby incorporated by reference.
  • the optimal chemical modification sites are 21, 23, 31 and 45, taken alone or in any combination.
  • a TNF-a variant of the present invention includes the R31 C mutation.
  • the variant TNF-a protein is purified or isolated after expression.
  • Variant TNF-a proteins may be isolated or purified in a variety of ways known to those skilled in the art depending on what other components are present in the sample.
  • DN-TNF- ⁇ proteins when administered, in addition to reducing immunogenicity, DN-TNF- ⁇ proteins surprisingly improve symptoms in gastrointestinal disorders that are characterized by reduced neuronal survival and reduced colonic cell motility. Such disorders may be associated with types of irritable bowel syndrome.
  • IBS has three clinical presentations— constipation predominant, diarrhea predominant or mixed. In one embodiment constipation predominant IBS may be due to reduced neuronal viability and motility.
  • DN-TNF molecules described herein, including XProl595 may be beneficial in therapy.
  • the mainstay of therapy is treating symptoms or using tricyclic anti-depressant or anticholinergic medications to reduce pain sensation from the bowel.
  • an embodiment includes the combination treatment a DN-TNF with any of the current treatments including, but not limited to tricyclic anti-depressants and/or anticholinergic medications.
  • irritable syndrome is exemplary of other gastrointestinal disorders characterized by reduced neuronal survival and decreased colonic cell motility, such as Chagas disease, Celiac disease, Hirschsprung's disease and the like.
  • Chagas disease commonly known as American trypanosomiasis, is caused by infection with the protozoan parasite Trypanosoma cruzi.
  • the patients develop chronic chagasic gastrointestinal disease characterized by enlargement of colon (megacolon).
  • motility disturbances like constipation Jabari S, et al. (2012), Cell Tissue Res. Aug; 349(2):473-81; da Silveira et al. (2007), Human Pathology, 38(8) 1256- 1264.
  • Celiac disease is a disease of the digestive system that damages the small intestine and interferes with the absorption of nutrients from food.
  • the disease is also featured by central and peripheral nervous system abnormalities, and the presence of anti-neuronal antibodies in the central and enteric nervous system have been demonstrated. It is usually characterized by an irritable bowel and diarrhea (Volta et al, Scan J Gastroentrol, 11, 1276-1281; 2002).
  • Hirschsprung's disease is a congenital disorder with aganglionic intestine. It causes symptoms like constipation, diarrhea, and vomiting and sometimes leads to serious colon complications, like enterocolitis and toxic megacolon (Uesaka et al. J Clin Invest, 118(5): 1890-8; 2008).
  • DN-TNF-a as disclosed herein may be used to treat a variety of GI disorders, including Crohn's disease, inflammatory bowel disorder (IBD) and the like, in a preferred embodiment the DN-TNF-a molecules find use in treating GI disorders not associated with inflammation, but rather that are associated with decreased neuronal survival and decreased colonic cell motility.
  • IBD inflammatory bowel disorder
  • treatment include amelioration or elimination of a disease or condition once it has been established or alleviation of the characteristic symptoms of such disease or condition.
  • a method as disclosed herein may also be used to, depending on the condition of the patient, prevent the onset of a disease or condition or of symptoms associated with a disease or condition, including reducing the severity of a disease or condition or symptoms associated therewith prior to affliction with said disease or condition.
  • Such prevention or reduction prior to affliction refers to administration of the compound or composition of the invention to a patient that is not at the time of administration afflicted with the disease or condition.
  • Preventing also encompasses preventing the recurrence or relapse-prevention of a disease or condition or of symptoms associated therewith, for instance after a period of improvement.
  • a DN-TNF-a protein as described herein is administered peripherally to a patient in need thereof to reduce symptoms associated with inflammatory bowel syndrome.
  • symptoms include, but are not limited to abdominal pain, diarrhea and constipation.
  • the treatment method includes administering a DN-TNF molecule as described herein to a patient suffering from inflammatory bowel syndrome.
  • the patient may be monitored for improvements by measuring a number of biomarkers, including activation of microglial cells or lipopolysaccharide (LPS) as is known in the art.
  • LPS lipopolysaccharide
  • levels of C-reactive protein may be measured according to methods known in the art as an indication of inflammation. As these markers have been found to be elevated in patients suffering from inflammatory disease, following treatment with a DN-TNF as described herein microglial activation, LPS or C-reactive protein levels are reduced as compared to levels prior to treatment.
  • Treatments that currently are available for gastrointestinal disorders may find use in combination with the DN-TNF molecules described herein.
  • GI therapies that may find use in combination with the DN-TNF molecules described herein include, but are not limited to additional antidiarrheal medications or antidepressant medications for pain control.
  • DN-TNFs such as XProl595 may be combined in a therapeutic regimen with agents, such as bardoxolone methyl or variants thereof.
  • treatment of the DN-TNF in a therapeutic regimen in combination with the co-therapies as described herein results in synergistic efficacy as compared to either of the treatments alone.
  • synergistic is meant that efficacy is more than the result of additive efficacy of the two treatments alone.
  • treatment of the DN-TNF in a therapeutic regimen includes the combination of steroidal anti-inflammatory molecules, such as but not limited to dexamethasone and the like or non-steroidal anti-inflammatory molecules.
  • steroidal anti-inflammatory molecules such as but not limited to dexamethasone and the like or non-steroidal anti-inflammatory molecules.
  • the pharmaceutical composition may be formulated in a variety of ways.
  • concentration of the therapeutically active variant TNF-a protein in the formulation may vary from about 0.1 to 100 weight %.
  • the concentration of the variant TNF-a protein is in the range of 0.003 to 1.0 molar, with dosages from 0.03, 0.05, 0.1, 0.2, and 0.3 millimoles per kilogram of body weight being preferred.
  • compositions of the present invention comprise a variant TNF-a protein in a form suitable for administration to a patient.
  • the pharmaceutical compositions are in a water soluble form, such as being present as pharmaceutically acceptable salts, which is meant to include both acid and base addition salts.
  • “Pharmaceutically acceptable acid addition salt” refers to those salts that retain the biological effectiveness of the free bases and that are not biologically or otherwise undesirable, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, / toluenesulfonic acid, salicylic acid and the like.
  • inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like
  • organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, o
  • “Pharmaceutically acceptable base addition salts” include those derived from inorganic bases such as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Particularly preferred are the ammonium, potassium, sodium, calcium, and magnesium salts.
  • Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine.
  • the pharmaceutical compositions may also include one or more of the following: carrier proteins such as serum albumin; buffers such as NaOAc; fillers such as microcrystalline cellulose, lactose, corn and other starches; binding agents; sweeteners and other flavoring agents; coloring agents; and polyethylene glycol.
  • carrier proteins such as serum albumin
  • buffers such as NaOAc
  • fillers such as microcrystalline cellulose, lactose, corn and other starches
  • binding agents such as microcrystalline cellulose, lactose, corn and other starches
  • sweeteners and other flavoring agents coloring agents
  • polyethylene glycol polyethylene glycol.
  • Additives are well known in the art, and are used in a variety of formulations.
  • the variant TNF-a proteins are added in a micellular formulation; see U.S. Pat. No. 5,833,948, hereby incorporated by reference.
  • liposomes may be employed with the TNF-a proteins to effectively deliver the protein.
  • compositions may be administered.
  • the TNF-a compositions of the present invention may be administered in combination with other therapeutics, either substantially simultaneously or co-administered, or serially, as the need may be.
  • antibodies including but not limited to monoclonal and polyclonal antibodies, are raised against variant TNF-a proteins using methods known in the art.
  • these anti-variant TNF-a antibodies are used for immunotherapy.
  • methods of immunotherapy are provided.
  • immunotherapy is meant treatment of TNF-a related disorders with an antibody raised against a variant TNF-a protein.
  • immunotherapy can be passive or active. Passive immunotherapy, as defined herein, is the passive transfer of antibody to a recipient (patient). Active immunization is the induction of antibody and/or T-cell responses in a recipient (patient).
  • the variant TNF-a protein antigen may be provided by injecting a variant TNF-a polypeptide against which antibodies are desired to be raised into a recipient, or contacting the recipient with a variant TNF-a protein encoding nucleic acid, capable of expressing the variant TNF-a protein antigen, under conditions for expression of the variant TNF-a protein antigen.
  • a therapeutic compound is conjugated to an antibody, preferably an anti-variant TNF-a protein antibody.
  • the therapeutic compound may be a cytotoxic agent.
  • targeting the cytotoxic agent to tumor tissue or cells results in a reduction in the number of afflicted cells, thereby reducing symptoms associated with cancer, and variant TNF- ⁇ protein related disorders.
  • Cytotoxic agents are numerous and varied and include, but are not limited to, cytotoxic drugs or toxins or active fragments of such toxins. Suitable toxins and their corresponding fragments include diphtheria A chain, exotoxin A chain, ricin A chain, abrin A chain, curcin, crotin, phenomycin, enomycin and the like. Cytotoxic agents also include radiochemicals made by conjugating radioisotopes to antibodies raised against cell cycle proteins, or binding of a radionuclide to a chelating agent that has been covalently attached to the antibody.
  • variant TNF-a proteins are administered as therapeutic agents, and can be formulated as outlined above.
  • variant TNF-a genes (including both the full- length sequence, partial sequences, or regulatory sequences of the variant TNF-a coding regions) may be administered in gene therapy applications, as is known in the art.
  • variant TNF-a genes can include antisense applications, either as gene therapy (i.e. for incorporation into the genome) or as antisense compositions, as will be appreciated by those in the art.
  • the nucleic acid encoding the variant TNF-a proteins may also be used in gene therapy.
  • genes are introduced into cells in order to achieve in vivo synthesis of a therapeutically effective genetic product, for example for replacement of a defective gene.
  • Gene therapy includes both conventional gene therapy where a lasting effect is achieved by a single treatment, and the administration of gene therapeutic agents, which involves the one time or repeated administration of a therapeutically effective DNA or mRNA.
  • Antisense RNAs and DNAs can be used as therapeutic agents for blocking the expression of certain genes in vivo.
  • oligonucleotides can be imported into cells where they act as inhibitors, despite their low intracellular concentrations caused by their restricted uptake by the cell membrane. (Zamecnik et al, Proc. Natl. Acad. Sci. U.S.A. 83:4143-4146 (1986), incorporated by reference).
  • the oligonucleotides can be modified to enhance their uptake, e.g. by substituting their negatively charged phosphodiester groups by uncharged groups.
  • nucleic acids there are a variety of techniques available for introducing nucleic acids into viable cells.
  • the techniques vary depending upon whether the nucleic acid is transferred into cultured cells in vitro, or in vivo in the cells of the intended host.
  • Techniques suitable for the transfer of nucleic acid into mammalian cells in vitro include the use of liposomes, electroporation, microinjection, cell fusion, DEAE-dextran, the calcium phosphate precipitation method, etc.
  • the currently preferred in vivo gene transfer techniques include transfection with viral (typically retroviral) vectors and viral coat protein- liposome mediated transfection (Dzau et al, Trends in Biotechnology 11 :205-210 (1993), incorporated by reference).
  • the nucleic acid source with an agent that targets the target cells, such as an antibody specific for a cell surface membrane protein or the target cell, a ligand for a receptor on the target cell, etc.
  • an agent that targets the target cells such as an antibody specific for a cell surface membrane protein or the target cell, a ligand for a receptor on the target cell, etc.
  • proteins which bind to a cell surface membrane protein associated with endocytosis may be used for targeting and/or to facilitate uptake, e.g. capsid proteins or fragments thereof tropic for a particular cell type, antibodies for proteins which undergo internalization in cycling, proteins that target intracellular localization and enhance intracellular half-life.
  • the technique of receptor- mediated endocytosis is described, for example, by Wu et al, J. Biol. Chem.
  • variant TNF-a genes are administered as DNA vaccines, either single genes or combinations of variant TNF-a genes. Naked DNA vaccines are generally known in the art. Brower, Nature Biotechnology, 16:1304-1305 (1998). Methods for the use of genes as DNA vaccines are well known to one of ordinary skill in the art, and include placing a variant TNF-a gene or portion of a variant TNF-a gene under the control of a promoter for expression in a patient in need of treatment.
  • the variant TNF-a gene used for DNA vaccines can encode full-length variant TNF-a proteins, but more preferably encodes portions of the variant TNF-a proteins including peptides derived from the variant TNF-a protein.
  • a patient is immunized with a DNA vaccine comprising a plurality of nucleotide sequences derived from a variant TNF-a gene.
  • a DNA vaccine comprising a plurality of nucleotide sequences derived from a variant TNF-a gene.
  • expression of the polypeptide encoded by the DNA vaccine induces cytotoxic T-cells, helper T-cells and antibodies, which recognize and destroy or eliminate cells expressing TNF-a proteins.
  • the DNA vaccines include a gene encoding an adjuvant molecule with the DNA vaccine.
  • adjuvant molecules include cytokines that increase the immunogenic response to the variant TNF-a polypeptide encoded by the DNA vaccine. Additional or alternative adjuvants are known to those of ordinary skill in the art and find use in the invention.
  • compositions are contemplated wherein a TNF-a variant of the present invention and one or more therapeutically active agents are formulated.
  • Formulations of the present invention are prepared for storage by mixing TNF-a variant having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington: The Science and Practice of Pharmacy (22nd ed.) eds. Loyd V. Allen, Jr., et al, Pharmaceutical Press, 2012, and Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed., 1980, both of which are incorporated entirely by reference), in the form of lyophilized formulations or aqueous solutions. Lyophilization is well known in the art, see, e.g., U.S. Pat. No.
  • Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as histidine, phosphate, citrate, acetate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl orbenzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and w-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as
  • the pharmaceutical composition that comprises the TNF-a variant of the present invention may be in a water-soluble form.
  • the TNF-a variant may be present as pharmaceutically acceptable salts, which is meant to include both acid and base addition salts.
  • “Pharmaceutically acceptable acid addition salt” refers to those salts that retain the biological effectiveness of the free bases and that are not biologically or otherwise undesirable, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, / toluenesulfonic acid, salicylic acid and the like.
  • inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like
  • organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, o
  • “Pharmaceutically acceptable base addition salts” include those derived from inorganic bases such as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Particularly preferred are the ammonium, potassium, sodium, calcium, and magnesium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine,
  • the formulations to be used for in vivo administration are preferably sterile. This is readily accomplished by filtration through sterile filtration membranes or other methods.
  • any of a number of delivery systems are known in the art and may be used to administer TNF-a variants of the present invention. Examples include, but are not limited to, encapsulation in liposomes, microparticles, microspheres (e.g. PLA/PGA microspheres), and the like.
  • an implant of a porous, non-porous, or gelatinous material, including membranes or fibers, may be used.
  • Sustained release systems may comprise a polymeric material or matrix such as polyesters, hydrogels, poly(vinylalcohol), polylactides, copolymers of L-glutamic acid and ethyl-L-glutamate, ethylene -vinyl acetate, lactic acid-glycolic acid copolymers such as the LUPRON DEPOT®, and poly-D-(-)-3-hydroxyburyric acid. It is also possible to administer a nucleic acid encoding the TNF-a of the current invention, for example by retroviral infection, direct injection, or coating with lipids, cell surface receptors, or other transfection agents. In all cases, controlled release systems may be used to release the TNF-a at or close to the desired location of action.
  • a nucleic acid encoding the TNF-a of the current invention for example by retroviral infection, direct injection, or coating with lipids, cell surface receptors, or other transfection agents.
  • controlled release systems may be used to release the TNF
  • Dosage forms for the topical or transdermal administration of a DN-TNF -protein disclosed herein include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants.
  • the DN-TNF-protein may be mixed under sterile conditions with a pharmaceutically- acceptable carrier, and with any preservatives, buffers, or propellants which may be required.
  • Powders and sprays can contain, in addition to the DN-TNF-protein, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances.
  • Sprays can additionally contain customary propellants, such as
  • chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons such as butane and propane.
  • the administration of the variant TNF-a proteins of the present invention is done peripherally in a variety of ways including, but not limited to, orally, subcutaneously, intravenously, intranasally, transdermally, intraperitoneally, intramuscularly, intrapulmonary, vaginally, rectally, or intraocularly.
  • the variant TNF-a protein may be directly applied as a solution, salve, cream or spray.
  • the TNF-a molecules of the present may also be delivered by bacterial or fungal expression into the human system (e.g., WO 04046346 A2, hereby incorporated by reference).
  • Subcutaneous administration may be preferable in some circumstances because the patient may self-administer the pharmaceutical composition.
  • Many protein therapeutics are not sufficiently potent to allow for formulation of a therapeutically effective dose in the maximum acceptable volume for subcutaneous administration. This problem may be addressed in part by the use of protein formulations comprising arginine-HCl, histidine, and polysorbate.
  • a TNF-a variant of the present invention may be more amenable to subcutaneous administration due to, for example, increased potency, improved serum half-life, or enhanced solubility.
  • Intravenous As is known in the art, protein therapeutics are often delivered by IV infusion or bolus.
  • the TNF-a variants of the present invention may also be delivered using such methods.
  • administration may be by intravenous infusion with 0.9% sodium chloride as an infusion vehicle.
  • Pulmonary delivery may be accomplished using an inhaler or nebulizer and a formulation comprising an aerosolizing agent.
  • AERx® inhalable technology commercially available from Aradigm, or InhanceTM pulmonary delivery system commercially available from Nektar Therapeutics may be used.
  • TNF-a variants of the present invention may be more amenable to intrapulmonary delivery.
  • TNF-a variants of the present invention may also be more amenable to intrapulmonary administration due to, for example, improved solubility or altered isoelectric point.
  • TNF-a variants of the present invention may be more amenable to oral delivery due to, for example, improved stability at gastric pH and increased resistance to proteolysis.
  • Transdermal patches may have the added advantage of providing controlled delivery of the DN-TNF -protein to the body. Dissolving or dispersing DN-TNF -protein in the proper medium can make such dosage forms. Absorption enhancers can also be used to increase the flux of DN- TNF-protein across the skin. Either providing a rate controlling membrane or dispersing DN-TNF- protein in a polymer matrix or gel can control the rate of such flux.
  • an effective amount of the compositions of the present invention ranges from about 0.000001 mg per kilogram body weight per day to about 10,000 mg per kilogram body weight per day.
  • the dosage ranges are from about 0.0001 mg per kilogram body weight per day to about 2000 mg per kilogram body weight per day.
  • An exemplary treatment regime entails administration once every day or once a week or once a month.
  • a DN-TNF protein may be administered on multiple occasions. Intervals between single dosages can be daily, weekly, monthly or yearly. Alternatively, A DN-TNF protein may be administered as a sustained release formulation, in which case less frequent administration is required.
  • Dosage and frequency vary depending on the half-life of the agent in the subject.
  • the dosage and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic.
  • a relatively low dosage is administered at relatively infrequent intervals over a long period of time. Some subjects continue to receive treatment for the rest of their lives.
  • a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, and preferably until the subject shows partial or complete amelioration of symptoms of disease.
  • the patent can be administered a prophylactic regime.
  • an effective amount (e.g., dose) of a DN-TNF protein described herein will provide therapeutic benefit without causing substantial toxicity to the subject.
  • Toxicity of the agent described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the LD50 (the dose lethal to 50% of the population) or the LD100 (the dose lethal to 100% of the population). The dose ratio between toxic and therapeutic effect is the therapeutic index.
  • the data obtained from these cell culture assays and animal studies can be used in formulating a dosage range that is not toxic for use in human.
  • the dosage of the agent described herein lies suitably within a range of circulating concentrations that include the effective dose with little or no toxicity.
  • the dosage can vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the exact formulation, route of administration and dosage can be chosen by the individual physician in view of the subject's condition. See, e.g., Fingle and Woodbury, Chapter 1 in Goodman and Gilman's The Pharmacological Basis of Therapeutics, 5th Ed., MacMillan Publishing Co., New York (1975), pages 1-46.
  • Example 1- NPY knockdown is associated with attenuated TNF production
  • Colitis was induced as per the documented protocols (Wirtz S, Neufert C, Weigmann B, Neurath MF. Chemically induced mouse models of intestinal inflammation. Nat Protoc. 2007;2(3):541-6.) in mice by administering 3% (w/v) dextran sodium sulfate (DSS, MP Biomedicals, CA) in drinking water for a week.
  • DSS dextran sodium sulfate
  • two groups of mice also received an intraperitoneal injection of one of the TNF inhibitors etanercept or XProl595 (both from Xencor, Monrovia, CA) @ 10 mg/ kg body weight) on days 1, 4 and 7. The mice were euthanized on day 8.
  • the experimental design is given in Figure 1A.
  • the distal colon was collected in liquid nitrogen to be used later for real-time PCR and Western blotting or cryo fixed (OCT) or paraffin embedded for hematoxylin-eosin staining or immunostaining.
  • Enzyme Linked Immunosorbent Assay ELISA.
  • Primary enteric neurons from WT and NPY-/- mice 129S-NpytmlRpa/J, Stock no. 004545, Jackson Laboratory, Maine, US) were cultured as described previously (Anitha et al.).
  • the colon from eight- week old WT and NPY-/- mice was harvested and cultured in RPMI medium for 24 h. TNF levels in the culture supernatants were determined by ELISA (Ray BioTech Inc, Norcross, GA).
  • Luciferase assay Enteric neurons were transfected with full length (-1078) and various deletion sequences of the NPY promoter (-278, -728, -867 and -1078 bp) in a pGL2-luciferase reporter system kindly provided by Dr. Denise Belsham (University of Toronto, Ontario, Canada) (Mayer CM, Cai F, Cui H, Gillespie JM, MacMillan M, Belsham DD. Analysis of a repressor region in the human neuropeptide Y gene that binds Oct-1 and Pbx-1 in GT1-7 neurons.
  • Site-directed mutagenesis was performed using the
  • ChIP Chromatin immunoprecipitation
  • TESS Transcription Element Search System
  • TER Transepithelial electrical Resistance
  • Caco2-BBE cells were seeded on transwell permeable filters (pore size 0.4 ⁇ ) at 40,000 cells/ 0.33 cm2, (Corning, Lowell, MA).
  • TER was measured using Millicel-ERS (Electrical Resistance System from Millipore, Billerica, MA) before and after basolateral addition of NPY (0.1 ⁇ ) at 1, 2, 24 and 48 h.
  • FITC-dextran (4 kD) was added to the apical compartment of Caco2-BBE monolayer. At 0 and 24 h, 100 ⁇ of the basolateral medium was sampled for FD4 translocation (mg of FITC-dextran release/ ml) by measuring the absorbance (Excitation ⁇ 450 nm, Emission ⁇ 510 nm).
  • Colonic motility assessment was evaluated by isometric muscle recording with electrical field stimulation as described previously (Chandrasekharan et al.) The longitudinal muscle strips with intact innervation were used for the experiment.
  • Example 2- TNF inhibitors attenuate NPY expression in enteric neurons, and TNF activates NPY promoter.
  • enteric neuronal cell line Studies on primary cells and an enteric neuronal cell line (Anitha M, Joseph I, Ding X, et al. Characterization of fetal and postnatal enteric neuronal cell lines with improvement in intestinal neural function. Gastroenterology. 2008;134(5): 1424-35) demonstrated that enteric neurons express both TNF receptors TNFR1 and TNFR2, and TNF induced NPY expression in enteric neuronal cells ( Figure 4D, p ⁇ 0.01). Further studies utilizing the neuronal cell line indicated that both TNF inhibitors etanercept (p ⁇ 0.05) and XProl595 ( Figure 4D, p ⁇ 0.01) inhibited NPY expression.
  • TNF-responsive region 728 and -867 bp
  • AP-1 Activator Protein- 1
  • Transcription Factor ATF
  • ATF Transcription Factor, ATF
  • ChIP assays showed increased phospho-c-Jun binding to the -769bp region of the NPY promoter after TNF stimulation.
  • real-time PCR demonstrated a significant increase in the phospho-c-jun promoter occupancy (2.2 ⁇ 0.5, p ⁇ 0.05, Figure 4F) in TNF-stimulated cells compared to control.
  • mutations were introduced in the c-jun binding site (consensus sequence TGAGTCA) on NPY promoter.
  • Serum FD-4 levels were lower in NPY-/- mice compared to WT, suggestive of decreased epithelial paracellular permeability in these mice ( Figure 5A, p ⁇ 0.05).
  • ex vivo permeability studies on isolated distal colonic strips from WT and NPY-/- mice using an Ussing chamber were performed.
  • a significantly higher short circuit current (ISc) was observed in WT mice (12.39 ⁇ 0.5 ⁇ ) compared to NPY-/- (3 ⁇ 0.2 ⁇ , Figure 5B, p ⁇ 0.05), suggestive of increased permeability in WT mice.
  • Example 4- NPY increases permeability of the epithelial monolayer via pore-forming Claudin-2
  • NPY induced similar Akt activation in T84 cells was also observed. Changes in other claudins like claudins- 1 and -4 on NPY treatment was not observed. Taken together the results indicate that in addition to mediating TNF effects, NPY can also have direct effects on permeability via PI3-K.
  • TNF levels are upregulated in human IBD. Having demonstrated TNF -regulation of NPY promoter in vitro, whether administration of TNF inhibitors can attenuate NPY expression during experimental colitis in mice was examined.
  • the in vivo study design utilizing a dextran sodium sulfate (DSS) model of colitis is represented in Figure 6A. It has previously been demonstrated that NPY is up regulated in the colon during two experimental colitis models, and that NPY-/- mice are resistant to colitis.
  • DSS dextran sodium sulfate
  • mice receiving intraperitoneal administration of TNF inhibitors exhibited attenuated NPY expression in the enteric ganglia as demonstrated by immunostaining and quantified by Metamorph (Figure 6B-C, p ⁇ 0.05).
  • enteric ganglia were isolated by laser capture micro dissection and assessed NPY expression by real-time PCR. There was a 2 fold up regulation ( ⁇ 0.1, p ⁇ 0.05) of NPY in the enteric ganglia of DSS-treated mice, which was significantly reduced by TNF inhibitors (Figure 6D, p ⁇ 0.01).
  • mice receiving the TNF inhibitors also exhibited less inflammation as assessed from loss of body weight (Figure 6E), colon length (Figure 6F) and histological damage (Figure 6G-H).
  • Figure 6E loss of body weight
  • Figure 6F colon length
  • Figure 6G-H histological damage
  • Etanercept is a recombinant fusion protein that acts as a decoy receptor that binds TNF, thereby reducing its availability.
  • XProl595 is a dominant negative inhibitor of TNF that depletes the TNF pool (composed of active TNF homotrimers) by replacing it with heterotrimers that do not bind to the receptor.
  • a comparison of the inhibitors revealed that soluble TNF inhibition was more beneficial considering changes in colon length and ( Figure 6E, p ⁇ 0.05) and histological damage (Figure 6G-H, p ⁇ 0.05).
  • Example 6- TNF inhibitors reduced oxidative stress and neuronal apoptosis in colitic mice, thereby improving neuronal survival
  • Oxidative stress is a characteristic feature of IBD and inducible nitric oxide synthase (iNOS) expression is an established marker of inflammation and oxidative stress. It has been previously demonstrated that NPY-/- mice exhibit less nitrosative stress and hence attenuated inflammation compared to WT. In this study it was observed that NPY levels are up regulated in biopsies from IBD patients (unpublished data) and also there was increased oxidative stress in biopsies as evident from increased catalase expression (unpublished data).
  • Colonic motility dysfunctions are commonly associated with IBD. Also inducible nitric oxide synthase (iNOS)-induced over production of nitric oxide has been implicated in the suppression of colonic contractility during IBD. Having demonstrated the protective role of TNF inhibitors against neuronal apoptosis, it was next assessed if enhanced neuronal survival would improve colonic motility. It has been previously demonstrated that WT mice exhibit severe colonic motility impairments during acute DSS colitis. It was found that TNF inhibitors significantly improved neuronal relaxation responses (tetrodotoxin-sensitive) from longitudinal muscle strips in mice.
  • iNOS inducible nitric oxide synthase
  • Non-adrenergic non-cholinergic (NANC) relaxation improved with etanercept treatment (22.57 ⁇ 7.25 %), and was even better in XProl595-treated mice (31.57 ⁇ 6.34 %, p ⁇ 0.05, Figure 8A).
  • the mice exposed to DSS alone (4.98 ⁇ 2.0%) exhibited significantly reduced relaxation due to extensive inflammation-induced neuronal loss.
  • the representative tracings for relaxation are given in Figure 8B.
  • colonic contraction induced after treatment of muscle strips with L-NAME, nitric oxide inhibitor
  • XProl595- treated mice p ⁇ 0.05, Figure 8C
  • Representative tracings for contraction are given in Figure 8D.
  • Colonic motility is coordinated by complex neuron-smooth muscle interactions; hence the relaxation and contraction responses of the colonic smooth muscle were also investigated. It was found that smooth muscle relaxation (induced by sodium
  • nitroprusside, SNP significantly improved in TNF-inhibitor treated mice as compared to the DSS- treated mice (p ⁇ 0.05, Figure 8E).
  • smooth muscle contraction responses significantly improved only in XProl595-treated mice (p ⁇ 0.05, Figure 8F).
  • the representative tracings are given in Figure 8G.

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Abstract

L'invention concerne un procédé de traitement d'un trouble gastro-intestinal associé à une diminution de la survie des cellules nerveuses entériques et de la motilité des cellules du côlon.
PCT/US2014/059190 2013-10-04 2014-10-03 Méthodes de traitement de troubles gastro-intestinaux WO2015051337A2 (fr)

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WO2017106363A1 (fr) * 2015-12-14 2017-06-22 Parkinson's Institute Affinement du diagnostic et du traitement de troubles neurologiques complexes à plusieurs symptômes
EP3307253A4 (fr) * 2015-06-12 2018-11-21 Georgia State University Research Foundation, Inc. Compositions et méthodes pour traiter la tolérance aux opioïdes
JP2019500412A (ja) * 2015-11-26 2019-01-10 ジャン, シソンJIANG, Shisong 望ましくない液性免疫反応を低減するための投薬の組み合わせ

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US7662367B2 (en) * 2000-03-02 2010-02-16 Xencor, Inc. Pharmaceutical compositions for the treatment of TNF-α related disorders
US20060258604A1 (en) * 2005-05-10 2006-11-16 Warren Strober Compositions and methods for the treatment of inflammatory bowel disease utilizing NF-kappaB decoy polynucleotides
EP1920770A4 (fr) * 2005-07-01 2010-09-29 Ajinomoto Kk Agent thérapeutique contre l'affection abdominale inflammatoire et inhibiteur de la production de tnf- alpha
US8007790B2 (en) * 2006-04-03 2011-08-30 Stowers Institute For Medical Research Methods for treating polycystic kidney disease (PKD) or other cyst forming diseases
WO2008043107A2 (fr) * 2006-10-06 2008-04-10 Alba Therapeutics Corporation Utilisation d'antagonistes des jonctions occlusives pour traiter les affections abdominales inflammatoires

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3307253A4 (fr) * 2015-06-12 2018-11-21 Georgia State University Research Foundation, Inc. Compositions et méthodes pour traiter la tolérance aux opioïdes
US10519207B2 (en) 2015-06-12 2019-12-31 Georgia State University Research Foundation, Inc. Compositions and methods for treating opioid tolerance
JP2019500412A (ja) * 2015-11-26 2019-01-10 ジャン, シソンJIANG, Shisong 望ましくない液性免疫反応を低減するための投薬の組み合わせ
WO2017106363A1 (fr) * 2015-12-14 2017-06-22 Parkinson's Institute Affinement du diagnostic et du traitement de troubles neurologiques complexes à plusieurs symptômes

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