WO2016115092A1 - Procédés de ciblage de mécanisme vasculaire hôte à des fins de protection thérapeutique contre la fièvre hémorragique - Google Patents

Procédés de ciblage de mécanisme vasculaire hôte à des fins de protection thérapeutique contre la fièvre hémorragique Download PDF

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WO2016115092A1
WO2016115092A1 PCT/US2016/012985 US2016012985W WO2016115092A1 WO 2016115092 A1 WO2016115092 A1 WO 2016115092A1 US 2016012985 W US2016012985 W US 2016012985W WO 2016115092 A1 WO2016115092 A1 WO 2016115092A1
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hemorrhagic fever
antagonist
subject
agonist
virus
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Sujan Shresta
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La Jolla Institute For Allergy And Immunology
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A61K31/427—Thiazoles not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/4439—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. omeprazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/444—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a six-membered ring with nitrogen as a ring heteroatom, e.g. amrinone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/241—Tumor Necrosis Factors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • Certain embodiments of the invention relate to compositions, and methods of using the same, for the treatment, prevention, reduction of severity of, or delay of the onset of, a viral infection, or a symptom thereof, caused by a virus selected from the family of Arenaviridae, Filoviridae, Bunyaviridae, Flaviviridae, and Rhabdoviridae.
  • embodiments of the invention relate to methods of treatment, prevention, reduction of severity of, or delay of the onset of, hemorrhagic fever.
  • Hemorrhagic fever can be caused by five distinct families of RNA viruses, and by many different strains within any one species.
  • dengue virus an NIAID Category A pathogen
  • DENV DENV serotypes
  • DF acute dengue fever
  • DHF/DSS dengue hemorrhagic fever/dengue shock syndrome
  • the invention is based, in part, on a role of the vascular endothelial growth factor (VEGF) pathway, the angiopoietin (Ang-1 and Ang-2 in particular) pathway, tight junction proteins and adherens junction proteins in the pathogenesis of viral hemorrhagic fever, for example dengue hemorrhagic fever / dengue shock syndrome (DHF/DSS).
  • VEGF vascular endothelial growth factor
  • Ang-1 and Ang-2 angiopoietin
  • DHF/DSS dengue hemorrhagic fever / dengue shock syndrome
  • DENV impacts 3.6 billion people worldwide, but no antivirals or vaccines are available to treat and prevent DENV infections or DENV induced hemorrhagic fever.
  • presented herein are methods for treating hemorrhagic fever.
  • a method comprises modulating an activity or expression of an adherens junction protein.
  • a method for treating hemorrhagic fever comprises administering to a subject, an agonist or antagonist of an adherens junction protein in an amount sufficient to treat a hemorrhagic fever.
  • an adherens junction protein is a cadherin, VE-cadherin, p120, gamma-catenin, or alpha-catenin.
  • a method for treating hemorrhagic fever comprises modulating an activity or expression of a tight junction protein in a subject.
  • a method for treating hemorrhagic fever comprises administering to a subject an agonist or antagonist of a tight junction protein in an amount sufficient to treat a hemorrhagic fever.
  • a tight junction protein is ZO-1 , ZO-2, ZO-3, an occluden, a claudin, or JAM-1 .
  • a method for treating hemorrhagic fever comprises modulating a VEGF pathway in a subject.
  • a method for treating hemorrhagic fever comprises administering to a subject an agonist or antagonist of a VEGF pathway in an amount sufficient to treat a hemorrhagic fever.
  • a method for treating hemorrhagic fever in a subject comprising administering to the subject an effective amount of a VEGF antagonist sufficient to treat the hemorrhagic fever.
  • a VEGF antagonist comprises one of apatinib, bevacizumab, pazopanib, sunitinib, sorafenib, axitinib, vandetanib, cabozantinib, ramucirumab, ponatinib, regorafenib or ziv-aflibercept, ZM 323881 HCI.
  • a method for treating hemorrhagic fever in a subject comprises modulating an Ang-1/Ang-2 pathway in a subject.
  • the method comprises administering to a subject an agonist or antagonist of the Ang-1/Ang-2 pathway in an amount sufficient to treat a hemorrhagic fever.
  • an Ang-1 antagonist comprises ml_4-3.
  • an Ang-2 antagonist comprises L1 -7(N) or Angy- 2-1 .
  • a method for treating hemorrhagic fever in a subject comprises administering an agonist or antagonist of a vascular endothelial protein tyrosine phosphatase (VE-PTP) in an amount sufficient to treat the hemorrhagic fever.
  • VE-PTP vascular endothelial protein tyrosine phosphatase
  • a method for treating hemorrhagic fever in a subject comprises administration of an effective amount of a Tie-2 agonist or antagonist. In some embodiments a method for treating hemorrhagic fever in a subject comprises administering to the subject an effective amount of a Tie-2 agonist or antagonist sufficient to treat a hemorrhagic fever.
  • a Tie-2 antagonist comprises one of SB-203580, 4-(6-Methoxy-2-naphthyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1 H-imidazole; Disodium succinate; 5-[4-[[[2-[[(1 S)-1 -Cyclohexylethyl]amino]-2-oxoethyl][(4- methylphenoxy)carbonyl]amino]methyl]phenyl]-3-pyridinecarboxylic acid, MGCD-265, 4-(4- pyrrolidinylphenyl)-1 ,3-thiazole-2-ylamine; or 2-Amino-4-(4-pyrrolidin-1 -ylphenyl)-1 ,3- thiazole.
  • a method for treating hemorrhagic fever in a subject comprising administering to the subject an effective amount of a TNFa antagonist sufficient to treat the hemorrhagic fever.
  • a TNFa antagonist comprises one of Etanercept, Infliximab, Adalimumab or Golimumab.
  • a method for treating hemorrhagic fever in a subject comprises modulating two or more of a VEGF pathway, an Ang-1/Ang-2 pathway and a TNF pathway in combination.
  • a method for treating hemorrhagic fever in a subject comprises administering to a subject a TNFa agonist or antagonist, and a Tie-2 agonist or antagonist in an amount sufficient to treat a hemorrhagic fever.
  • a method for treating hemorrhagic fever in a subject comprises administering a VEGF agonist or antagonist, and a Tie-2 agonist or antagonist in an amount sufficient to treat the hemorrhagic fever.
  • a method for treating hemorrhagic fever in a subject comprises administering a TNFa agonist or antagonist, and a VEGF agonist or antagonist in amounts sufficient to treat a hemorrhagic fever.
  • a method for treating hemorrhagic fever in a subject comprises administering to a subject a TNFa agonist or antagonist, and an Ang-1 agonist or antagonist in an amount sufficient treat the hemorrhagic fever.
  • a method for treating hemorrhagic fever in a subject comprises administering to the subject a TNFa agonist or antagonist, and an Ang-2 agonist or antagonist in an amount sufficient to treat the hemorrhagic fever.
  • a method for treating hemorrhagic fever in subject comprises administering to the subject a VEGF agonist or antagonist, and an Ang-1 agonist or antagonist in amounts sufficient treat the hemorrhagic fever.
  • a method for treating hemorrhagic fever in a subject comprises administering to the subject, a VEGF agonist or antagonist, and an Ang-2 agonist or antagonist in amounts sufficient to treat the hemorrhagic fever.
  • a hemorrhagic fever comprises a viral hemorrhagic fever caused by one or more of Arenaviridae (Lassa virus, Lujo virus, Junin virus, Machupo virus, Sabia virus or Guanarito virus), Bunyaviridae (Hantavirus, Nairovirus, Garissa virus, llesha virus, Orthobunyavirus or Phlebovirus), Filoviradae (Ebola virus, Marburg virus), Flaviviridae (Dengue virus, Yellow fever virus, Omsk hemorrhagic fever virus or Kyasanur Forest disease virus) or Rhabdoviridae.
  • Arenaviridae Lassa virus, Lujo virus, Junin virus, Machupo virus, Sabia virus or Guanarito virus
  • Bunyaviridae Hyantavirus, Nairovirus, Garissa virus, llesha virus, Orthobunyavirus or Phlebovirus
  • Filoviradae Ebola virus, Marburg virus
  • a hemorrhagic fever comprises Lassa fever, South American hemorrhagic fevers including Argentine hemorrhagic fever, Venezuelan hemorrhagic fever or Brazilian hemorrhagic fever, Whitwater Arroyo virus fever, Flexal virus fever, Ebola hemorrhagic fever, Marburg hemorrhagic fever, Crimean-Congo hemorrhagic fever, Rift Valley fever, hemorrhagic fevers with renal syndrome including Hantaan virus hemorrhagic fever, Seoul virus hemorrhagic fever, Dobrava virus hemorrhagic fever, Puumala virus hemorrhagic fever, hantavirus pulmonary syndrome-associated hemorrhagic fevers, including Bayou virus hemorrhagic fever, Black Creek Canal virus hemorrhagic fever, New York virus hemorrhagic fever, Sin Nombre virus hemorrhagic fever, Andes virus hemor
  • a hemorrhagic fever comprises Dengue fever. In some embodiments the hemorrhagic fever is caused by antibody dependent enhancement of Dengue fever. In some aspects the treatment reduces, decreases, inhibits, delays, eliminates or prevents the probability, severity, frequency, or duration of one or more symptoms associated with or caused by the hemorrhagic fever. In certain embodiments the symptoms comprise one or more of pain, disseminated
  • an agonists and/or antagonists is combined into a single molecule.
  • the single molecule comprises a bi-specific or tri-specific antibody or aptamer.
  • a method for treating hemorrhagic fever in a subject comprises modulating the inflammatory response in a subject in combination with reducing, decreasing, inhibiting, delaying, eliminating or preventing vascular leakage in the subject.
  • a method for treating hemorrhagic fever in a subject comprising modulating the inflammatory response in the subject in combination with modulating angiogenesis in the subject.
  • a method for treating hemorrhagic fever in a subject comprises treating antibody-dependent-enhancement of infection in the subject.
  • a subject has vascular leakage resulting from antibody-dependent- enhancement of infection.
  • a subject has cytokine storm. In certain embodiments a subject a subject has secondary viral infection.
  • a composition comprising one or more of a TNFa agonist or antagonist, an Ang-1 agonist or antagonist, an Ang-2 agonist or antagonist, a VEGF agonist or antagonist and a Tie-2 agonist or antagonist.
  • a composition comprises a TNFa agonist or antagonist and an Ang-1 agonist or antagonist.
  • a composition comprises a TNFa agonist or antagonist and an Ang-2 agonist or antagonist.
  • a composition further comprises a VEGF agonist or antagonist.
  • a composition comprises a VEGF agonist or antagonist and an Ang-1 agonist or antagonist.
  • a composition comprises a VEGF agonist or antagonist and an Ang-2 agonist or antagonist. In certain embodiments a composition comprises a Tie-2 agonist or antagonist. In certain embodiments a composition comprises a TNFa agonist or antagonist and a Tie-2 agonist or antagonist. In certain embodiments a composition comprises a VEGF agonist or antagonist and a Tie-2 agonist or antagonist. In certain embodiments a composition comprises a VEGF agonist or antagonist and a TNFa agonist or antagonist.
  • composition herein is a pharmaceutical composition.
  • a hemorrhagic fever is caused by a virus of the family Flaviviridae.
  • a hemorrhagic fever is caused by a virus of the genus Flavivirus.
  • a hemorrhagic fever is caused by DENV.
  • an agonist or antagonist is an antibody, a small molecule, a protein, a peptide, an antisense nucleic acid or an aptamer, including an antibody-small molecule conjugate, a bispecific antibody or bispecific molecule.
  • FIG. 1 Schematic diagram of the VEGF and Ang-1/Ang-2 signaling pathways in endothelial cells.
  • VEGF binding to the VEGF receptor activates SRC, which in turn causes the phosphorylation and internalization of VE-cadherin after dissociation from vascular endothelial protein tyrosine phosphatase (VE-PTP), ultimately resulting in increased vascular permeability and cell-cell junction destabilization.
  • VE-PTP vascular endothelial protein tyrosine phosphatase
  • Ang-1 acts as a Tie-2 receptor agonist and promotes endothelial survival via PI3K/AKT signaling while inhibiting N F-KB mediated inflammation and SRC-dependent vascular leakage.
  • Ang-2 functions as an Ang-1 antagonist and mediates increases in vascular permeability.
  • FIG. 1 Elevated levels of VEGF and Ang-2 in DENV-infected mice.
  • AG129 mice were infected with 2 x 10 4 PFU of DENV2 strain, S221 i.v., in the presence of 5 ⁇ g of anti-DENV monoclonal antibody 2H2.
  • Levels of VEGF (panel A) and Ang-2 (panel B) in serum collected from na ' ive and infected mice at 2, 3, and 4 days following infection were detected by ELISA.
  • *** p ⁇ 0.001 Each symbol represents an individual mouse.
  • FIG. 3 Absence of ZO-1 expression in DENV-infected mice.
  • AG129 mice were infected with DENV via the ADE mode as described in Fig. 2.
  • the small intestine from na ' ive control and DENV-infected mice were harvested on day 3 p.i. and processed for staining with anti-ZO-1 antibody (red).
  • Phalloidin was used to stain F-actin (green), and nuclei were stained with Hoechst.
  • a single blood vessel from the dotted rectangle in the Merged panel was magnified from the original multi-panel stitch mega image using the ZEN software. Isosurface rendered Images in the last two panels on the right are 3D
  • mice were infected with DENV2 via the ADE route as described in Fig. 2.
  • One group was administered 1200 ⁇ g sunitinib intraperitoneally (i.p.) once daily on days 1 and 2 (left panel), while two other groups received the same amount of sunitinib once on either day 2 or 3 after infection (right panel).
  • Control group was injected with the drug carrier (mixture of kolliphor, ethanol, DMSO, and water) once daily on days 1 and 2 after infection. Survival was assessed. ** p ⁇ 0.05; ** p ⁇ 0.01 .
  • the number of mice (n) is indicated for each group
  • FIG. 5A Sunitinib treatment can protect mice from DENV-induced vascular leakage.
  • AG129 mice were infected with DENV as described in Fig. 2.
  • Infected mice were treated with 1200 ⁇ g sunitinib or vehicle only (i.p.; once per day) on 1 and 2 days after infection.
  • Evans Blue was injected on day 3.5 p.i., followed by harvesting of the small intestine. Evans Blue in the small intestine was extracted with formamide and quantified spectrophotometrically. * p ⁇ 0.05.
  • FIG. 5B Sunitinib treatment does not impact virus levels in the liver of DENV-infected mice.
  • AG129 mice were infected with DENV as described in Fig. 2.
  • One group of the infected mice were treated with 1200 ⁇ g sunitinib i.p. once daily on 1 and 2 days after infection.
  • the other group of infected mice was similarly injected with the drug carrier.
  • Viral RNA levels in the liver on day 3 p.i. were quantitated by qRT-PCR
  • FIG. 6 Treatment with a Tie-2 kinase inhibitor can protect the host against DENV.
  • AG129 mice were infected with DENV as described in Fig. 2. Mice were treated with 1200, 900, or 600 ⁇ g Tie-2 inhibitor (Selleckchem) i.p. once daily on 1 , 2, and 3 days after infection. Control group was similarly injected with the drug carrier (mixture of kolliphor, ethanol, DMSO, and water). Survival was monitored. * p ⁇ 0.05
  • mice were infected with DENV as described in Fig. 2. Mice were administered 30 g or 100 g of anti-TNF monoclonal antibody i.p. once on day 2 after infection. Both treatment groups survive the DENV challenge, whereas all untreated mice succumb to infection by day 5 (data not shown). Mice were bled retrorbitally on days 2 and 3 after infection to collect serum samples. Ang-2 in the sera was detected by ELISA. * p ⁇ 0.05, ** p ⁇ 0.01 , *** p ⁇ 0.001
  • FIG. 8 The LysM-Cre + lfnar1 f f mouse model of DENV infection.
  • Figure 9 A Schematic of the experimental design for testing the effects of the VEGF pathway inhibitors on DENV disease pathogenesis.
  • Figure 10 A Schematic of the experimental design for testing the effects of the Ang-1/Ang-2 pathway modulators on DENV disease pathogenesis in mice and cell cultures.
  • Figure 11 A Schematic of the experimental design for testing the efficacy of anti-TNF, in combination with the VEGF and/or Ang1-Ang-2 pathway modulator.
  • FIG. 12 Dual treatment of DENV-infected AG 129 mice with anti-TNF antibody and sunitinib. AG129 mice were infected with DENV as described in Fig. 2. Top panel: To define minimal therapeutic and subtherapeutic doses of anti-TNF treatment against DENV, mice were treated with 20, 10, or 5 ⁇ g of neutralizing anti-TNF (clone XT3.1 1 , BioXcell) i.p. once on day 2 after infection. Control group was similarly injected with isotype control antibody (anti-horseradish peroxidase rat lgG1 ; clone HRPN from BioXcell). Bottom panel: To explore whether combination therapy targeting the inflammatory and
  • the invention is based, at least in part, on a role for the pathways of angiogenic factors VEGF pathway, the angiopoietin (Ang-1 and Ang-2 in particular) pathway, tight junction proteins and adherens junction proteins in DHF/DSS.
  • Angiopoietins are part of a family of vascular growth factors.
  • angiopoietins are involved with controlling microvascular permeability, vasodilation, and vasoconstriction by signaling smooth muscle cells surrounding vessels.
  • Angiopoietins comprise ANGPT1 (Ang-1 ), ANGPT2 (Ang-2), ANGPT4, and in some aspects, related proteins such as ANGPTL1 , ANGPTL2, ANGPTL3, ANGPTL4, ANGPTL5, ANGPTL6, and ANGPTL7.
  • the inventors discovered a role for VEGF, Ang-1 and Ang-2 in DHF/DSS using small molecule inhibitors and a model of ADE-mediated dengue disease in AG129 mice lacking type I and II IFN receptors.
  • the present inventors have identified regulators of endothelial integrity that can be targeted for development of a novel host-based therapeutic approach against hemorrhagic fever including from dengue virus infection.
  • the present inventors have found that DENV-infected mice have elevated levels of vascular endothelial growth factor (VEGF) and angiopoietin-2 (Ang-2) relative to uninfected mice, and treatment of infected mice with sunitinib, a small molecule inhibitor of the VEGF receptor (VEGFR), or a small molecule inhibitor of Tie-2, a tyrosine kinase receptor for Ang-1 and 2, can protect against the lethal DHF/DSS-like disease.
  • VEGF vascular endothelial growth factor
  • Ang-2 angiopoietin-2
  • Tie-2 a small molecule inhibitor of Tie-2
  • tyrosine kinase receptor for Ang-1 and 2 can protect against the lethal DHF/DSS-like disease.
  • TNFa blockade prevents lethal DHF/DSS-like disease in mice, and that treatment of DENV-infected mice with neutralizing anti-TNFa antibodies results in decreased Ang-2 levels.
  • methods comprising blockade of both angiogenic and inflammatory pathways to provide therapeutic activity against DENV.
  • Therapeutics that block vascular leakage and permit DHF/DSS patients to recover without complex fluid replacement are urgently needed.
  • the present invention provides the use of inexpensive small molecules (some of which have already been tested in humans) that modulated the angiogenic and/or inflammatory pathways as therapeutics for treatment of DHF/DSS.
  • the present methods by focusing on a host-targeted approach, circumvent the challenges associated with developing virus-targeted therapeutics and vaccines that must protect against all four DENV serotypes and multiple genotypes within each serotype of an RNA virus, including drug resistance and partial immune responses.
  • tight junction proteins or adherens junction proteins can be targeted for treatment of hemorrhagic fever and that modulating the expression or activity of tight junction proteins or adherens junction proteins may protect against hemorrhagic fever.
  • tight junction proteins include Zo-1 (TJP1 ), Zo-2 (TJP2), Zo-3 (TJP3), an occluden, occludin (OCLN), Junctional Adhesion Molecules (JAM-A, JAM-B and JAM-C), Jam1 (F1 1 R), a claudin (e.g., CLDN1 -CLDN25), Coxsackie Virus And Adenovirus Receptor (CAR), Par-3, Par-6, MAGI, MUPP1 , and PATJ.
  • Non-limiting examples of adherens junction proteins include a cadherin (e.g., Cadherin 1 (E- cadherin), Cadherin 2 (N-cadherin)), Cadherin 4 (R-cadherin), Cadherin 5 (VE-cadherin), Cadherin 13 (T-cadherin), Cadherin 3 (P-cadherin), Cadherin 1 1 (OB-cadherin), Cadherin 15 (M-cadherin), Cadherin 12 (N-cadherin 2), Cadherin 10 (T2-cadherin), Cadherin 16 (KSP- cadherin), Cadherin 18 Type 2, Cadherin 8 Type 2, Cadherin 9 Type 2, Cadherin 7 Type 2, Cadherin 19 Type 2, Cadherin 20 Type 2, Cadherin 22 Type 2, p120 (Catenin (Cadherin- Associated Protein, Delta 1 ), gamma-catenin (Catenin (Cadherin-Associated Protein), Beta 1 ), and alpha-cat
  • a composition comprises a VE-PTP agonist or antagonist.
  • a method comprises administering a VE-PTP agonist or antagonist to a subject.
  • the present invention provides modulating expression or activity of VE-PTP to treat hemorrhagic fever including methods for treating hemorrhagic fever by administering an agonist or antagonist of VE-PTP in an amount sufficient to treat the hemorrhagic fever.
  • a non-limiting examples of a VE-PTP antagonist include AKB-9778.
  • a composition comprises a TNF antagonist.
  • a method comprises administering a TNF antagonist (e.g., a TNFa antagonist) to a subject.
  • a TNF antagonist is a drug (e.g., a pharmaceutical drug, compound, molecule, reagent or protein) that inhibits or suppresses a physiologic response to tumor necrosis factor (TNF, TNFa).
  • TNF antagonists include etanercept, infliximab, adalimumab, golimumab, certolizumab pegol, anti-TNFa antibodies (e.g., TNFa neutralizing antibodies), certain xanthine derivatives, (e.g.
  • pentoxifylline pentoxifylline
  • bupropion derivatives thereof or combinations thereof.
  • a composition comprises a TNF agonist.
  • a method comprises administering a TNF agonist to a subject.
  • a TNF agonist is a drug (e.g., a pharmaceutical drug, compound, molecule, reagent or protein) that stimulates a pro-inflammatory response by stimulating signaling through a TNF receptor.
  • a TNF agonist includes TNFa, LTa, homotrimers thereof, heterotrimers thereof, agonist antibodies that bind to and signal through TNFR80 or TNFR60, derivatives thereof, binding fragments thereof, and
  • a composition comprises a Tie-2 agonist.
  • a method comprises administering a Tie-2 agonist to a subject.
  • a Tie-2 agonist is a drug (e.g., a pharmaceutical drug, compound, molecule, reagent or protein) that binds a Tie-2 receptor (e.g., as a ligand) that stimulates tyrosine phosphorylation of Tie-2.
  • a Tie-2 agonist include Ang-1 ,
  • a Tie-2 agonist comprises Ang-1 . In some embodiments a Tie-2 agonist comprises Ang-2 (e.g., at high doses with corresponding low levels of Ang-1 ).
  • a composition comprises an Ang-2 antagonist.
  • a method comprises administering an Ang-2 antagonist to a subject.
  • a Tie-2 agonist comprises an Ang-2 antagonist.
  • an Ang-2 antagonist can bind to a Tie-2 ligand (e.g., a Tie-2 antagonist, e.g., Ang-2) and blocks binding of the ligand to a membrane bound Tie-2 receptor.
  • a Tie-2 antagonist e.g., a Tie-2 antagonist, e.g., Ang-2
  • an Ang-2 antagonist comprises an Ang-2 binding protein (e.g., a soluble receptor, fc-fusion protein or antibody).
  • an Ang-2 antagonist comprises the soluble Fc fusion protein L1 -7(N) (Oliner J, Min H, Leal J, et al. (2004) Cancer Cell. 6:507-16).
  • an Ang-2 antagonist comprises Angy-2-1
  • a composition comprises a Tie-2 antagonist.
  • a method comprises administering a Tie-2 antagonist to a subject.
  • a Tie-2 antagonist is a drug (e.g., a pharmaceutical drug, compound, molecule, reagent or protein (e.g., ligand or antibody)) that inhibits tyrosine phosphorylation of Tie-2 by a Tie-2 receptor agonist (e.g., Ang-1 ).
  • a Tie-2 antagonist is a ligand or antibody that binds a Tie-2 receptor (e.g., binds an extracellular portion of Tie- 2).
  • a Tie-2 antagonist is a drug (e.g., a pharmaceutical drug, compound, molecule, reagent or protein) that inhibits or blocks Ang-1 binding to Tie-2. In some embodiments, a Tie-2 antagonist does not inhibit or compete with binding of Ang-1 to Tie-2.
  • a Tie-2 antagonist that binds to a Tie-2 receptor include Ang-2, L1 -7[N], humanized or chimeric derivatives of a Tie-2 blocking antibody, binding fragments of a Tie-2 blocking antibody, derivatives thereof or combinations thereof.
  • a Tie-2 antagonist comprises Ang-2, a derivative or binding fragment thereof.
  • a Tie-2 antagonist comprises a small compound Tie-2 inhibitor.
  • a Tie-2 antagonist is a drug (e.g., a pharmaceutical drug, small compound) that inhibits or blocks tyrosine kinase activity of a Tie-2 receptor stimulated by a Tie-2 receptor agonist (e.g., Ang-1 ).
  • a Tie-2 receptor agonist e.g., Ang-1
  • Any suitable tyrosine kinase inhibitor that inhibits Tie-2 induced tyrosine kinase activity can be used for a composition or method herein.
  • Non- limiting examples of a small compound Tie-2 antagonist include SB-203580, S1577
  • a composition comprises an Ang-1 antagonist.
  • a method comprises administering an Ang-1 antagonist to a subject.
  • a Tie-2 antagonist comprises an Ang-1 antagonist.
  • an Ang-1 antagonist comprises an Ang-1 binding protein (e.g., a soluble receptor, fc-fusion protein or antibody).
  • an Ang-1 antagonist can bind to Ang-1 and blocks binding of Ang-1 to a membrane bound Tie-2 receptor.
  • an Ang-1 antagonist comprises ml_4-3, an Ang-1 neutralizing peptibody (Oliner J, Min H, Leal J, et al. (2004) Cancer Cell. 6:507-16).
  • a composition comprises a VEGF agonist.
  • a method comprises administering a VEGF agonist to a subject.
  • a VEGF agonist stimulates signaling through a VEGF receptor (e.g., VEGFR- 1 , VEGFR-2 and/or VEGFR-3).
  • a VEGF agonist binds to a VEGF receptor and induces vasculogenesis and/or angiogenesis (e.g., in vascular endothelium).
  • Non-limiting examples of a VEGF agonist include natural and modified functional variants of VEGF ligands (e.g., VEGF-A, VEGF-B, VEGF-C, VEGF-D, and VEGF-E), agonistic antibodies that bind to and signal through a VEGF receptor, derivatives thereof and combinations thereof.
  • VEGF ligands e.g., VEGF-A, VEGF-B, VEGF-C, VEGF-D, and VEGF-E
  • agonistic antibodies that bind to and signal through a VEGF receptor, derivatives thereof and combinations thereof.
  • a composition comprises a VEGF antagonist.
  • a method comprises administering a VEGF antagonist to a subject.
  • a VEGF antagonist inhibits and/or blocks signaling through a VEGF receptor (e.g., VEGFR-1 , VEGFR-2 and/or VEGFR-3).
  • a VEGF antagonist inhibits or blocks binding of a VEGF ligand (e.g., VEGF-A, VEGF-B, VEGF-C, VEGF-D, and VEGF-E) to a VEGF receptor.
  • a VEGF antagonist binds to a VEGF receptor.
  • a VEGF antagonist inhibits and/or blocks vasculogenesis and/or angiogenesis (e.g., in vascular endothelium) induced by a VEGF ligand.
  • a VEGF antagonist include soluble VEGF receptors and modified variants thereof that bind a VEGF ligand, antagonistic antibodies that bind to and/or block signaling through a VEGF receptor, antagonistic antibodies that bind to a VEGF ligand and/or block binding of a VEGF ligand to a VEGF receptor, derivatives thereof and combinations thereof.
  • VEGF antagonists include apatinib, bevacizumab, pazopanib, sunitinib, sorafenib, axitinib, vandetanib, cabozantinib, ramucirumab, ponatinib, regorafenib or ziv-aflibercept, ZM 323881 HCI, lenvatinib, motesanib, pazopanib, nintedanib (BIBF 1 120), afatinib, derivatives thereof and combinations thereof.
  • An antibody as referred to herein, can be a polyclonal or monoclonal antibody, or binding fragment thereof.
  • Antibodies sometimes are IgG, IgM, IgA, IgE, or an isotype thereof (e.g., lgG1 , lgG2a, lgG2b or lgG3), sometimes are polyclonal or monoclonal, and sometimes are chimeric, humanized or bispecific versions of an antibody.
  • an antibody or portion thereof comprises a chimeric antibody, Fab, Fab', F(ab')2, Fv fragment, scFv, diabody, aptamer, synbody, camelid, the like and/or a combination thereof.
  • Methods of the invention include treatment methods, which result in any therapeutic or beneficial effect.
  • treatment or “treating” refers to decreasing, reducing inhibiting, suppressing, preventing, controlling or limiting one or more adverse (e.g., physical) symptoms, disorders, illnesses, diseases or complications caused by or associated with hemorrhagic fever pathology (e.g., pain, disseminated intravascular coagulation, bone marrow dysfunction, bleeding, headache, muscle or joint pain, nausea, vomiting, rash, vascular leakage, plasma leakage, fluid accumulation, respiratory distress, organ impairment or damage, fatigue or restlessness, fever, rash, headache, pain behind the eyes, muscle or joint pain, nausea, vomiting, loss of appetite).
  • adverse e.g., physical
  • symptoms e.g., disseminated intravascular coagulation, bone marrow dysfunction
  • bleeding headache, muscle or joint pain
  • treatment methods include reducing, decreasing, inhibiting, delaying or preventing onset, progression, frequency, duration, severity, probability or susceptibility of one or more adverse symptoms, disorders, illnesses, diseases or complications caused by or associated with hemorrhagic fever pathology (e.g., pain, disseminated intravascular coagulation, bone marrow dysfunction, bleeding, headache, muscle or joint pain, nausea, vomiting, rash, vascular leakage, plasma leakage, fluid accumulation, respiratory distress, organ impairment or damage, fatigue or restlessness, fever, rash, headache, pain behind the eyes, muscle or joint pain, nausea, vomiting, loss of appetite).
  • hemorrhagic fever pathology e.g., pain, disseminated intravascular coagulation, bone marrow dysfunction, bleeding, headache, muscle or joint pain, nausea, vomiting, rash, vascular leakage, plasma leakage, fluid accumulation, respiratory distress, organ impairment or damage, fatigue or restlessness, fever, rash, headache, pain behind the eyes, muscle or joint pain, nausea, vomiting, loss of appetite).
  • treatment methods include improving, accelerating, facilitating, enhancing, augmenting, or hastening recovery of a subject from a hemorrhagic fever pathogenesis, or one or more adverse symptoms, disorders, illnesses, diseases or complications caused by or associated with hemorrhagic fever pathology (e.g., pain, disseminated intravascular coagulation, bone marrow dysfunction, bleeding, headache, muscle or joint pain, nausea, vomiting, rash, vascular leakage, plasma leakage, fluid accumulation, respiratory distress, organ impairment or damage, fatigue or restlessness, fever, rash, headache, pain behind the eyes, muscle or joint pain, nausea, vomiting, loss of appetite).
  • adverse symptoms, disorders, illnesses, diseases or complications caused by or associated with hemorrhagic fever pathology e.g., pain, disseminated intravascular coagulation, bone marrow dysfunction, bleeding, headache, muscle or joint pain, nausea, vomiting, rash, vascular leakage, plasma leakage, fluid accumulation, respiratory distress, organ impairment or damage, fatigue or restlessness, fever,
  • treatment methods include stabilizing pathogenesis, or an adverse symptom, disorder, illness, disease or complication caused by or associated with hemorrhagic fever pathology.
  • hemorrhagic fever refers to hemorrhagic pathology which is caused by infection of a subject by a virus of the family Arenaviridae, Filoviridae, Bunyaviridae, Flaviviridae, or Rhabdoviridae.
  • a hemorrhagic fever is a viral hemorrhagic fever.
  • Non-limiting examples of a virus that can cause hemorrhagic fever include Arenaviridae (Lassa virus, Lujo virus, Junin virus, Machupo virus, Sabia virus or Guanarito virus), Bunyaviridae (Hantavirus, Nairovirus, Garissa virus, llesha virus, Orthobunyavirus or Phlebovirus), Filoviradae (Ebola virus, Marburg virus), Flaviviridae (Dengue virus, Yellow fever virus, Omsk hemorrhagic fever virus or Kyasanur Forest disease virus) and Rhabdoviridae.
  • a virus that causes hemorrhagic fever is a virus of the the family Flaviviridae; genus Flavivirus.
  • a virus that causes hemorrhagic fever is a DENV.
  • a method of preventing or treating hemorrhagic fever caused by a virus of the family Arenaviridae, Filoviridae, Bunyaviridae, Flaviviridae, and/or Rhabdoviridae In some embodiments presented herein is a method of preventing or treating hemorrhagic fever caused by a virus of the genus Flavivirus. In some embodiments presented herein is a method of preventing or treating hemorrhagic fever caused by DENV.
  • Non-limiting examples of hemorrhagic fever include Lassa fever, South American hemorrhagic fevers including Argentine hemorrhagic fever, Venezuelan hemorrhagic fever or Brazilian hemorrhagic fever, Whitwater Arroyo virus fever, Flexal virus fever, Ebola hemorrhagic fever, Marburg hemorrhagic fever, Crimean-Congo hemorrhagic fever, Rift Valley fever, hemorrhagic fevers with renal syndrome including Hantaan virus hemorrhagic fever, Seoul virus hemorrhagic fever, Dobrava virus hemorrhagic fever, Puumala virus hemorrhagic fever, hantavirus pulmonary syndrome-associated hemorrhagic fevers, including Bayou virus hemorrhagic fever, Black Creek Canal virus hemorrhagic fever, New York virus hemorrhagic fever, Sin Nombre virus hemorrhagic fever, Andes
  • a therapeutic or beneficial effect of treatment is therefore any objective or subjective measurable or detectable improvement or benefit provided to a particular subject.
  • a therapeutic or beneficial effect can, but need not be, complete ablation of all or any particular adverse symptom, disorder, illness, disease or complication caused by or associated with hemorrhagic fever pathology (e.g., pain, disseminated intravascular coagulation, bone marrow dysfunction, bleeding, headache, muscle or joint pain, nausea, vomiting, rash, vascular leakage, plasma leakage, fluid accumulation, respiratory distress, organ impairment or damage, fatigue or restlessness, fever, rash, headache, pain behind the eyes, muscle or joint pain, nausea, vomiting, loss of appetite).
  • hemorrhagic fever pathology e.g., pain, disseminated intravascular coagulation, bone marrow dysfunction, bleeding, headache, muscle or joint pain, nausea, vomiting, rash, vascular leakage, plasma leakage, fluid accumulation, respiratory distress, organ impairment or damage, fatigue or restlessness, fever, rash, headache, pain behind
  • treatment may be achieved when there is an incremental improvement or a partial reduction in an adverse symptom, disorder, illness, disease or complication caused by or associated with hemorrhagic fever pathology, or an inhibition, decrease, reduction, suppression, prevention, limit or control of worsening or progression of one or more adverse symptoms, disorders, illnesses, diseases or complications caused by or associated with hemorrhagic fever pathology, over a short or long duration (hours, days, weeks, months, etc.).
  • a therapeutic or beneficial effect also includes reducing or eliminating the need, dosage frequency or amount of a second active treatment such as another drug or other agent (e.g., anti-viral) used for treating a subject having or at risk of having a hemorrhagic fever pathology.
  • a second active treatment such as another drug or other agent (e.g., anti-viral) used for treating a subject having or at risk of having a hemorrhagic fever pathology.
  • reducing an amount of an adjunct therapy for example, a reduction or decrease of a treatment for a hemorrhagic fever.
  • Adverse symptoms and complications associated with hemorrhagic fever pathology include, for example, pain, disseminated intravascular coagulation, bone marrow dysfunction, bleeding, headache, muscle or joint pain, nausea, vomiting, rash, vascular leakage, plasma leakage, fluid accumulation, respiratory distress, organ impairment or damage, fatigue or restlessness, fever, rash, headache, pain behind the eyes, muscle or joint pain, nausea, vomiting, loss of appetite, etc., and the like.
  • Other symptoms of hemorrhagic fever pathogenesis are known to one of skill in the art and treatment thereof in accordance with the invention is provided. Thus, the aforementioned symptoms and complications are treatable in accordance with the invention.
  • a method comprises preventing and/or treating an adverse symptom and/or complication associated with hemorrhagic fever pathology. In some embodiments a method comprises reducing, decreasing, inhibiting, delaying, eliminating, and/or preventing the severity, frequency, and/or duration of one or more symptoms associated with, or caused by, a hemorrhagic fever.
  • agonists or antagonists can be administered in a sufficient or effective amount.
  • a "sufficient amount” or “effective amount” or an “amount sufficient” or an “amount effective” refers to an amount that provides, in single (e.g., primary) or multiple (e.g., booster) doses, alone or in combination with one or more other compounds, treatments, therapeutic regimens or agents (e.g., a drug), a long term or a short term detectable or measurable improvement in a given subject or any objective or subjective benefit to a given subject of any degree or for any time period or duration (e.g., for minutes, hours, days, months, years, or cured).
  • an amount sufficient, or an amount effective is provided in a single administration. In some embodiments, an amount sufficient, or an amount effective, is provided in multiple administrations. In some embodiments, an amount sufficient, or an amount effective, is achieved by agonists or antagonists alone, or in a composition or method that comprises a second active component. In addition, an amount sufficient or an amount effective need not be sufficient or effective if given in single or multiple doses without a second or additional administration or dosage, since additional doses, amounts or duration above and beyond such doses, or additional antigens, compounds, drugs, agents, treatment or therapeutic regimens may be included in order to provide a given subject with a detectable or measurable improvement or benefit to the subject.
  • An amount sufficient or an amount effective need not be therapeutically or prophylactically effective in each and every subject treated, nor a majority of subjects treated in a given group or population.
  • An amount sufficient or an amount effective means sufficiency or effectiveness in a particular subject, not a group of subjects or the general population. As is typical for such methods, different subjects will exhibit varied responses to treatment.
  • subject refers to an animal, typically a mammalian animal (mammal), such as a nonhuman primate (apes, gibbons, gorillas, chimpanzees, orangutans, macaques), a domestic animal (dogs and cats), a farm animal (poultry such as chickens and ducks, horses, cows, goats, sheep, pigs), experimental animal (mouse, rat, rabbit, guinea pig) and humans.
  • mammalian animal such as a nonhuman primate (apes, gibbons, gorillas, chimpanzees, orangutans, macaques), a domestic animal (dogs and cats), a farm animal (poultry such as chickens and ducks, horses, cows, goats, sheep, pigs), experimental animal (mouse, rat, rabbit, guinea pig) and humans.
  • mammalian animal such as a nonhuman primate (apes, gibbons, gorillas,
  • Any suitable mammal can be treated by a method described herein.
  • mammals include humans, non-human primates (e.g., apes, gibbons, chimpanzees, orangutans, monkeys, macaques, and the like), domestic animals (e.g., dogs and cats), farm animals (e.g., horses, cows, goats, sheep, pigs) and experimental animals (e.g., mouse, rat, rabbit, guinea pig).
  • Subjects include animal disease models, for example, a mouse model, and other animal models of pathogen (e.g., Dengue Virus) infection known in the art.
  • a mammal is a human.
  • a mammal can be any age or at any stage of development (e.g., an adult, teen, child, infant, or a mammal in utero).
  • a mammal can be male or female.
  • a mammal can be a pregnant female.
  • a mammal can be an animal disease model, for example, animal models used for the study of viral infections (e.g., DENV infection).
  • a subject or mammal is "at risk" of a virus infection (e.g., an infection by a virus of the family Flaviviridae, an infection by a virus of the genus
  • Flavivirus or infection by a DENV.
  • a mammal that is at risk may have increased risk factors for a virus infection, non-limiting examples of which include immunocompromised individuals or immune deficient subjects (e.g., bone marrow transplant recipients, irradiated individuals, subjects having certain types of cancers, particularly those of the bone marrow and blood cells (e.g., leukemia, lymphoma, multiple myeloma), subjects with certain types of chronic infections (e.g., HIV, e.g., AIDS), subjects treated with immunosuppressive agents, subjects suffering from malnutrition and aging, subjects taking certain medications (e.g. disease-modifying anti-rheumatic drugs, immunosuppressive drugs, glucocorticoids) and subjects undergoing chemotherapy), the like or combinations thereof).
  • immunocompromised individuals or immune deficient subjects e.g., bone marrow transplant recipients, irradiated individuals, subjects having certain types of cancers, particularly those of the bone marrow and blood cells (e.g
  • a subject at risk is, will be, or has been in a location or environment suspected of containing a virus.
  • a subject at risk can be a medical professional that is providing care to another who is suspected of being infected with, or known to be infected with a virus.
  • a subject at risk is any subject that has been exposed to a virus.
  • a subject in need of a treatment or composition described herein is a subject at risk of a virus infection.
  • a subject in need of a treatment or composition described herein is a subject infected with, or suspected of being infected with, a virus.
  • a subject in need of a treatment or composition described herein is a subject experiencing one or more symptoms associated with a viral infection (e.g., a hemorrhagic pathology).
  • symptoms associated with a virus infection include fever (e.g., a body temperature greater than 38.6 °C or
  • a composition described herein is used to treat a symptom of a viral infection.
  • subjects appropriate for treatment include those having or at risk of having hemorrhagic fever pathology.
  • Target subjects therefore include subjects that have been exposed to or contacted with a viral hemorrhagic fever (e.g., a DENV), or that have an ongoing infection or have developed one or more adverse symptoms caused by or associated with hemorrhagic fever pathology, regardless of the type, timing or degree of onset, progression, severity, frequency, duration of the symptoms.
  • Treatment of an infection can be at any time during the hemorrhagic fever.
  • Agonists or antagonists can be administered as a combination (e.g., with a second active), or separately, concurrently or in sequence (sequentially) in accordance with the methods as a single or multiple dose e.g., one or more times hourly, daily, weekly, monthly or annually or between about 1 to 10 weeks, or for as long as appropriate, for example, to achieve a reduction in the onset, progression, severity, frequency, duration of one or more symptoms or complications associated with or caused by hemorrhagic fever pathology, or an adverse symptom, condition or complication associated with or caused by a hemorrhagic fever.
  • a method can be practiced one or more times (e.g., 1 -10, 1 -5 or 1 -3 times) an hour, day, week, month, or year.
  • a non-limiting dosage schedule is 1 -7 times per week, for 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more weeks, and any numerical value or range or value within such ranges.
  • compositions for use according to the methods of the invention described herein can be chosen by a caregiver (e.g., a medical professional, a physician) in view of the patient's condition. See e.g., Fingl et al. 1975, in "The Pharmacological Basis of Therapeutics," Ch. 1 , p. 1 ; which is
  • any suitable route of administration can be used for administration of a compound described herein.
  • Methods of the invention may be practiced by any mode of administration or delivery, or by any route, systemic, regional and local administration or delivery.
  • Exemplary administration and delivery routes include intravenous (i.v.), intraperitoneal (i.p.), intrarterial, intramuscular, parenteral, subcutaneous, intra-pleural, topical, dermal, intradermal, transdermal, transmucosal, intra-cranial, intraspinal, rectal, oral (alimentary), mucosal, inhalation, respiration, intranasal, intubation, intrapulmonary, intrapulmonary instillation, buccal, sublingual, intravascular, intrathecal, intracavity, iontophoretic, intraocular, ophthalmic, optical, intraglandular, intraorgan, or intralymphatic.
  • routes of administration include topical or local (e.g., transdermal ⁇ or cutaneously, (e.g., on the skin or epidermus), in or on the eye, intranasally, transmucosally, in the ear, inside the ear (e.g., behind the ear drum)), enteral (e.g., delivered through the gastrointestinal tract, e.g., orally (e.g., as a tablet, capsule, granule, liquid, emulsification, lozenge, or combination thereof), sublingual, by gastric feeding tube, and the like), by parenteral administration (e.g., parenterally, e.g.,
  • intracardiac into the heart
  • intracavernous injection intralesional (into a skin lesion)
  • intraosseous infusion into the bone marrow
  • intrathecal into the spinal canal
  • intrauterine intravaginal, intravesical infusion, intravitreal
  • a composition herein is provided to a subject.
  • a composition that is provided to a subject can be provided to a subject for self-administration or to another (e.g., a caregiver, a medical professional) for administration to a subject.
  • a composition described herein can be provided as an instruction written by a medical practitioner that authorizes a patient to be provided a composition or treatment described herein (e.g., a prescription).
  • a composition can be provided to a subject wherein the subject self-administers a composition orally, intravenously or by way of an inhaler, for example.
  • a dose can be administered in an effective amount or an amount sufficient to treat, prevent or slow a virus infection or to treat, prevent or slow one or more adverse symptoms and/or complications.
  • An exact dose can be determined by a caregiver or medical professional by methods known in the art (e.g., by analyzing data and/or the results of a clinical trial).
  • Doses can be based upon current existing protocols, empirically determined, using animal disease models or optionally in human clinical trials. Initial study doses can be based upon animal studies set forth herein, for a mouse, which weighs about 30 grams, and the amount of agonist or antagonist administered that is determined to be effective.
  • Exemplary non-limiting amounts are in a range of about 0.1 mg/kg to about 100 mg/kg, and any numerical value or range or value within such ranges. Greater or lesser amounts (doses) can be administered, for example, 0.01 -500 mg/kg, and any numerical value or range or value within such ranges.
  • the dose can be adjusted according to the mass of a subject, and will generally be in a range from about 1 ⁇ g/kg-500 mg/kg, 1 -10 ⁇ g/kg, 10-25 ⁇ g/kg, 25-50 ⁇ g/kg, 50-100 ⁇ g/kg, 100-500 ⁇ g/kg, 500-1 ,000 ⁇ g/kg, 1 -5 mg/kg, 5-10 mg/kg, 10-20 mg/kg, 20-50 mg/kg, 50-100 mg/kg, 100-250 mg/kg, 250-500 mg/kg, or more, two, three, four, or more times per hour, day, week, month or annually.
  • a typical range will be from about 0.3 mg/kg to about 50 mg/kg, 0-25 mg/kg, or 1 .0-10 mg/kg, or any numerical value or range or value within such ranges.
  • Doses can vary and depend upon whether the treatment is prophylactic or therapeutic, whether a subject has been previously exposed to, infected with our suffered from a hemorrhagic fever, the onset, progression, severity, frequency, duration probability of or susceptibility of the symptom, condition, pathology or complication, or vaccination or immunization to which treatment is directed, the clinical endpoint desired, previous or simultaneous treatments, the general health, age, gender, race or immunological competency of the subject and other factors that will be appreciated by the skilled artisan. The skilled artisan will appreciate the factors that may influence the dosage and timing required to provide an amount sufficient for providing a therapeutic or prophylactic benefit.
  • compositions, agonists or antagonists disclosed herein will be administered as soon as practical, typically within 1 -2, 2-4, 4-12, 12- 24 or 24-72 hours after a subject is suspected of having a hemorrhagic fever, or within 1 -2, 2-4, 4-12, 12-24 or 24-48 hours after onset or development of one or more adverse symptoms, conditions, pathologies, complications, etc., associated with or caused by a hemorrhagic fever pathology.
  • the dose amount, number, frequency or duration may be proportionally increased or reduced, as indicated by the status of the subject. For example, whether the subject has a pathogen infection, whether the subject has been exposed to, contacted or infected with pathogen or is merely at risk of pathogen contact, exposure or infection, whether the subject is a candidate for or will be vaccinated or immunized.
  • the dose amount, number, frequency or duration may be proportionally increased or reduced, as indicated by any adverse side effects, complications or other risk factors of the treatment or therapy.
  • compositions including pharmaceutical compositions, e.g., a pharmaceutically acceptable carrier or excipient.
  • pharmaceutical compositions are useful for, among other things, administration to a subject in vivo or ex vivo.
  • the term "pharmaceutically acceptable” and “physiologically acceptable” mean a biologically acceptable formulation, gaseous, liquid or solid, or mixture thereof, which is suitable for one or more routes of administration, in vivo delivery or contact.
  • Such formulations include solvents (aqueous or non-aqueous), solutions (aqueous or non-aqueous), emulsions (e.g., oil-in-water or water-in-oil), suspensions, syrups, elixirs, dispersion and suspension media, coatings, isotonic and absorption promoting or delaying agents, compatible with pharmaceutical administration or in vivo contact or delivery.
  • Aqueous and non-aqueous solvents, solutions and suspensions may include suspending agents and thickening agents.
  • Such pharmaceutically acceptable carriers include tablets (coated or uncoated), capsules (hard or soft), microbeads, powder, granules and crystals.
  • Supplementary active compounds e.g., preservatives, antibacterial, antiviral and antifungal agents
  • compositions can be formulated to be compatible with a particular route of administration.
  • pharmaceutical compositions include carriers, diluents, or excipients suitable for administration by various routes.
  • routes of administration for contact or in vivo delivery which a composition can optionally be formulated include inhalation, respiration, intranasal, intubation, intrapulmonary instillation, oral, buccal, intrapulmonary, intradermal, topical, dermal, parenteral, sublingual,
  • iontophoretic intraocular, ophthalmic, optical, intravenous (i.v.), intramuscular,
  • Formulations suitable for parenteral administration comprise aqueous and nonaqueous solutions, suspensions or emulsions of the active compound, which preparations are typically sterile and can be isotonic with the blood of the intended recipient.
  • Non-limiting illustrative examples include water, saline, dextrose, fructose, ethanol, animal, vegetable or synthetic oils.
  • Co-solvents may be added to an agonist or antagonist composition or formulation.
  • Non-limiting examples of co-solvents contain hydroxyl groups or other polar groups, for example, alcohols, such as isopropyl alcohol; glycols, such as propylene glycol, polyethylene glycol, polypropylene glycol, glycol ether; glycerol; polyoxyethylene alcohols and polyoxyethylene fatty acid esters.
  • Non-limiting examples of co-solvents contain hydroxyl groups or other polar groups, for example, alcohols, such as isopropyl alcohol; glycols, such as propylene glycol, polyethylene glycol, polypropylene glycol, glycol ether; glycerol;
  • polyoxyethylene alcohols and polyoxyethylene fatty acid esters.
  • Supplementary compounds e.g., preservatives, antioxidants, antimicrobial agents including biocides and biostats such as antibacterial, antiviral and antifungal agents
  • Pharmaceutical compositions may therefore include preservatives, anti-oxidants and antimicrobial agents.
  • Preservatives can be used to inhibit microbial growth or increase stability of ingredients thereby prolonging the shelf life of the pharmaceutical formulation.
  • Suitable preservatives include, for example, EDTA, EGTA, benzalkonium chloride or benzoic acid or benzoates, such as sodium benzoate.
  • Antioxidants include, for example, ascorbic acid, vitamin A, vitamin E, tocopherols, and similar vitamins or provitamins.
  • An antimicrobial agent or compound directly or indirectly inhibits, reduces, delays, halts, eliminates, arrests, suppresses or prevents contamination by or growth, infectivity, replication, proliferation, reproduction, of a pathogenic or non- pathogenic microbial organism.
  • Classes of antimicrobials include antibacterial, antiviral, antifungal and antiparasitics.
  • Antimicrobials include agents and compounds that kill or destroy (-cidal) or inhibit (-static) contamination by or growth, infectivity, replication, proliferation, reproduction of the microbial organism.
  • anti-bacterials include penicillins (e.g., penicillin G, ampicillin, methicillin, oxacillin, and amoxicillin), cephalosporins (e.g., cefadroxil, ceforanid, cefotaxime, and ceftriaxone), tetracyclines (e.g., doxycycline, chlortetracycline, minocycline, and tetracycline), aminoglycosides (e.g., amikacin, gentamycin, kanamycin, neomycin, streptomycin, netilmicin, paromomycin and tobramycin), macrolides (e.g., azithromycin, clarithromycin, and erythromycin), fluoroquinolones (e.g., ciprofloxacin, lomefloxacin, and norfloxacin), and other antibiotics including chloramphenicol, clindamycin,
  • penicillins e.g.
  • anti-virals include reverse transcriptase inhibitors; protease inhibitors; thymidine kinase inhibitors; sugar or glycoprotein synthesis inhibitors; structural protein synthesis inhibitors; nucleoside analogues; and viral maturation inhibitors.
  • anti-virals include nevirapine, delavirdine, efavirenz, saquinavir, ritonavir, indinavir, nelfinavir, amprenavir, zidovudine (AZT), stavudine (d4T), larnivudine (3TC), didanosine (DDI), zalcitabine (ddC), abacavir, acyclovir, penciclovir, ribavirin, valacyclovir, ganciclovir, 1 ,-D-ribofuranosyl-1 ,2,4-triazole-3
  • compositions and methods of the invention are known in the art (see, e.g., Remington: The Science and Practice of Pharmacy (2003) 20 th ed., Mack Publishing Co., Easton, PA;
  • range expressly includes all possible subranges, all individual numerical values within that range, and all numerical values or numerical ranges include integers within such ranges and fractions of the values or the integers within ranges unless the context clearly indicates otherwise.
  • This construction applies regardless of the breadth of the range and in all contexts throughout this patent document.
  • reference to a range of 90-100% includes 91 -99%, 92-98%, 93-95%, 91 -98%, 91 -97%, 91 - 96%, 91 -95%, 91 -94%, 91 -93%, and so forth.
  • Reference to a range of 90-100% includes 91 %, 92%, 93%, 94%, 95%, 95%, 97%, etc., as well as 91 .1 %, 91 .2%, 91 .3%, 91 .4%, 91 .5%, etc., 92.1 %, 92.2%, 92.3%, 92.4%, 92.5%, etc., and so forth.
  • Reference to a range of 1 -5 fold therefore includes 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, fold, etc., as well as 1 .1 , 1 .2, 1 .3, 1 .4, 1 .5, fold, etc., 2.1 , 2.2, 2.3, 2.4, 2.5, fold, etc., and so forth.
  • reference to a series of ranges of 2-72 hours, 2-48 hours, 4-24 hours, 4-18 hours and 6-12 hours includes ranges of 2-6 hours, 2, 12 hours, 2-18 hours, 2- 24 hours, etc., and 4-27 hours, 4-48 hours, 4-6 hours, etc.
  • a series of range formats are used throughout this document.
  • the use of a series of ranges includes combinations of the upper and lower ranges to provide a range. Accordingly, a series of ranges include ranges which combine the values of the boundaries of different ranges within the series. This construction applies regardless of the breadth of the range and in all contexts throughout this patent document.
  • reference to a series of ranges such as 5-10, 10-20, 20-30, 30-40, 40-50, 50-75, 75-100, 100-150, and 150-171 includes ranges such as 5-20, 5-30, 5-40, 5-50, 5-75, 5-100, 5-150, 5-171 , and 10-30, 10-40, 10-50, 10-75, 10-100, 10-150, 10-171 , and 20-40, 20-50, 20-75, 20-100, 20-150, 20-171 , and so forth.
  • the invention is generally disclosed herein using affirmative language to describe the numerous embodiments and aspects.
  • the invention also specifically includes embodiments in which particular subject matter is excluded, in full or in part, such as substances or materials, method steps and conditions, protocols, or procedures. For example, in certain embodiments or aspects of the invention, materials and/or method steps are excluded. Thus, even though the invention is generally not expressed herein in terms of what the invention does not include aspects that are not expressly excluded in the invention are nevertheless disclosed herein.
  • vascular leakage is a hallmark of DHF/DSS; however, the precise nature and pathogenic mechanisms of DENV-induced vascular leakage are poorly understood.
  • the angiogenic factors vascular endothelial growth factor (VEGF) and angiopoietins (Ang-1 and Ang-2 in particular) are important mediators of vascular integrity and remodelling (Fig. 1 ).
  • mice were passively administered 5 ⁇ g of DENV-specific mouse monoclonal antibody (clone 2H2; DENV1 -4 cross- reactive lgG2a) before infection with 2 x 10 4 PFU of the DENV2 strain S221 .
  • mice inoculated with a high viral challenge dose were more clinically relevant than mice inoculated with a high viral challenge dose in the absence of enhancing antibodies, as severe dengue disease was mostly observed in individuals with secondary DENV infection and infants born to DENV-immune mothers (i.e. patients with DENV-specific antibodies).
  • levels of VEGF and Ang-2 were measured in the serum of mice on days 2, 3, and 4 after infection (Fig. 2).
  • VEGF and Ang-2 levels were higher in DENV-infected mice relative to uninfected mice on day 2 post-infection (p.i.) and the levels varied widely in the infected mice on day 4, when some of the mice had already died and all were severely ill.
  • ZO-1 a tight junction protein that has been studied in the context of human DENV infection.
  • incubation of human umbilical vein endothelial cells (HUVECs) with sera from DHF/DSS patients resulted in downregulation of ZO-1 expression.
  • ZO-1 expression was detectable in the small intestine capillaries of na ' rve but not DENV-infected mice (Fig. 3).
  • sunitinib can protect a host against DENV, and that treatment with sunitinib can be delayed at least until day 2 after infection.
  • sunitinib treatment can impact a DENV-induced increase in vascular permeability was assessed.
  • Sunitinib-treated mice with DENV infection exhibited lower levels of vascular leakage than vehicle-treated control animals in the small intestine, as determined by the Evans Blue extravasation assay (Fig. 5A), indicating that sunitinib treatment can prevent DENV-induced vascular leakage.
  • Fig. 5A Evans Blue extravasation assay
  • the potentially therapeutic effect of sunitinib administration at late time points after infection suggested that the VEGF pathway does not influence DENV levels.
  • viral load in the liver on day 3 after infection with DENV correlated with survival.
  • the inventors measured DENV RNA levels in sunitinib-treated vs. untreated mice, and observed that sunitinib treatment did not impact the liver viral titer on day 3 p.i. (Fig. 5B).
  • sunitinib affects multiple kinases, these results suggested that the VEGF pathway likely regulates endothelial barrier function during DENV infection, and therefore is involved in the host response to DENV infection and that inhibiting this pathway prevents vascular leakage and protect against DENV-induced disease.
  • Wild-type mice are resistant to parenteral infection with DENV, as the virus is able to block type I and type II interferon (IFN) receptor signaling in human but not murine cells.
  • IFN interferon
  • the antiviral IFN response must be disrupted in mice to make them susceptible to DENV infection and manifest signs of severe disease. Therefore, the inventor has developed a model of DHF/DSS-like disease first in 129/Sv mice lacking the type I and II IFN receptors (also known as AG129) upon infection with DENV alone or via the ADE route.
  • mice This mouse model reproduces key pathophysiological features of DHF/DSS, including similar cellular and tissue tropism and lethal vascular leakage, cytokine storm, low platelet count, elevated hematocrit, and hemorrhage.
  • the inventor created a similar dengue disease model in single-deficient IFNAR " _ mice.
  • the relevance of the IFNAR " " mouse model was further increased by crossing them with HLA transgenic mice expressing human MHC class I or class II molecule.
  • LysM-Cre+lfna ⁇ ' mice have been used to develop the most immunocompetent animal model of DHF/DSS developed to date (Fig. 8). These mice were made by crossing C57BL/6 LysM- Cre + mice to C57BL/6 Ifna ⁇ ' mice. These mice lack the type I IFN receptor on
  • macrophages a small subset of DCs, and neutrophils, and have substantial deletion of the receptor on monocytes and splenic macrophages.
  • macrophages are the main targets of DENV in mice, the lack of type I IFN receptor on these cells allowed for DENV replication.
  • the lack of the type I IFN receptor on only a subset of cells rendered the LysM- Cre + lfnai il mice susceptible to DENV-induced DHF/DSS-like disease; the rest of their immune system, including T, B, and dendritic cells, was remarkably intact.
  • LysM-Cre ⁇ mice uniformly succumb to infection with 10 6 PFU of the DENV2 strain S221 in the presence of enhancing levels of exogenously injected mouse DENV-immune sera (Fig. 8). In the absence of DENV-immune sera, a high viral challenge dose (10 7 PFU of DENV2 strain S221 ) was required to induce a lethal disease in these mice (data not shown).
  • the ADE model of DENV infection is used in LysM-Cre+lfnar 1 ⁇ m ⁇ ce.
  • the ADE model is clinically relevant (i.e. DENV alone), as DHF/DSS was observed mostly after sequential infections in DENV-endemic countries. Additionally, the ADE model required a lower viral challenge dose that may be more physiologically relevant to human infection than the primary infection model.
  • Key results based on the LysM-Cre + Ifnat 1 " mouse model are confirmed using another recently generated model of DENV infection in the Itgax- Cre + lfnai il mice, which lack the type I IFN receptor in dendritic cells.
  • DENV strains are used to infect tissue culture cells to validate mechanism of action of each pathway modulator at a cellular level.
  • Cell culture models of DENV infection include both macrophage and endothelial cells, and represent both mouse and human cells, and primary and transformed cells.
  • Both direct (virus alone) and ADE (virus + DENV-specific antibody) routes of infection are tested in studies with macrophages and liver sinusoidal endothelial cells (LSECs) (which express Fey receptors) where the ADE mode impacts the angiogenesis pathways.
  • LSECs liver sinusoidal endothelial cells
  • the ADE route is included in studies with other endothelial cell types.
  • Apatinib (MedChem Express) is a tyrosine kinase inhibitor that inhibits VEGF-R2 with IC 50 of 1 nM
  • ZM 323881 HCI (MedChem Express) is another VEGF-R2 inhibitor with IC 50 of ⁇ 2 nM.
  • sunitinib which affects at least eight tyrosine kinase receptors
  • apatinib and ZM 323881 HCI may be more selective in blocking VEGF-R2 than sunitinib.
  • Apatinib, ZM 323881 HCI, sunitinib, and anti-mouse VEGF-R2 antibody (clone DC101 ; BioXcell) or anti-mouse VEGF-A antibody (clone 2G1 1 -2A05; BioLegend) that inhibits mouse VEGF binding to mouse VEGF-R2 are administered to block the VEGF pathway in mice with DENV infection.
  • Anti-human VEGF-R2 (clone 89106; R&D Systems) or anti-human VEGF (clone MAB293; R&D Systems) in human cell culture studies.
  • mice are injected i.p. with each drug at -1 , 1 , 24, 48, 72, and 96 hours after DENV infection and monitored for survival. Different doses of each drug are tested based on the data (Fig. 4).
  • a control group is injected with appropriate vehicle or isotype control antibody.
  • mice body weight is measured, and health of mice evaluated on a scale of 1 -5 for appearance of coat (smooth coat to very ruffled coat), mobility (active/scurrying to no movement/spastic movement) and attitude (alert to extremely lethargic). These are recorded once a day at 3 p.m. by the same staff involved in the experiment. Mice in control groups are expected to become sick by day 3 after viral challenge and succumb to infection by day 5-6. Experiments are terminated on day 30 p.i.
  • the dosing regimen (in terms of amount, frequency, interval, and timing of drug administration) for each drug is optimized.
  • the maximum tolerated dose (MTD) and the treatment dose at which half of the animals survive to day 30 under day 1 , 2, or 3 p.i. treatment regimen (ED 50 ) is determined.
  • MTD is selected based on the results of a dose-escalation study in which groups of mice receive vehicle control or increasing doses of a drug, followed by evaluation of the mice for morbidity, mortality, and weight loss on a daily basis, clinical pathology (hematology and clinical chemistry) on days 3 and 7, and gross necropsy on all animals, whether they die on-study or survive to day 7.
  • the therapeutic index (Tl) (calculated as the nominal MTD divided by the ED 50 ) of each drug is then defined.
  • mice are euthanized and their organs and sera harvested to measure viral titers in both lymphoid and non-lymphoid tissues.
  • Cytokine storm is assessed by quantifying serum levels of cytokines associated with DHF/DSS in humans and mouse models (TNF, IL-6, IL-8, and IL-10) by ELISA.
  • vascular permeability in key organs such as the liver and intestine is quantitated by the Evans Blue assay and confocal imaging of tight junction and adherens junction protein expression in endothelial cells (Fig. 3).
  • Fig. 3 For quantitation of vascular leakage via imaging, co- localization of CD31 (an endothelial cell marker) with ZO-1 and other tight junction (occludin, ZO-2, ZO-3, claudin-1 , and JAM-1 ) and adherens junction (VE-cadherin) proteins can be examined.
  • the assays for assessing vascular leakage and cytokine storm which are most readily evident 12 to 24 hours prior to death, are performed using day 3 p.i. and moribund samples.
  • VEGF pathway antagonists prevent/restore the endothelial barrier function
  • drug-treated animals should exhibit little or no vascular leakage.
  • the expression pattern of various tight and adherens junction proteins in untreated vs. drug-treated mice is used to provide insights into particular junctional protein pathways that are targeted by each drug.
  • serum levels of key cytokines in untreated vs. drug-treated mice are used to indicate whether cytokine release is affected by the action of the VEGF pathway blocker.
  • tissues of mice are evaluated for inflammation/recruitment of immune cells and identification of cell types producing key cytokines via histopathologic evaluation and immunohistochemistry- and flow cytometry- based approaches. Differences in the patterns of inflammation and cytokine storm in drug- treated vs. vehicle-treated mice is used to provide important insights into interaction between angiogenesis and inflammatory pathways during DENV infection in vivo.
  • HUVECs primary human macrovascular endothelial cells
  • HMEC-1 a human dermal microvascular endothelial cell line
  • HPMEC-ST1 .6R a human pulmonary microvascular endothelial cell line
  • HUVEC and HMEC-1 or HPMEC-ST1 .6R endothelial cell culture models of DENV infection are used.
  • the drug effects on viral replication, cellular activation/differentiation i.e.
  • E-selectin VCAM-1 , ICAM-1 , procoagulant molecules such as von Willebrand factor, tissue factor, PAI-1 and PAF that are found at elevated levels in DHF/DSS patients relative to DF cases, and markers of apoptosis and necrosis
  • procoagulant molecules such as von Willebrand factor, tissue factor, PAI-1 and PAF that are found at elevated levels in DHF/DSS patients relative to DF cases, and markers of apoptosis and necrosis
  • production of cytokines/angiogenesis factors i.e. TNF, IL-6, IL-8, IL-10, VEGF, Ang-1 , and Ang-2
  • barrier function is assessed.
  • the endothelial cell barrier function is assessed via assays that measure transwell permeability of fluorescently labeled 10-kDa dextran and transendothelial electrical resistance (TEER) and expression of key endothelial cell junction proteins (e.g., ZO-1 and VE-cadherin) by immunohistochemistry. Confocal microscopy-based data on the integrity of endothelial cell junctions can be confirmed via electron microscopy. Finally, the mouse and cell culture data are linked by comparing the effects (in terms of endothelial barrier function) of treating the endothelial cell cultures with serum from untreated vs. drug-treated mice with DENV infection and culture supernatant from untreated vs. drug-treated macrophage cultures.
  • LSEC liver sinusoidal endothelial cells
  • mouse LSECs are isolated from LysM-Cre+lfna ⁇ ' mice and SK Hep1 (an immortalized human cell line that may be of LSEC origin and that has been reported to support DENV infection used to develop a primary and transformed LSEC model of DENV infection, respectively).
  • SK Hep1 cells do not support significant levels of DENV infection, human LSECs are immortalized. Mechanisms of drug affect on viral infection, production of cytokines/angiogenesis factors, cellular activation/differentiation, and barrier function in LSECs is assessed.
  • BMM mouse bone marrow-derived macrophages
  • DENV infection is used in mouse bone marrow-derived macrophages (BMM), and DENV infection, and to validate production of cytokines/angiogenesis factors, and cellular activation/differentiation compared in untreated vs. drug-treated BMM.
  • BMM are chosen because macrophages are the primary targets of DENV in our mouse model, and cells of the monocyte/macrophage/dendritic cell lineage are likely the major cell type supporting DENV replication in.
  • macrophages can express VEGF-R2 under certain conditions, significant effects of drug treatment in the BMM model of DENV infection may be observed.
  • similar studies are performed using primary human macrophage model of DENV infection (e.g. models of DENV infection using human blood monocyte-derived MO and M2 macrophages)
  • VEGF pathway antagonists transduce VEGF-R2 signaling and thus abrogate/minimize DENV-induced disruption of endothelial barrier function.
  • Ang-1 binds Tie-2 and stimulated phosphorylation and downstream signaling to stabilize blood vessels, while Ang-2 competed with Ang-1 for Tie-2 binding and reduced Tie-2 phosphorylation.
  • Ang-1 and Ang-2 act as endogenous Tie-2 agonists and antagonists, respectively, and therapeutic agents that agonize (but not antagonize) Tie- 2 function are generally expected to promote endothelial cell integrity and barrier function.
  • Tie-2 antagonism (instead of Tie-2 agonism) protects against DENV-induced lethality in mice highlights the complexity and recently recognized actions of the Ang-1 /Ang-2 pathway that appear to be context-dependent.
  • Ang-2 can also function as a Tie-2 agonist especially when Ang-2 levels are high and Ang-1 levels are low/absent, and Tie-1 , which is structurally homologous to Tie-2 but is considered an orphan receptor that does not bind either Ang-1 or Ang-2, can inhibit Tie-2 signaling by forming a heterodimeric complex with Tie-2.
  • Tie-2 kinase inhibitor (Selleckchem Catalog No. S1577) is an optimized compound of SB-203580, blocks Tie-2 activity with an IC 50 of 0.25 ⁇ and >10 fold selectivity over VEGF-R2 and PDGFR, and was used in the studies in Figure 7.
  • Another small molecule Tie-2 inhibitor (Santa Cruz Biotechnology catalog No.
  • CAS 1020412-97-8 has an IC 50 of 1 ⁇ , and it was tested to be selective against 17 different tyrosine kinases (including the receptor tyrosine kinases VEGF-R2, EGFR, and c-MET) up to 20 ⁇ compound concentrations.
  • tyrosine kinases including the receptor tyrosine kinases VEGF-R2, EGFR, and c-MET
  • Anti-mouse Ang-2 clone Angy-2-1 ; AdipoGen
  • anti-human Ang-2 clone Angy-1 -4; AdipoGen
  • Vasculotide (GenScript or custom synthesis), a synthetic PEGylated peptide that binds to Tie-2 without displacing Ang-1 or Ang-2, and recombinant human Ang-1 (R&D Systems) that can stabilize endothelial barrier function in a mouse model of sepsis are used as agonists.
  • Tie-2 kinase inhibitor Fig. 7
  • Fig. 7 Due to the unexpected results obtained with a Tie-2 kinase inhibitor (Fig. 7) and the complexities of the Ang-1 /Ang-2 pathway, it may be evaluated how each treatment impacts the expression of both receptors (Tie-1 and Tie-2) and ligands (Ang-1 and Ang-2) and activation of Tie-1 and Tie-2 in mice and cell cultures.
  • Tie-1 and Tie-2 activation the phosphorylation status of Tie-1 , Tie-2, and the downstream components, including AKT, ERK, and eNOS are determined.
  • mice demonstrates an intersection of the TNF and Ang-1/Ang-2 pathways during DENV infection in mice (Fig. 8) and the present inventor has discovered that simultaneously targeting both TNF and one of the angiogenic pathways (either VEGF or Ang-1 /Ang-2) provided a benefit over anti-TNF alone in protecting the host against DHF/DSS.
  • LysM- Cre+lfna ⁇ ' mice are treated with 10 ⁇ g or 5 ⁇ g of anti-TNF antibody, together with partially protective doses of the VEGF or Ang-1 /Ang-2 pathway modulators and evaluated for survival, weight loss, and health.
  • Anti-TNF treatment reduces Ang-2 levels in DENV-infected AG129 mice (Fig. 8) and combination therapy of anti-TNF and sunitinib can provide protection against DENV-induced lethality in AG129 mice (Fig. 12, bottom panel).
  • the drugs can be administered at different time points after infection. Based on results showing that day 2 p.i. is the latest time point for an effective anti-TNF or sunitinib treatment, the combination therapy may prolong the dosing window to at least day 3 p.i.
  • the mechanism of action of the combined drugs in these mice can be dissected, as described above. Based on the present data on sunitinib-treatment of DENV-infected AG129 mice (Figs.
  • the combination treatment may protect against DENV-induced lethality by improving endothelial barrier function with minimal impact on viral replication.
  • the activation status of VEGF-R2, Tie-1 , and Tie-2 in tissues also indicate how the combination treatment impacts the VEGF and Ang-1 /Ang-2 signaling.

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Abstract

L'invention concerne des compositions et des procédés pour la prévention et le traitement de la fièvre hémorragique, d'infections virales et des symptômes de celles-ci. De même, l'invention concerne des compositions et des procédés pour le traitement d'une infection virale causée par un virus choisi dans la famille des Arenaviridae, Filoviridae, Bunyaviridae, Flaviviridae et Rhabdoviridae. Les procédés présentés ici sont fondés, en partie, sur la modulation du mécanisme d'action du facteur de croissance endothélial vasculaire (VEGF), du mécanisme d'action de l'angiopoïétine (Ang -1 et Ang -2 en particulier), ainsi que des protéines de jonction cellulaire serrée et protéines de jonction adhérente, qui sont impliqués dans la pathogenèse de la fièvre hémorragique virale.
PCT/US2016/012985 2015-01-12 2016-01-12 Procédés de ciblage de mécanisme vasculaire hôte à des fins de protection thérapeutique contre la fièvre hémorragique WO2016115092A1 (fr)

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US11814425B2 (en) 2006-04-07 2023-11-14 Eye Point Pharmaceuticals, Inc. Antibodies that bind human protein tyrosine phosphatase beta (HPTPbeta) and uses thereof
US10894824B2 (en) 2018-09-24 2021-01-19 Aerpio Pharmaceuticals, Inc. Multispecific antibodies that target HPTP-β (VE-PTP) and VEGF
US11873334B2 (en) 2018-09-24 2024-01-16 EyePoint Pharmaceuticals, Inc. Method of treating ocular conditions by administering an antibody that activates Tie2 and binds VEGF

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