WO2015124570A1 - Méthodes et composition pharmaceutique pour le traitement de l'infection par le virus de la grippe a - Google Patents

Méthodes et composition pharmaceutique pour le traitement de l'infection par le virus de la grippe a Download PDF

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WO2015124570A1
WO2015124570A1 PCT/EP2015/053318 EP2015053318W WO2015124570A1 WO 2015124570 A1 WO2015124570 A1 WO 2015124570A1 EP 2015053318 W EP2015053318 W EP 2015053318W WO 2015124570 A1 WO2015124570 A1 WO 2015124570A1
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mice
platelet
virus
influenza
par4
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PCT/EP2015/053318
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English (en)
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Béatrice Riteau
Martine Jandrot-Perrus
Ba Vuong LE
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INSERM (Institut National de la Santé et de la Recherche Médicale)
Institut National De La Recherche Agronomique (Inra)
Université Claude Bernard - Lyon 1
Université Paris Xiii Paris-Nord
Université Paris Diderot - Paris 7
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Publication of WO2015124570A1 publication Critical patent/WO2015124570A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
    • 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
    • A61K31/415—1,2-Diazoles
    • A61K31/416—1,2-Diazoles condensed with carbocyclic ring systems, e.g. indazole
    • 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
    • A61K31/4353—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 ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4365—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 ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system having sulfur as a ring hetero atom, e.g. ticlopidine
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7076—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic acid
    • 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
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • 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
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses

Definitions

  • the present invention relates to methods and pharmaceutical compositions for the treatment of influenza A virus infection.
  • Influenza is one of the most common infectious diseases in humans, occurring as seasonal epidemic and sporadic pandemic outbreaks.
  • influenza A viruses IAV
  • IAV influenza A viruses
  • the pathogenesis of influenza is a complex process involving both viral determinants and the immune system (Foucault et al, 201 1; Fukuyama and Kawaoka, 2011; Kuiken et al, 2012).
  • dysregulation of cytokine production contributes to collateral damages of the lungs, possibly leading to organ failure and death (Cheung et al, 2002; La Gruta et al, 2007).
  • a deleterious pulmonary inflammation is typically observed during infection with highly pathogenic IAV subtypes (de Jong et al, 2006; Kobasa et al, 2007).
  • the endothelium which line the interior surface of blood vessels is proposed as to orchestrate the crescendo in cytokine accumulation, although all the actors playing this piece are not identified (Teijaro et al., 2011).
  • platelets Upon endothelium injury, platelets are immediately recruited by inflamed endothelial cells in the absence of denudation via the release from Weibel Palade bodies of P-selectin and high molecular weight von Willebrand factor and platelet glycoprotein lb. When injury is more severe, platelets adhere and are activated by subendothelial proteins (Rumbaut and Thiagarajan, 2010). Simultaneously, Protease- Activated Receptor (PARs) mediates activation of platelets by thrombin. These events lead to the conformational change of the platelet glycoprotein Ilb/IIIa (GPIIb/IIIa) receptor for fibrinogen that bridges platelets leading to their aggregation and reinforcement of activation.
  • PARs Protease- Activated Receptor
  • platelet activation is strongly associated with enhanced inflammatory responses. Activated platelets release potent inflammatory molecules and play a key role in leukocyte recruitment (Duerschmied et al, 2013). Platelet activation is finely tuned but its dysfunction is pathogenic and contributes to inflammatory disorders (Cohen, 2002; Degen et al, 2007; Medcalf, 2007). Thus, uncontrolled platelet activation could contribute to the pathogenesis of IAV infections by feeding a harmful inflammatory response in the respiratory tract. However, at present the role of platelets in the context of IAV infection has never been investigated.
  • the present invention relates to methods and pharmaceutical compositions for the treatment of influenza A virus infection.
  • the present invention is defined by the claims.
  • the hallmark of severe influenza virus infections is excessive inflammation of the lungs. Platelets are activated during influenza, but their role in influenza virus pathogenesis and inflammatory responses is unknown. The inventors used targeted gene deletion approaches and pharmacological interventions to investigate the role of platelets during influenza virus infection in mice. Lungs of infected mice were massively infiltrated by aggregates of activated platelets. Platelet activation promoted IAV pathogenesis. Activating protease-activated receptor 4 (PAR4), a platelet receptor for thrombin that is crucial for platelet activation, exacerbated influenza-induced acute lung injury and death.
  • PAR4 protease-activated receptor 4
  • mice treated with a specific GPIIbllla antagonist, eptifibatide had the same effect.
  • mice treated with other anti-platelet compounds were also protected from severe lung injury and lethal infections induced by several influenza strains.
  • the intricate relationship between hemostasis and inflammation has major consequences in influenza virus pathogenesis, and anti-platelet drugs might be explored to develop new anti-inflammatory treatment against influenza virus infections.
  • an object of the present invention relates to a method for the treatment of influenza A virus (IAV) infection in a subject in need thereof comprising administering the subject with a therapeutically effective amount of at least one anti-platelet agent.
  • IAV infection has its general meaning in the art and refers to the disease caused by an infection with an influenza A virus.
  • IAV infection is caused by influenza virus A that is HIM, H2N2, H3N2 or H5N1.
  • the subject can be human or any other animal (e.g., birds and mammals) susceptible to influenza infection (e.g. domestic animals such as cats and dogs; livestock and farm animals such as horses, cows, pigs, chickens, etc.).
  • said subject is a mammal including a non-primate (e.g., a camel, donkey, zebra, cow, pig, horse, goat, sheep, cat, dog, rat, and mouse) and a primate (e.g., a monkey, chimpanzee, and a human).
  • a subject is a non-human animal.
  • a subject is a farm animal or pet.
  • a subject is a human.
  • a subject is a human infant.
  • a subject is a human child.
  • a subject is a human adult.
  • a subject is an elderly human.
  • therapeutic treatments include the reduction or amelioration of the progression, the reduction or amelioration of the severity and/or duration of influenza infections, and more particularly the reduction or amelioration of the inflammatory burden of the lungs (e.g. blockade of the IAV-induced inflammation), resulting from the administration of at least one anti-platelet agent of the present invention.
  • the antiplatelet agents of the present invention are used in the community setting to treat people who already have influenza so as to reduce the severity of the infection (i.e. by reduction the inflammatory burden of the lungs (e.g. blockade of the IAV-induced inflammation), reduce the number of days that they are sick and prevent fatal outcome.
  • the anti-platelet agents of the present invention are used in a prophylactic treatment.
  • prophylactic treatment refers to any medical or public health procedure whose purpose is to prevent, rather than treat or cure a disease.
  • the terms “prevent”, “prevention” and “preventing” refer to the reduction in the risk of acquiring or developing a given condition, or the reduction or inhibition of the recurrence or said condition in a subject who is not ill, but who has been or may be near a subject with the disease.
  • the antiplatelet agents of the present invention are used to prevent the inflammatory burden of the lungs (e.g.
  • an "anti-platelet agent” refers to members of a class of pharmaceuticals that inhibit platelet function, for example, by inhibiting the activation, aggregation, adhesion or granular secretion of platelets.
  • the anti-platelet agent is not a PAR-1 antagonist, nor aspirin.
  • the anti-platelet agent is selected from the group consisting of GPIIb/IIIa antagonists (e.g., tirofiban, eptifibatide, and abciximab), thromboxane-A2 -receptor antagonists (e.g., ifetroban), thromboxane-A2-synthetase inhibitors, phosphodiesterase-III (PDE-III) inhibitors (e.g., dipyridamole, cilostazol), and PDE V inhibitors (such as sildenafil), and pharmaceutically acceptable salts or prodrugs thereof.
  • the GPIIb/IIIa antagonist is not tirofiban.
  • the anti-platelet agent of the present invention is selected from the group consisting of ADP (adenosine diphosphate) receptor antagonists, in particular antagonists of the purinergic receptors P2Y1 and P2Y12.
  • P2Y12 receptor antagonists include ticlopidine, clopidogrel, Prasugrel, AR-C69931MX, Cangrelor, MRS2179 1 including pharmaceutically acceptable salts or prodrugs thereof.
  • the anti-platelet agent of the present invention is a PAR-4 antagonist.
  • PAR4 has its general meaning in the art and refers to protease-activated receptor ' which is a low-affinity receptor that mediates thrombin signaling at high concentrations (C-C. Wu et a/, Eur. J. Pharmacol, 2006, 546, 142-147).
  • PAR-4 antagonists are well known in the art (Giuseppe Cirino, Beatrice Severino Thrombin receptors and their antagonists: an update on the patent literature. Expert Opinion on Therapeutic Patents Jul 2010, Vol. 20, No. 7, Pages 875-884). Compounds that function as PAR-4 antagonists are disclosed, for example, in EP667345B1, EP1166785A1, JP 2002080367, EP1331233, and US2006106032, all incorporated by reference.
  • thePAR-4 antagonist is a small organic molecule.
  • the PAR4 antagonists is the indazole derivative YD-3 (ethyl 4- (1- benzyl-lH-indazol-3-yl)benzoate) (Wu CC, Huang SW, Hwang TL, YD-3, a novel inhibitor of protease-induced platelet activation. Br J Pharmacol 2000;130: 1289-96) which has the general formula of:
  • the PAR4 antagonist is selected from the group consisting of :
  • the PAR4 antagonist is selected from the group consisting of imidazothiadiazole and imidazopyridazine derivatives as described in WO2013163241. In some embodiments, the PAR4 antagonist is selected from the group consisting of:
  • the PAR4 antagonist is selected from the group consisting of pepducins.
  • the PAR4 antagonist is P4pall0, whose sequence is pal- S GRRYGH ALR-NH2 (SEQ ID NO: l) (Kuliopulos A, Covic L. G protein coupled receptor (GPCR) agonists and antagonists and methods of activating and inhibiting GPCR using the same.
  • GPCR G protein coupled receptor
  • the anti-platelet agent of the present invention is an antibody which acts as a GPIIb/IIIa antagonist, a thromboxane- A2 -receptor antagonist, an ADP (adenosine diphosphate) receptor antagonist (in particular a antagonist of the purinergic receptors P2Y1 and P2Y12), or a PAR4 antagonist.
  • antibody includes both naturally occurring and non-naturally occurring antibodies. Specifically, “antibody” includes polyclonal and monoclonal antibodies, and monovalent and divalent fragments thereof. Furthermore, “antibody” includes chimeric antibodies, wholly synthetic antibodies, single chain antibodies, and fragments thereof. The antibody may be a human or non human antibody. A non human antibody may be humanized by recombinant methods to reduce its immunogenicity in man.
  • the antibody is a monoclonal antibody. In one embodiment of the antibodies or portions thereof described herein, the antibody is a polyclonal antibody. In one embodiment of the antibodies or portions thereof described herein, the antibody is a humanized antibody. In one embodiment of the antibodies or portions thereof described herein, the antibody is a chimeric antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a light chain of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a heavy chain of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a Fab portion of the antibody.
  • the portion of the antibody comprises a F(ab')2 portion of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a Fc portion of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a Fv portion of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a variable domain of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises one or more CDR domains of the antibody.
  • monoclonal antibodies are prepared according to conventional methodology. Monoclonal antibodies may be generated using the method of Kohler and Milstein (Nature, 256:495, 1975). To prepare monoclonal antibodies useful in the invention, a mouse or other appropriate host animal is immunized at suitable intervals (e.g., twice-weekly, weekly, twice-monthly or monthly) with the relevant antigenic forms (e.g. PAR4 polypeptides). The animal may be administered a final "boost" of antigen within one week of sacrifice. It is often desirable to use an immunologic adjuvant during immunization.
  • suitable intervals e.g., twice-weekly, weekly, twice-monthly or monthly
  • suitable antigenic forms e.g. PAR4 polypeptides
  • the animal may be administered a final "boost" of antigen within one week of sacrifice. It is often desirable to use an immunologic adjuvant during immunization.
  • Suitable immunologic adjuvants include Freund's complete adjuvant, Freund's incomplete adjuvant, alum, Ribi adjuvant, Hunter's Titermax, saponin adjuvants such as QS21 or Quil A, or CpG- containing immunostimulatory oligonucleotides.
  • Other suitable adjuvants are well-known in the field.
  • the animals may be immunized by subcutaneous, intraperitoneal, intramuscular, intravenous, intranasal or other routes. A given animal may be immunized with multiple forms of the antigen by multiple routes.
  • the recombinant protein may be provided by expression with recombinant cell lines, in particular in the form of human cells expressing the receptor (e.g.
  • lymphocytes are isolated from the spleen, lymph node or other organ of the animal and fused with a suitable myeloma cell line using an agent such as polyethylene glycol to form a hydridoma. Following fusion, cells are placed in media permissive for growth of hybridomas but not the fusion partners using standard methods, as described (Coding, Monoclonal Antibodies: Principles and Practice: Production and Application of Monoclonal Antibodies in Cell Biology, Biochemistry and Immunology, 3rd edition, Academic Press, New York, 1996).
  • cell supernatants are analyzed for the presence of antibodies of the desired specificity, i.e., that selectively bind the antigen.
  • Suitable analytical techniques include ELISA, flow cytometry, immunoprecipitation, and western blotting. Other screening techniques are well-known in the field. Preferred techniques are those that confirm binding of antibodies to conformationally intact, natively folded antigen, such as non-denaturing ELISA, flow cytometry, and immunoprecipitation.
  • an antibody from which the pFc' region has been enzymatically cleaved, or which has been produced without the pFc' region designated an F(ab')2 fragment, retains both of the antigen binding sites of an intact antibody.
  • an antibody from which the Fc region has been enzymatically cleaved, or which has been produced without the Fc region designated an Fab fragment, retains one of the antigen binding sites of an intact antibody molecule.
  • Fab fragments consist of a covalently bound antibody light chain and a portion of the antibody heavy chain denoted Fd.
  • the Fd fragments are the major determinant of antibody specificity (a single Fd fragment may be associated with up to ten different light chains without altering antibody specificity) and Fd fragments retain epitope-binding ability in isolation.
  • CDRs complementarity determining regions
  • FRs framework regions
  • CDR1 through CDRS complementarity determining regions
  • the second proposal was that if an amino acid in the framework of the human immunoglobulin is unusual and the donor amino acid at that position is typical for human sequences, then the donor amino acid rather than the acceptor may be selected.
  • the third proposal was that in the positions immediately adjacent to the 3 CDRs in the humanized immunoglobulin chain, the donor amino acid rather than the acceptor amino acid may be selected.
  • the fourth proposal was to use the donor amino acid reside at the framework positions at which the amino acid is predicted to have a side chain atom within 3 A of the CDRs in a three dimensional model of the antibody and is predicted to be capable of interacting with the CDRs.
  • the above methods are merely illustrative of some of the methods that one skilled in the art could employ to make humanized antibodies. One of ordinary skill in the art will be familiar with other methods for antibody humanization.
  • humanized forms of the antibodies some, most or all of the amino acids outside the CDR regions have been replaced with amino acids from human immunoglobulin molecules but where some, most or all amino acids within one or more CDR regions are unchanged. Small additions, deletions, insertions, substitutions or modifications of amino acids are permissible as long as they would not abrogate the ability of the antibody to bind a given antigen.
  • Suitable human immunoglobulin molecules would include IgGl, IgG2, IgG3, IgG4, IgA and IgM molecules.
  • a "humanized" antibody retains a similar antigenic specificity as the original antibody.
  • Fully human monoclonal antibodies also can be prepared by immunizing mice transgenic for large portions of human immunoglobulin heavy and light chain loci. See, e.g., U.S. Pat. Nos. 5,591,669, 5,598,369, 5,545,806, 5,545,807, 6,150,584, and references cited therein, the contents of which are incorporated herein by reference.
  • mice have been genetically modified such that there is a functional deletion in the production of endogenous (e.g., murine) antibodies.
  • the animals are further modified to contain all or a portion of the human germ-line immunoglobulin gene locus such that immunization of these animals will result in the production of fully human antibodies to the antigen of interest.
  • monoclonal antibodies can be prepared according to standard hybridoma technology. These monoclonal antibodies will have human immunoglobulin amino acid sequences and therefore will not provoke human anti-mouse antibody (KAMA) responses when administered to humans.
  • KAMA human anti-mouse antibody
  • the present invention also provides for F(ab') 2 Fab, Fv and Fd fragments; chimeric antibodies in which the Fc and/or FR and/or CDRl and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; chimeric F(ab')2 fragment antibodies in which the FR and/or CDRl and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; chimeric Fab fragment antibodies in which the FR and/or CDRl and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; and chimeric Fd fragment antibodies in which the FR and/or CDRl and/or CDR2 regions have been replaced by homologous human or non-human sequences.
  • the present invention also includes so-called single chain antibodies.
  • the various antibody molecules and fragments may derive from any of the commonly known immunoglobulin classes, including but not limited to IgA, secretory IgA, IgE, IgG and IgM.
  • IgG subclasses are also well known to those in the art and include but are not limited to human IgGl, IgG2, IgG3 and IgG4.
  • the antibody according to the invention is a single domain antibody.
  • the term "single domain antibody” (sdAb) or “VHH” refers to the single heavy chain variable domain of antibodies of the type that can be found in Camelid mammals which are naturally devoid of light chains. Such VHH are also called “nanobody®”. According to the invention, sdAb can particularly be llama sdAb.
  • the agent is an aptamer which acts as a GPIIb/IIIa antagonist, a thromboxane- A2 -receptor antagonist, an ADP (adenosine diphosphate) receptor antagonist (in particular a antagonist of the purinergic receptors P2Y1 and P2Y12), or a PAR4 antagonist.
  • Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity.
  • Such ligands may be isolated through Systematic Evolution of Ligands by Exponential enrichment (SELEX) of a random sequence library.
  • the random sequence library is obtainable by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer, eventually chemically modified, of a unique sequence.
  • the anti-platelet agent of the present invention is an inhibitor of gene expression wherein the gene is typically selected from the group of genes encoding for GPIIb/IIIa receptort, a thromboxane-A2 -receptor, an ADP (adenosine diphosphate) receptor (in particular the purinergic receptors P2Y1 and P2Y12), or PAR4.
  • An "inhibitor of gene expression” refers to a natural or synthetic compound that has a biological effect to inhibit the expression of a gene.
  • said inhibitor of gene expression is a siRNA, an antisense oligonucleotide or a ribozyme.
  • anti-sense oligonucleotides including anti-sense RNA molecules and anti-sense DNA molecules, would act to directly block the translation of mineralocorticoid receptor mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of mineralocorticoid receptor, and thus activity, in a cell.
  • antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the mRNA transcript sequence encoding mineralocorticoid receptor can be synthesized, e.g., by conventional phosphodiester techniques.
  • Methods for using antisense techniques for specifically inhibiting gene expression of genes whose sequence is known are well known in the art (e.g. see U.S.
  • Small inhibitory RNAs can also function as inhibitors of expression for use in the present invention, mineralocorticoid receptor gene expression can be reduced by contacting a subject or cell with a small double stranded RNA (dsRNA), or a vector or construct causing the production of a small double stranded RNA, such that mineralocorticoid receptor gene expression is specifically inhibited (i.e. RNA interference or RNAi).
  • dsRNA small double stranded RNA
  • RNAi small double stranded RNA
  • Antisense oligonucleotides, siRNAs, shRNAs and ribozymes of the invention may be delivered in vivo alone or in association with a vector.
  • a "vector" is any vehicle capable of facilitating the transfer of the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid to the cells and typically cells expressing mineralocorticoid receptor.
  • the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector.
  • the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid sequences.
  • Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rous sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus.
  • retrovirus such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rous sarcoma virus
  • adenovirus adeno-associated virus
  • SV40-type viruses polyoma viruses
  • Epstein-Barr viruses Epstein-Barr viruses
  • papilloma viruses herpes virus
  • vaccinia virus
  • a "therapeutically effective amount” refers to an amount sufficient to elicit the desired biological response.
  • the desired biological response is to reduce or ameliorate the severity, duration, progression, of the infection, and more particular to reduce or ameliorate the inflammatory burden of the lungs.
  • the precise amount of the anti-platelet agent administered to a subject will depend on the mode of administration, the type and severity of the infection and on the characteristics of the subject, such as general health, age, sex, body weight and tolerance to drugs. The skilled artisan will be able to determine appropriate dosages depending on these and other factors.
  • an "effective amount" of the second agent will depend on the type of drug used. Suitable dosages are known for approved agents and can be adjusted by the skilled artisan according to the condition of the subject, the type of conditio n(s) being treated and the amount of a compound described herein being used. In cases where no amount is expressly noted, an effective amount should be assumed.
  • anti-platelet agents described herein can be administered to a subject in a dosage range from between approximately 0.01 to 100 mg/kg body weight/day for therapeutic or prophylactic treatment.
  • Dosages of the antiplatelet agents described herein can range from between about 0.01 to about 100 mg/kg body weight/day, about 0.01 to about 50 mg/kg body weight/day, about 0.1 to about 50 mg/kg body weight/day, or about 1 to about 25 mg/kg body weight/day. It is understood that the total amount per day can be administered in a single dose or can be administered in multiple dosing, such as twice a day (e.g., every 12 hours), tree times a day (e.g., every 8 hours), or four times a day (e.g., every 6 hours).
  • the anti-platelet agents of the present invention can be administered to a subject within, for example, 48 hours (or within 40 hours, or less than 2 days, or less than 1.5 days, or within 24 hours) of onset of symptoms (e.g., nasal congestion, sore throat, cough, aches, fatigue, headaches, and chills/sweats).
  • the therapeutic treatment can last for any suitable duration, for example, for 5 days, 7 days, 10 days, 14 days, etc.
  • the anti-platelet agents of the present invention are use in combination with an additional suitable therapeutic agent, for example, an antiviral agent or a vaccine.
  • an additional suitable therapeutic agent for example, an antiviral agent or a vaccine.
  • an effective amount can be achieved using a first amount of a anti-platelet agent and a second amount of an additional suitable therapeutic agent (e.g. an antiviral agent or vaccine).
  • neuraminidase inhibitors include oseltamivir, oseltamivir carboxylate (GS4071; see e.g. Eisenberg et al., Antimicrob Agents Chemother.
  • M2 inhibitors include include amino adamantane compounds such as amantadine (1-amino-adamantane), rimantadine (l-(l-aminoethyl)adamantane), spiro[cyclopropane-l,2'-adamantan]-2-amine, spiro[pyrrolidine-2,2'-adamantane], spiro[piperidine-2,2'-adamantane], 2-(2-adamantyl)piperidine, 3-(2-adamantyl)pyrrolidine, 2- (1-adamantyl) piperidine, 2-(l-adamantyl)pyrrolidine, and 2-(ladamantyl)-2-methyl- pyrrolidine; and M2-specific monoclonal antibodies (see e.g.
  • RNA polymerase inhibitors refers to an antiviral agent that inhibits the polymerase, protease, and/or endonuclease activity of the viral RNA polymerase complex or one of its subunits (i.e. PB1, PB2andPA).
  • RNA polymerase inhibitors include antiviral nucleoside analogs such as ribavirin, viramidine, 6- fluoro-3-hydroxy-2pyrazinecarboxamide (T-705), 2'-deoxy-2'-fluoroguanosine, pyrazofurin, 3-deazaguanine, carbodine (see e.g. Shannon et al, Antimicrob Agents Chemother. (1981) 20:769-76), and cyclopenenyl cytosine (see e.g. Shigeta et al, Antimicrob Agents Chemother. (1988) 32:906-11); and the endonuclease inhibitor flutimide (see e.g. Tomassini et al, Antimicrob Agents Chemother. (1996) 40: 1189-93).
  • antiviral nucleoside analogs such as ribavirin, viramidine, 6- fluoro-3-hydroxy-2pyrazinecarboxamide (T-705), 2'-deoxy-2'-
  • influenza-specific interfering oligonucleotides examples include siRNAs (see e.g. Zhou et al, Antiviral Res. (2007) 76: 186-93), antisense oligonucleotides, phosphorothioate oligonucleotides, ribozymes (see e.g. U.S. Pat. No. 6,258,585 to Draper), morpholino oligomers and peptide nucleic acids (see e.g. Schubert and Kurreck, Handb Exp Pharmacol. (2006) 173:261-87).
  • siRNAs see e.g. Zhou et al, Antiviral Res. (2007) 76: 186-93
  • antisense oligonucleotides examples include phosphorothioate oligonucleotides, ribozymes (see e.g. U.S. Pat. No. 6,258,585 to Draper), morpholino oligomers and
  • interferons include interferons.
  • An "interferon” or “IFN”, as used herein, is intended to include any molecule defined as such in the literature, comprising for example any types of IFNs (type I and type II) and in particular, IFN-alpha, IFN-beta, INF-omega and IFN-gamma.
  • the term interferon, as used herein, is also intended to encompass salts, functional derivatives, variants, muteins, fused proteins, analogs and active fragments thereof. In a preferred embodiment the interferon is interferon-alpha.
  • Interferon-alpha includes, but is not limited to, recombinant interferon-a2a (such as ROFERON® interferon available from Hoffman-LaRoche, Nutley, N.J.), interferon- a2b (such as Intron-A interferon available from Schering Corp., Kenilworth, N.J., USA), a consensus interferon, and a purified interferon-a product.
  • a combination therapy comprises active immunization with an influenza antigenic polypeptide (e.g. influenza hemagglutinin and the matrix 2 ectodomain polypeptides) or passive immunization with one or more neutralizing antibodies directed to an influenza antigenic polypeptide (e.g. antibodies raised against the influenza hemagglutinin and the matrix 2 ectodomain polypeptides).
  • an influenza antigenic polypeptide e.g. influenza hemagglutinin and the matrix 2 ectodomain polypeptides
  • the anti-platelet agents of the present invention are typically formulated into pharmaceutical compositions that further comprise a pharmaceutically acceptable carrier, diluent, adjuvant or vehicle.
  • the present invention relates to a pharmaceutical composition comprising a anti-platelet agent described above, and a pharmaceutically acceptable carrier, diluent, adjuvant or vehicle.
  • the present invention is a pharmaceutical composition comprising an effective amount of a compound of the present invention or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier, diluent, adjuvant or vehicle.
  • Pharmaceutically acceptable carriers include, for example, pharmaceutical diluents, excipients or carriers suitably selected with respect to the intended form of administration, and consistent with conventional pharmaceutical practices.
  • a pharmaceutically acceptable carrier may contain inert ingredients which do not unduly inhibit the biological activity of the compounds.
  • the pharmaceutically acceptable carriers should be biocompatible, e.g., non-toxic, non-inflammatory, non-immunogenic or devoid of other undesired reactions or side-effects upon the administration to a subject. Standard pharmaceutical formulation techniques can be employed.
  • the pharmaceutically acceptable carrier, adjuvant, or vehicle includes any and all solvents, diluents, or other liquid vehicle, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired.
  • Remington's Pharmaceutical Sciences, Sixteenth Edition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1980) discloses various carriers used in formulating pharmaceutically acceptable compositions and known techniques for the preparation thereof.
  • any conventional carrier medium is incompatible with the anti-platelet agents of the present invention, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutically acceptable composition, its use is contemplated to be within the scope of this invention.
  • materials which can serve as pharmaceutically acceptable carriers include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins (such as human serum albumin), buffer substances (such as twin 80, phosphates, glycine, sorbic acid, or potassium sorbate), partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes (such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, or zinc salts), colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, polyacrylates, waxes, polyethylene- polyoxypropylene-block polymers, methylcellulose, hydroxypropyl methylcellulose, wool fat, sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powder
  • compositions described herein may be administered orally, parenterally, by inhalation spray, rectally, nasally, buccally, or r via an implanted reservoir depending on the severity of the infection being treated.
  • parenteral as used herein includes, but is not limited to, subcutaneous, intravenous, intramuscular, intra-articular, intra- synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques.
  • the compositions are administered orally, intraperitoneally or intravenously.
  • Sterile injectable forms of the compositions described herein may be aqueous or oleaginous suspension.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension in a nontoxic parenterally-acceptable diluent or solvent, for example as a solution in 1,3-butanediol.
  • a nontoxic parenterally-acceptable diluent or solvent for example as a solution in 1,3-butanediol.
  • acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono- or di-glycerides.
  • Fatty acids such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically- acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions.
  • oils such as olive oil or castor oil
  • These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, such as carboxymethyl cellulose or similar dispersing agents which are commonly used in the formulation of pharmaceutically acceptable dosage forms including emulsions and suspensions.
  • a long-chain alcohol diluent or dispersant such as carboxymethyl cellulose or similar dispersing agents which are commonly used in the formulation of pharmaceutically acceptable dosage forms including emulsions and suspensions.
  • surfactants such as Tweens, Spans and other emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms may also be used for the purposes of formulation.
  • compositions described herein may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous suspensions or solutions.
  • carriers commonly used include, but are not limited to, lactose and corn starch.
  • Lubricating agents such as magnesium stearate, are also typically added.
  • useful diluents include lactose and dried cornstarch.
  • aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening, flavoring or coloring agents may also be added.
  • the pharmaceutical compositions described herein may be administered in the form of suppositories for rectal administration. These can be prepared by mixing the agent with a suitable non-irritating excipient which is solid at room temperature but liquid at rectal temperature and therefore will melt in the rectum to release the drug. Such materials include, but are not limited to, cocoa butter, beeswax and polyethylene glycols.
  • the pharmaceutical compositions may also be administered to the respiratory tract.
  • the respiratory tract includes the upper airways, including the oropharynx and larynx, followed by the lower airways, which include the trachea followed by bifurcations into the bronchi and bronchioli.
  • Pulmonary delivery compositions can be delivered by inhalation by the patient of a dispersion so that the active ingredient within the dispersion can reach the lung where it can, for example, be readily absorbed through the alveolar region directly into blood circulation.
  • Pulmonary delivery can be achieved by different approaches, including the use of nebulized, aerosolized, micellular and dry powder-based formulations; administration by inhalation may be oral and/or nasal. Delivery can be achieved with liquid nebulizers, aerosol-based inhalers, and dry powder dispersion devices. Metered-dose devices are preferred.
  • One of the benefits of using an atomizer or inhaler is that the potential for contamination is minimized because the devices are self contained.
  • Dry powder dispersion devices for example, deliver drugs that may be readily formulated as dry powders.
  • a pharmaceutical composition of the invention may be stably stored as lyophilized or spray- dried powders by itself or in combination with suitable powder carriers.
  • the delivery of a pharmaceutical composition of the invention for inhalation can be mediated by a dosing timing element which can include a timer, a dose counter, time measuring device, or a time indicator which when incorporated into the device enables dose tracking, compliance monitoring, and/or dose triggering to a patient during administration of the aerosol medicament.
  • Examples of pharmaceutical devices for aerosol delivery include metered dose inhalers (MDIs), dry powder inhalers (DPIs), and air-jet nebulizers.
  • the compounds for use in the methods of the invention can be formulated in unit dosage form.
  • unit dosage form refers to physically discrete units suitable as unitary dosage for subjects undergoing treatment, with each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, optionally in association with a suitable pharmaceutical carrier.
  • the unit dosage form can be for a single daily dose or one of multiple daily doses (e.g., about 1 to 4 or more times per day). When multiple daily doses are used, the unit dosage form can be the same or different for each dose.
  • FIGURES
  • Figure 1 Upon IAV infection, platelets infiltrate the lungs, and IAV particles are observed in platelets.
  • A Immunohistochemistry analysis of lungs from uninfected (NI) or infected mice inoculated with A/PR/8/34 virus, at a sublethal dose (75 pfu/mouse) or LDso (250 pfu/mouse; day 6 post-infection). Antibodies against the IAV nucleoprotein (NP) and CD41 were used to detect virus infected cells and platelets, respectively. The results are representative of three mice per group.
  • B Platelet numbers in BAL were assessed using a Vet ABCTM Hematology Analyzer on day 6 post-inoculation of mock or IAV-infected mice.
  • Black arrows indicate viral particles. Staining of a platelet-like granule is shown on the upper right panel. As a control for HA staining, electron microscopic immunogold labeling was performed on purified A/PR/8/34 viruses using the anti-HA antibody (lower right panel).
  • FIG. 2 Upon IAV infection, platelets are stimulated and contribute to influenza pathogenesis.
  • Serotonin and sP-selectin were measured by ELISA in the BAL and plasma of Mock (NI) or A/PR 8/34 virus-infected mice, respectively, on day 6 post- inoculation (75 pfu/mouse, sublethal dose or 250 pfu/mouse, LDso).
  • Data represent the means ⁇ SEM of 4 mice per group, * p ⁇ 0.05 for LD 50 vs. NI; ** p ⁇ 0.01 for LD 50 vs. NI.
  • FIG. 3 Platelet activation and inflammation.
  • A Platelet numbers in BAL of A/PR/8/34 virus infected mice (250 pfu/mouse, LDso) were assessed using a Vet ABCTM Hematology Analyzer, at the indicated time post-inoculation. The results are represented as the means ⁇ SEM of 4 mice per group. On days 0 and 6, additional results from Figure IB are included.
  • B-D sP-selectin in the plasma (B), ILl- ⁇ in the BAL (C) and ILl- ⁇ in the plasma (D) of infected mice (A/PR/8/34, 250 pfu/mouse, LDso) were determined by ELISA at the indicated times.
  • HE hematoxylin and eosin
  • FIG. 6 PAR4 antagonist protects mice against IAV infection and deleterious lung inflammation.
  • Thromboxane B2 was measured by ELISA in the BAL of infected mice (A/PR/8/34, 250 pfu/mouse, LDso) after treatment with pepducin or vehicle, on day 6 post- inoculation. Data represent the mean ⁇ SEM of 4-6 mice per group.
  • C Lung virus titers after infection of mice with A/PR/8/34 virus (250 pfu/mouse, LDso) treated with pepducin or vehicle. The results represent the means ⁇ SEM from 3 individual animals per group.
  • A Ultrastructural analysis of platelets in the lungs of infected mice (A/PR/8/34, 250 pfu/mouse, LDso), treated or not with eptifibatide, was performed by transmission electron microscopy. Note the aggregation of platelets in the lungs of infected mice, and their disaggregation after treatment of mice with eptifibatide. Sections show platelet aggregates with an interstitial localization.
  • B Thromboxane B2 (TXB2) was measured by ELISA in the BAL of infected mice (A/NL/602/09, 30,000 pfu/mouse LDso) after treatment with eptifibatide or vehicle.
  • mice The results represent the means ⁇ SEM of 3-5 mice per group.
  • sP- selectin was measured by ELISA in the plasma of A/PR/8/34 virus-infected mice (250 pfu/mouse, LDso) that were treated or not with eptifibatide, on day 6 post-inoculation.
  • Data represent the means ⁇ SEM of 4 mice per group. * p ⁇ 0.05 for pepducin vs. saline.
  • FIG. 8 Eptifibatide treatment prevents severe inflammation during influenza virus infections.
  • B Lung virus titers after infection of mice with the A/NL/602/09 virus (30,000 pfu/mouse, LDso) treated with eptifibatide or vehicle. Data represent the means ⁇ SEM from 3 individual animals per group.
  • FIG. 9 Histopathological analysis of lungs from infected mice after treatment with eptiflbatide.
  • A Histopathological analysis of lungs obtained from mice inoculated with A/PR/8/34 virus (250 PFU/mouse) and treated or not with eptiflbatide.
  • A/PR/8/34 virus 250 PFU/mouse
  • eptiflbatide the extended areas with interstitial and peribronchial inflammation and interstitial and alveolar hemorrhage.
  • the infected group treated with eptiflbatide note the limited areas with slight peribronchial inflammation but no major hemorrhage.
  • A549 cells and MDCK cells were purchased from ATCC.
  • IAV A/PR 8/34 virus (H1N1), A/HK 1/68 (H3N2) and A/NL/602/2009 (H1N1) (ATCC) were gifts from G.F. Rimmelzwaan (Erasmus, Netherlands).
  • the highly pathogenic avian A FPV/Bratislava/79 (H7N7) strain was from the Institute of Molecular Virology, Munster, Germany.
  • DAPI Life Technologies, Paris, France
  • Alexa Fluor® secondary antibodies Life Technologies
  • eptiflbatide Integrilin ® , GlaxoSmithKline, Marly- le-Roi, France
  • Clopidogrel Santa Cruz Biotechnology, Heidelberg, Germany
  • MRS 2179 Tocris Bioscience, Bristol, United Kingdom
  • PAR4 antagonist pepducin p4pal-10 Polypeptide Laboratories, France
  • PAR4 agonist peptide AYPGKF-NH 2 , Bachem, Weil-am-Rhein, Germany
  • PAR4 control peptide YAPGKF-NH2, Bachem
  • monoclonal anti-neutrophil Ly6G Cedarlane, Tebu-bio, Le Perray en Yvelines, France
  • polyclonal anti-platelet CD41 Bioss, Woburn, USA
  • monoclonal anti- viral HA Santa Cruz Biotechnology, Heidelberg, Germany
  • mice Female, 7-week-old BALB/c mice were used for H7N7 virus infections. Otherwise, 6- week-old C57BL/6 female mice (Charles River Laboratories, Arbresle, France) and GPIIIa " ' mice or wild-type littermates on a C57BL/6 background were used in this study. For the latter, heterozygous mice were crossed, and WT and KO offspring (males and females) were used. Polymerase chain reaction of tail-tip genomic DNA was performed (12) to determine the absence or presence of the GPIIIa gene. Infection experiments were performed as previously described (13).
  • mice were anesthetized with ketamine/xylazine (42.5/5 mg/kg) and inoculated intranasally with IAV, in a volume of 20 ⁇ .
  • Eptifibatide was injected intraperitoneally (500 ⁇ g/kg or 10 ⁇ g/200 ⁇ per mouse of -20 g body weight) every 3 days until the end of the experiment.
  • MRS 2179 was dissolved in saline buffer and administered once intravenously (50 mg/kg) on day 0.
  • Clopidogrel dissolved in saline buffer was injected intraperitoneally (30 mg/kg) every day until the end of the experiment.
  • mice were anesthetized every day for 3 days.
  • mice On the first day, the anesthetized mice were infected intranasally in the presence or absence of PAR4-AP or control peptide (100 ⁇ g/mouse, in a volume of 20 ⁇ ). Intranasal peptide treatments were also repeated on days 2 and 3 after infection.
  • PAR4 antagonist treatment pepducin p4pal-10 was given intraperitoneally (0.5 mg/kg) two days post-infection, and treatments were repeated on the next two days.
  • mice Upon inoculation, the survival rates were followed. Alternatively, mice were sacrificed at prefixed time points to perform BAL or harvest lungs. ELISA was performed according to the manufacturers' instructions. Virus titers were assessed as previously described (14). Lungs were also harvested for histology and immunohistochemistry as previously described (15). Evaluation of hemorrhagic foci by histopathological analysis
  • Lungs from mice inoculated with A/PR/8/34 virus (250 PFU/mouse) with or without eptifibatide treatment were fixed in 10% neutral buffered formalin and embedded in paraffin. Then, 4-6 ⁇ sections were cut and stained with hematoxylin and eosin (H&E) to evaluate histopathological changes. Staining was performed by incubation of the lung sections with Harris hematoxylin for 6 min, running tap water for 1 min, eosin Y for 10 min, 70% ethanol for 1 min, 95% ethanol for 1 min, 100% ethanol for 1 min and two rinses in 100% xylene for 1 min.
  • H&E hematoxylin and eosin
  • Histology and injury scoring were performed by a blinded investigator who analyzed the samples and determined the levels of injury according to a semiquantitative scoring system (counting inflammatory infiltration, vascular congestion, hemorrhage, fibrin deposits and epithelial cell apoptosis).
  • lung tissues were cut into 1-mm 3 pieces, fixed in 2% glutaraldehyde at 4°C, washed in 0.2 M cacodylate-HCl buffer containing 0.4 M saccharose and post-fixed in 0.3 M cacodylate-HCl buffer containing 2% osmium tetroxide for 1 hour. After dehydration in a graded alcohol series, tissue samples were impregnated with a 75% Epon A/25% Epon B/1.7% DMP30 mixture. Tissue embedding entailed polymerization at 60°C for 72 hours.
  • Cells from the BAL were centrifuged at 1,800 rpm for 5 minutes at room temperature and suspended in phosphate buffer saline (PBS) at a concentration of 5xl0 5 /ml. Then, 100 ⁇ of the solution was then used to centrifuge the cells onto coverslips (1,000 rpm for 5 minutes), using a Shandon Cytospin 4 centrifuge. The slides were then dipped in a box containing methanol and kept at -20°C for fixation and permeabilization. After 10 minutes, cells were extensively washed with PBS to remove the fixative. Cells were then incubated with primary antibodies to CD41 and viral HA for 1 hour at room temperature.
  • PBS phosphate buffer saline
  • Platelets were counted using the Vet ABCTM Hematology Analyzer (SCIL) using the mouse smart card 7030.
  • SCIL Vet ABCTM Hematology Analyzer
  • the automated cell counter differentiates mouse platelets based on their size in multiple sample fluids. Leukocytes and neutrophils in the BAL were visualized by May-Grunwald Giemsa stained cytospin preparations, as previously performed (13). Flow cytometry of blood platelets
  • the Kaplan-Meier test was used for survival rates.
  • the Mann- Whitney test was used for two-group comparisons of mean percentages in the flow cytometry experiments, lung virus titers, ELISA and total protein quantifications.
  • One-way ANOVA for non-parametric measures was used for multiple-group comparisons in dose-responses or kinetics experiments.
  • Dunn's multiple comparison test was employed as a post hoc test using NI as a control. Probabilities *p ⁇ 0.05, ** p ⁇ 0.01 , *** p ⁇ 0.001 were considered statistically significant.
  • Platelet recruitment to the lungs was first examined after infection of mice with a sublethal or a 50% lethal dose (LD 50 ) of IAV A/PR/8/34. Immunohistochemistry of the lungs, using monoclonal antibodies for IAV nucleoprotein (NP) and CD41, was used to detect virus- infected cells and platelets, respectively ( Figure 1A). At both doses, many IAV-infected cells and marked platelet infiltrates were detected in the lungs of infected mice compared to uninfected mice. To confirm these results, platelet counts in the BAL of infected versus uninfected mice (sublethal dose or LD 50 ) were assessed using a blood cell counter (Figure IB).
  • the platelet levels increased in a dose-dependent manner and were significantly higher than in those of uninfected mice, reaching 50 ⁇ 10 9 cells/L on day 6 post-inoculation (LD 50 ). Differences were not significant upon infection with IAV at the sublethal dose. Viral proteins are present within platelets
  • Platelets contribute to influenza pathogenesis
  • GPIIIa " ' mice which were then infected with IAV A/PR/8/34, and the survival rates were monitored. As shown in Figure 2D, compared to WT mice, GPIIIa ⁇ ⁇ mice were significantly more resistant to IAV-induced death. Time course of platelet activation, ILl-beta release and platelet binding to leukocytes
  • Platelets were counted in the BAL of infected mice (LD 50 ) at various times post- inoculation. Upon infection, platelet counts increased in a time-dependent manner (Figure 3 A), peaked on day 3 and stayed elevated until day 8. Plasmatic sP-selectin significantly increased during the course of infection and plateaued on days 3-8 ( Figure 3B). Increased ILl-beta was also detected in the BAL and blood of infected mice but with different lags ( Figure 3C-D). ILl-beta was released in the BAL paralleled platelet activation, whereas ILl- beta peaked in the blood on day 2 post-inoculation and then rapidly decreased.
  • PAR4 promotes pathogenesis of IAV infection in a platelet-dependent pathway
  • mice were inoculated with a sublethal dose of IAV A/PR/8/34 and stimulated with 100 ⁇ g/mouse of the PAR4 agonist peptide AYPGKF-NH 2 (PAR4-AP) or the inactive control peptide YAPGKF-NH 2 (Control-P).
  • PAR4-AP the PAR4 agonist peptide AYPGKF-NH 2
  • Control-P the inactive control peptide YAPGKF-NH 2
  • the anti-platelet drug eptifibatide protects mice from lethal influenza infection
  • mice were inoculated with IAV A/PR/8/34 (LD 50 ) and were treated or not with 500 ⁇ g/kg of eptifibatide every 3 days. This dosage is comparable to the lowest doses used clinically in humans (20-22).
  • Eptifibatide treatment had a dramatic effect on lung infiltration by platelets: platelet aggregation was totally prevented, and only isolated platelets were observed ( Figure 7A). Furthermore, this effect was accompanied by decreases in TXB2 and sP-selectin in the fluid of infected mice compared to controls ( Figure 7B), showing that inhibition of platelet aggregation also limited the extent of platelet activation.
  • Eptifibatide treatment protects mice from hemorrhage induced by influenza
  • Eptifibatide markedly reduced the severity of pulmonary injury induced by influenza virus infections, and a marked reduction in neutrophil infiltration was observed. (Figure 9A-B). More importantly, almost no hemorrhage was detected in the lungs of infected mice treated with eptifibatide.
  • the present study shows that platelets play an active role in fueling the dysregulation of inflammation and promoting pathogenesis of influenza virus infections. Histological analysis of lungs provided evidence that platelets massively infiltrate the lungs of infected mice. Additionally, infiltrated platelets stained positive for viral HA, based on immunofluorescence staining of BAL and immunogold labeling of ultrathin cryosections of lungs. The technical limitation of the staining did not allow us to determine whether platelets engulfed the entire virions, only IAV fragments or antigens. However, because platelets incorporate influenza viruses in vitro (23), our results suggest that platelets recruited to the lungs most likely take up IAV particles in vivo as well.
  • Platelets contribute to the host defense against bacterial infectious agents by limiting vascular lesions and inducing injury repair (28, 29). However, unbalanced platelet activation may have pathological consequences. In our influenza model, platelet activation and aggregation were deleterious. PAR4 and GPIIIa are both key molecules in platelet function. PAR4 is strictly required for platelet activation in mice, while GPIIIa is required for platelet aggregation. First, mice deficient in GPIIIa were protected from lung injury and death. Furthermore, stimulation of PAR4 increased lung inflammation and the severity of IAV infection. In contrast, PAR4 antagonists protected mice from death.
  • thrombin-mediated platelet activation most likely occurs through PAR4 activation, but thrombin activation of PARI may also be involved in the pathogenesis of IAV infection. Indeed, we recently found that PARI signaling contributes to IAV pathogenicity in mice (33). In this context, PARI cooperates with plasminogen, which controls pathogenesis, via fibrinolysis (34). Thus, investigations into the role of hemostasis dysregulation may help better understand IAV pathogenesis (35-37).
  • cytokine production induced by platelet activation was only observed at later time points after infection.
  • the virus Upon infection, the virus is recognized as foreign by highly conserved receptors known as pattern recognition receptors. Activation of these receptors results in the secretion of cytokines and chemokines, which corresponds to the early inflammatory response against IAV infection (35).
  • pattern recognition receptors Activation of these receptors results in the secretion of cytokines and chemokines, which corresponds to the early inflammatory response against IAV infection (35).
  • the amplification and intensity of inflammation depends on the replicative capacity of the virus.
  • a resolution phase of inflammation is engaged at later time points postinfection, and this partly determines the duration of inflammation. Resolution of inflammation is largely influenced by the vascular endothelium (43). Upon injury of the latter, platelets are activated.
  • Annexin v incorporated into influenza virus particles inhibits gamma interferon signaling and promotes viral replication. J Virol 2014;88: 11215-11228. 18. LeBouder F, Lina B, Rimmelzwaan GF, Riteau B. Plasminogen promotes influenza a virus replication through an annexin 2-dependent pathway in the absence of neuraminidase. J Gen Virol 2010;91 :2753-2761.

Abstract

L'invention concerne des méthodes et des compositions destinées au traitement d'une infection par le virus de la grippe A. Elle concerne en particulier une méthode de traitement d'une infection par le virus de la grippe A (IAV) chez un sujet qui en a besoin, la méthode consistant à administrer au sujet une quantité thérapeutiquement efficace d'au moins un anti-agrégant plaquettaire.
PCT/EP2015/053318 2014-02-18 2015-02-17 Méthodes et composition pharmaceutique pour le traitement de l'infection par le virus de la grippe a WO2015124570A1 (fr)

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US9963466B2 (en) 2016-03-07 2018-05-08 Vanderbilt University Substituted 5-membered heterocyclic analogs as protease activated receptor 4 (PAR-4) antagonists
US11306099B1 (en) 2016-03-11 2022-04-19 Angel Pharmaceutical Co., Ltd. Compounds and methods for modulating Bruton's Tyrosine Kinase

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