WO2009129164A1 - Combinaisons pharmaceutiques de fragments de tfpi et d'un antibiotique - Google Patents

Combinaisons pharmaceutiques de fragments de tfpi et d'un antibiotique Download PDF

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WO2009129164A1
WO2009129164A1 PCT/US2009/040322 US2009040322W WO2009129164A1 WO 2009129164 A1 WO2009129164 A1 WO 2009129164A1 US 2009040322 W US2009040322 W US 2009040322W WO 2009129164 A1 WO2009129164 A1 WO 2009129164A1
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tfpi
gram
set forth
animal
antibiotic
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PCT/US2009/040322
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English (en)
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Stephen Hardy
Yumin Dai
Sabine Schirm
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Novartis Ag
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    • 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
    • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
    • 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
    • A61K38/14—Peptides containing saccharide radicals; Derivatives thereof, e.g. bleomycin, phleomycin, muramylpeptides or vancomycin
    • 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
    • A61K38/55—Protease inhibitors
    • A61K38/57—Protease inhibitors from animals; from humans
    • 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
    • A61P31/04—Antibacterial agents

Definitions

  • Clotting is an example of the most primitive immune system. Clots entrap bacteria and prevent the spread of infection through the systemic circulation. However, Gram-positive pathogens may have adapted to use clotting to their advantage. These bacteria bind to fibrin through their fibrin binding proteins and thereby evade phagocytosis. There is therefore a continuing need in the art for effective methods and compositions for treating Gram-positive bacterial infections and/or improving the current methods of treating these infections.
  • compositions for treating a Gram-positive bacterial infection in an animal comprising a C-terminal polypeptide of TFPI and an antibiotic in a pharmaceutically acceptable carrier.
  • said C-terminal polypeptide of TFPI consists essentially of the amino acid sequence set forth in SEQ ID NO:3, 4, 5 or 8.
  • said C-terminal polypeptide of TFPI consists of the amino acid sequence set forth in SEQ ID NO:3, 4, 5 or 8.
  • said antibiotic is vancomycin or erythromycin.
  • the pharmaceutical composition further comprises TFPI or ala-TFPI.
  • said ala-TFPI consists of the amino acid sequence set forth in SEQ ID NO:2.
  • said Gram-positive bacterial infection results from infection by Staphylococcus aureus or Streptococcus pneumonia. In one embodiment, said Gram-positive bacterial infection results in severe community acquired pneumonia or sepsis.
  • methods of treating a Gram-positive bacterial infection in an animal comprising administering a therapeutically effective amount of the pharmaceutical composition set forth above to a subject in need thereof.
  • a therapeutically effective amount of a pharmaceutical composition as set forth above in the manufacture of a medicament for the treatment of a Gram- positive bacterial infection in an animal.
  • compositions for treating a Gram-positive bacterial infection in an animal comprising a C-terminal polypeptide of TFPI and an antibiotic for sequential or simultaneous administration.
  • said C-terminal polypeptide of TFPI consists essentially of the amino acid sequence set forth in SEQ ID N0:3, 4, 5 or 8.
  • said C-terminal polypeptide of TFPI consists of the amino acid sequence set forth in SEQ ID N0:3, 4, 5 or 8.
  • said antibiotic is vancomycin or erythromycin.
  • the pharmaceutical combination further comprises TFPI or ala-TFPI.
  • said ala-TFPI consists of the amino acid sequence set forth in SEQ ID N0:2.
  • said Gram-positive bacterial infection results from infection by Staphylococcus aureus or Streptococcus pneumonia. In one embodiment, said Gram-positive bacterial infection results in severe community acquired pneumonia or sepsis.
  • Disclosed herein is the use of a therapeutically effective amount of a pharmaceutical combination set forth above in the manufacture of a medicament for the treatment of a Gram- positive bacterial infection in an animal.
  • methods of treating a Gram-positive bacterial infection in an animal comprising administering a therapeutically effective amount of the pharmaceutical combination set forth above to a subject in need thereof.
  • cytokine induction by Gram-positive bacteria comprising administering a C-terminal polypeptide of TFPI and an antibiotic to a plurality of cells having cytokine induction due to a Gram- positive bacteria
  • said C-terminal polypeptide of TFPI consists essentially of the amino acid sequence set forth in SEQ ID NO:3, 4, 5 or 8.
  • said C-terminal polypeptide of TFPI consists of the amino acid sequence set forth in SEQ ID NO:3, 4, 5 or 8.
  • said antibiotic is vancomycin or erythromycin.
  • the method further comprises administering TFPI or ala-TFPI to the plurality of cells.
  • said Gram-positive bacterial infection results from infection by Staphylococcus aureus or Streptococcus pneumonia.
  • said Gram-positive bacterial infection results in severe community acquired pneumonia or sepsis.
  • a C-terminal polypeptide of TFPI and an antibiotic for simultaneous, separate or sequential administration to a subject suffering from a Gram-positive bacterial infection.
  • a C-terminal polypeptide of TFPI and an antibiotic for combined use in treating a Gram-positive bacterial infection in an animal Disclosed herein is a combination of a C-terminal polypeptide of TFPI and an antibiotic for use in treating a Gram- positive bacterial infection in an animal.
  • a C-terminal polypeptide of TFPI and an antibiotic for use in a method of treating a Gram- positive bacterial infection in an animal.
  • a C-terminal polypeptide of TFPI for use in a method of treating a Gram-positive bacterial infection in an animal, wherein the C-terminal polypeptide of TFPI is administered simultaneously, separately or sequentially with an antibiotic.
  • an antibiotic for use in a method of treating a Gram-positive bacterial infection in an animal, wherein the antibiotic is administered with a C-terminal polypeptide of TFPI.
  • a C-terminal polypeptide of TFPI in the manufacture of medicament for treating a Gram-positive bacterial infection in an animal, wherein said medicament is prepared for administration with an antibiotic.
  • Disclosed herein is the use of an antibiotic in the manufacture of medicament for treating a Gram-positive bacterial infection in an animal, wherein said medicament is prepared for administration with a C-terminal polypeptide of TFPI.
  • a C-terminal polypeptide of TFPI in the manufacture of a medicament for treating a Gram-positive bacterial infection in an animal, wherein the animal has previously been treated with or will subsequently be treated with an antibiotic.
  • Disclosed herein is the use of an antibiotic in the manufacture of medicament for treating a Gram-positive bacterial infection in an animal, wherein the animal has previously been treated with or will subsequently be treated with a C-terminal polypeptide of TFPI.
  • a C-terminal polypeptide of TFPI, TFPI or ala-TFPI and an antibiotic for simultaneous, separate or sequential administration to an animal suffering from a Gram-positive bacterial infection.
  • a C-terminal polypeptide of TFPI, TFPI or ala-TFPI and an antibiotic for combined use in treating a Gram-positive bacterial infection in an animal.
  • Disclosed herein is a combination of a C-terminal polypeptide of TFPI, TFPI or ala-TFPI and an antibiotic for use in treating a Gram-positive bacterial infection in an animal.
  • a C-terminal polypeptide of TFPI, TFPI or ala-TFPI and an antibiotic for use in a method of treating a Gram-positive bacterial infection in an animal.
  • a C-terminal polypeptide of TFPI for use in a method of treating a Gram-positive bacterial infection in an animal, wherein the C-terminal polypeptide of TFPI is administered with an antibiotic and TFPI or ala-TFPI.
  • an antibiotic for use in a method of treating a Gram-positive bacterial infection in an animal, wherein the antibiotic is administered with a C-terminal polypeptide of TFPI and TFPI or ala-TFPI.
  • TFPI or ala-TFPI for use in a method of treating a Gram-positive bacterial infection in an animal, wherein the TFPI or ala-TFPI is administered with a C-terminal polypeptide of TFPI and an antibiotic.
  • a C-terminal polypeptide of TFPI in the manufacture of medicament for treating a Gram-positive bacterial infection in an animal, wherein said medicament is prepared for administration with an antibiotic and TFPI or ala-TFPI.
  • Disclosed herein is the use of an antibiotic in the manufacture of medicament for treating a Gram-positive bacterial infection in an animal, wherein said medicament is prepared for administration with a C-terminal polypeptide of TFPI and TFPI or ala-TFPI.
  • TFPI or ala-TFPI in the manufacture of medicament for treating a Gram-positive bacterial infection in an animal, wherein said medicament is prepared for administration with an antibiotic and a C- terminal polypeptide of TFPI.
  • a C-terminal polypeptide of TFPI in the manufacture of a medicament for treating a Gram-positive bacterial infection in an animal, wherein the animal has previously been treated with or will subsequently be treated with an antibiotic and TFPI or ala-TFPI.
  • Disclosed herein is the use of an antibiotic in the manufacture of medicament for treating a Gram-positive bacterial infection in an animal, wherein the animal has previously been treated with or will subsequently be treated with a C-terminal polypeptide of TFPI and TFPI or ala-TFPl.
  • TFPI or ala-TFPI in the manufacture of medicament for treating a Gram-positive bacterial infection in an animal, wherein the animal has previously been treated with or will subsequently be treated with a C-terminal polypeptide of TFPI and an antibiotic.
  • a C-terminal polypeptide of TFPI in the manufacture of a medicament for treating a Gram-positive bacterial infection in an animal, wherein the animal has previously been treated with or will subsequently be treated with TFPI or ala-TFPI, and wherein the animal has previously been treated with or will subsequently be treated with an antibiotic.
  • Disclosed herein is the use of an antibiotic in the manufacture of medicament for treating a Gram-positive bacterial infection in an animal, wherein the animal has previously been treated with or will subsequently be treated with a C-terminal polypeptide of TFPI, and wherein the animal has previously been treated with or will subsequently be treated with TFPI or ala-TFPI.
  • TFPI or ala-TFPI in the manufacture of medicament for treating a Gram-positive bacterial infection in an animal, wherein the animal has previously been treated with or will subsequently be treated with a C-terminal polypeptide of TFPI 1 and wherein the animal has previously been treated with or will subsequently be treated with an antibiotic.
  • said C-terminal polypeptide of TFPI consists essentially of the amino acid sequence set forth in SEQ ID NO:3, 4, 5 or 8. In one embodiment of the above uses, said C-terminal polypeptide of TFPI consists of the amino acid sequence set forth in SEQ ID NO:3, 4, 5 or 8. In one embodiment of the above uses, said antibiotic is vancomycin or erythromycin. In one embodiment of the above uses, said TFPI or ala-TFPI is ala-TFPi, and wherein said ala-TFPI consists of the amino acid sequence set forth in SEQ ID N0:2. In one embodiment of the above uses, said Gram-positive bacterial infection results from infection by Staphylococcus aureus or Streptococcus pneumonia. In one embodiment, said Gram-positive bacterial infection results in severe community acquired pneumonia or sepsis.
  • a commercial package for the treatment of a Gram-positive bacterial infection comprising TFPI or ala-TFPI, a C-terminal polypeptide of TFPI, and an antibiotic for simultaneous, separate or sequential administration.
  • said C-terminal polypeptide of TFPI consists essentially of the amino acid sequence set forth in SEQ ID N0:3, 4, 5 or 8.
  • said C-terminal polypeptide of TFPI consists of the amino acid sequence set forth in SEQ ID N0:3, 4, 5 or 8.
  • said antibiotic is vancomycin or erythromycin.
  • said TFPI or ala-TFPI is ala-TFPI, and wherein said ala-TFPI consists of the amino acid sequence set forth in SEQ ID N0:2.
  • said Gram- positive bacterial infection results from infection by Staphylococcus aureus or Streptococcus pneumonia, In one embodiment, said Gram-positive bacterial infection results in severe community acquired pneumonia or sepsis.
  • Combinations comprise: 1) C-terminal polypeptides of TFPI in combination with an antibiotic; and 2) C-terminal polypeptides of TFPI in combination with TFPI or a TFPl analog and an antibiotic.
  • Tissue Factor Pathway Inhibitor is a powerful anticoagulant thought to have anti-inflammatory activity [1]. TFPI can be used to inhibit angiogenesis associated with, for example, tumors [2J.
  • the protein has several principal domains: three serine protease inhibitor domains of the Kunitz type (K1, K2 and K3), an N-terminal domain (NTD), and a C-terminal domain (CTD).
  • K1 domain inhibits clotting factor Vila- tissue factor (TF) complex.
  • TF tissue factor
  • CTD C-terminal domain
  • the K1 domain inhibits clotting factor Vila- tissue factor (TF) complex.
  • the K2 domain inhibits factor Xa.
  • the CTD is also involved in celi association, heparin binding, and optimal Xa inhibition.
  • TFPI refers to the mature serum glycoprotein having the 276 amino acid residue sequence shown in SEQ ID NO:1 and a molecular weight of about 38,000 Daltons without glycosylation.
  • the native protein has a molecular weight of 45,400 Daltons when glycosylate is present [4].
  • the cloning of the TFPi cDNA is described in reference 5.
  • TFPI used herein may be non-glycosylated or glycosylated.
  • TFPI analog is a derivative of TFPI modified with one or more amino acid additions or substitutions, for example from one to eighty (generally conservative in nature and preferably in non-Kunitz domains or in the C-terminal portion of the protein), one or more amino acid deletions, for example from one to eighty (e.g., TFPI fragments), or the addition of one or more chemical moieties to one or more amino acids, so long as the modifications do not destroy TFPI biological activity.
  • the activity that is not destroyed can include TFPI's anticoagulant activity and/or its anti- bacteria I activity, as well as its activity in the prothrombin assay.
  • TFPI analogs comprise all three Kunitz domains.
  • TFPI and TFPI analogs can be either glycosylated or non-glycosylated.
  • a TFPI analog should retain its C-terminal domain (CTD), as this region is where the anti-bacterial activity has been localized.
  • CCD C-terminal domain
  • At least 50% [e.g., >60%, >70%, >80%, >90%, >92%, >94%, >96%, >98%, >99%, or more) by number of the TFPI analog molecules in a composition should be uncleaved at the thrombin cleavage site present between amino acids 254 and 255 of TFPI.
  • a preferred TFPI analog is N-L-alanyl-TFPI (ala-TFPI), whose amino acid sequence is shown in SEQ ID NO:2.
  • AIa-TFPI is also known under the international drug name "tifacogin".
  • the amino terminal alanine residue of ala- TFPI was engineered into the TFPI sequence to improve Ecoli expression [6].
  • Endogenous TFPI is secreted and expressed with a signal peptide.
  • the amino terminal methionine is part of the signal peptide and not part of the mature TFPI.
  • Other analogs of TFPI are described in reference [7].
  • TFPI analogs possess some measure of the activity of TFPI as determined by a bioactivity assay (for example, see refs. [8] & [9] as described below).
  • TFPI has three thrombin cleavage sites; (i) between Lys-86 & Thr-87, between K1 & K2; (ii) between Arg-107 & Gly-108 (the reactive site toward factor Xa in K2); and (iii) between Lys-254 & Thr-255 in the C-terminal basic region. It has been found that anti-bacterial activity of TFPI resides in the CTD, and in particular in the region proximal to and/or downstream of the thrombin cleavage site between Lys-254 and Thr-255 in SEQ ID NO:1.
  • TFPI analog wherein the analog lacks the thrombin cleavage site found near the
  • TFPI C-terminus of natural TFPI
  • analog lacks the thrombin cleavage site present between amino acids Lys-254 and Thr-255 of natural TFPI
  • a TFPI analog wherein the analog comprises (i) at least one Kunitz domain and (ii) a C-terminal region, but wherein the analog does not have a thrombin cleavage site between its most C-terminal Kunitz domain and the C-terminal region
  • thrombin cleavage site between its most C-terminal Kunitz domain and the C-terminal region
  • thrombin a TFPI analog, wherein the analog cannot be cleaved by thrombin to give a N-terminal polypeptide that includes a Kunitz domain and a C-terminal polypeptide that does not include a Kunitz domain
  • a TFPI analog wherein the analog contains fewer than two (i.e., one or none) Lys-Thr dipeptides.
  • the natural cleavage site can be removed in various ways.
  • the lysine and/or the threonine can be substituted with different amino acids to give a dipeptide that is not recognized by thrombin.
  • the lysine and/or the threonine can be deleted.
  • one or more amino acids can be inserted between the lysine and the threonine.
  • the TFPI analog can be incubated with thrombin in a test digestion to confirm that the natural C-terminus cleavage no longer takes place.
  • TFPI analog wherein the analog includes Kunitz domain 3, but lacks the C-terminus domain
  • TFPI analog wherein the analog is a TFPI that has been truncated by up to q ⁇ q is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40) amino acids from the C-terminus.
  • C-terminus truncation of TFPI has been reported previously, but this has usually been in combination with deletion of K3.
  • TFPI has three thrombin cleavage sites: (i) between Lys-86 & Thr-87; (ii) between Arg-107 & Gly-108; and (iii) between Lys-254 & Thr-255. It has been found that the anti-bacteriat activity of TFPI resides in the C-terminal basic region and, in particular, in the region proximal to and/or downstream of the thrombin cleavage site between Lys-254 and Thr-255 in SEQ ID NO:1. ⁇ International Patent Publication No. WO2007014199).
  • the C-terminal polypeptides of TFPI based on the CTD of TFPI may be used, e.g., in pharmaceutical compositions as anti-bacterial agents, methods of treating of Gram-positive bacterial infections, methods of modulating a cytokine response to Gram-positive bacteria and in the manufacture of medicaments for treating such infections.
  • TFPI C-terminal polypeptides of TFPI in combination with antibiotics, e.g., vancomycin and erythromycin
  • antibiotics e.g., vancomycin and erythromycin
  • the synergistic activity of such peptides in combination with TFPI or ala-TFPI and antibiotics e.g., vancomycin and erythromycin.
  • TFPI C-terminal polypeptides of TFPI: (1) a polypeptide consisting of amino acid sequence SEQ ID NO:3, 4, 5 or 8; (2) a polypeptide comprising amino acid sequence SEQ ID NCW 1 4, 5 or 8, provided that the polypeptide is not TFPI or a TFPI analog; (3) a polypeptide comprising amino acid sequence SEQ ID NO:3, 4, 5 or 8, provided that the amino acid (if one is present) to the N-terminus of SEQ ID NO:3, 4, 5 or 8 is not Lys; (4) a polypeptide comprising an amino acid sequence that is at least 50% ⁇ e.g., >60%, >70%, >80%, >85%, >90%, >92%, >94%, >96%, >98%, or more) identical to SEQ ID NO:3, 4, 5 or 8; (5) a polypeptide comprising amino acid sequence SEQ ID N0:3, 4, 5 or 8, provided that at least one of the amino acids in said SEQ ID N0:3, 4, 5
  • Antimicrobial activity has also been seen in peptides derived from the CTD, but not including the most C-terminal residues of TFPI.
  • C-terminal polypeptides of TFPI comprising a fragment of at least 3 (ag., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14) consecutive amino acids of amino acid sequence SEQ ID NO:8 ⁇ the 14-mer overlap of SEQ ID NOs:3 and 4).
  • the polypeptide may or may not itself be a fragment of TFPI (e.g., of SEQ ID NO:1) or a TFPI analog.
  • C-terminal polypeptides of TFPI comprising a fragment of at least three (e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, or more) consecutive amino acids of amino acid sequence SEQ ID NO:9.
  • the polypeptide may or may not itself be a fragment of TFPI (e.g., of SEQ ID NO:1).
  • Preferred fragments of SEQ ID NO:9 are also fragments of SEQ ID NO:8.
  • C-terminal polypeptides of TFPI comprising a fragment of SEQ ID NO:1, provided that (a) the fragment includes at least 3 (e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14) consecutive amino acids of amino acid sequence SEQ ID NO:8, and (b) the polypeptide is not TFPI or a TFPI analog.
  • C-terminal polypeptides of TFPI preferably consist of no more than 250 amino acids (e.g., no more than 225, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 10O 1 95, 90, 80, 70, 60, 50, 45, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 , 10, 9, 8, 7, 6 or even 5 amino acids).
  • Polypeptides consisting of between 5 and 90 amino acids are preferred (e,g., consisting of between 5 and 80, 5 and 70, 5 and 60 amino acids, efc). Particularly preferred are polypeptides consisting of between 8 and 25 amino acids.
  • C-terminal polypeptides of TFPI preferably consists of at least three amino acids (e.g., at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or at least 50 amino acids).
  • C-terminal polypeptides of TFPI having formula NH2-A-B-C-COOH, wherein: A is a polypeptide sequence consisting of a amino acids; C is a polypeptide sequence consisting of c amino acids; B is polypeptide sequence which is a fragment of at least b consecutive amino acids from the amino acid sequence SEQ ID N0:3, 4, 5 or 8, where b is 3 or more ⁇ e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21).
  • the value of a is generally at least 1 (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
  • the value of c is generally at least 1 (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
  • a+c is at least 1 (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
  • a+c is at most 1000 (e.g., at most 900, 800, 700, 600, 500, 450, 400, 350, 300, 250, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2).
  • the amino acid sequence of -A- typically shares less than ro% sequence identity to the a amino acids which are N-terminal of sequence -B- in SEQ ID NO:2.
  • the amino acid sequence of -C- typically shares less than ⁇ % sequence identity to the c amino acids which are C-terminal of sequence -B- in the sequence.
  • the values of m and n are both 60 or less ⁇ e.g., 50, 40, 30, 20, 10 or less).
  • the values of m and n may be the same as or different from each other.
  • the C-terminal polypeptides of TFPI do not consist of SEQ ID NO:10, which was disclosed by Hembrough ef a/, in reference [10] as having anti-tumor and anti-angiogenic activity, but not as having anti-bacterial activity.
  • C-terminal polypeptides of TFPI may, compared to SEQ ID NO:3, 4, 5, 8 or 9, include one or more (e.g., 1, 2, 3, 4, 5, 6, efc.) conservative amino acid substitutions, i.e., replacements of one amino acid with another which has a related side chain.
  • amino acids are generally divided into four families: (1) acidic, i.e., aspartate, glutamate; (2) basic, i.e., lysine, arginine, histidine; (3) non-polar, i.e., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar i.e., glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids.
  • the C-termina! polypeptides of TFPI may have one or more (e.g., 1, 2, 3, 4, 5, 6, etc.) single amino acid deletions relative to a reference sequence.
  • the C-terminal polypeptides of TFPI may include one or more (e.g., 1, 2, 3, 4, 5, 6 etc.) insertions (e.g., each of 1, 2 or 3 amino acids) relative to a reference sequence.
  • C-terminal polypeptides of TFPI disclosed herein can be prepared in many ways known in the art, e.g., by chemical synthesis (in whole or in part), by digesting TFPI using proteases, by translation from RNA, by purification from cell culture (e.g., from recombinant expression), etc.
  • a preferred method for production of peptides ⁇ 40 amino acids long involves in vitro chemical synthesis [11,12].
  • Solid-phase peptide synthesis is particularly preferred, such as methods based on tBoc or Fmoc [13] chemistry.
  • Enzymatic synthesis [14] may also be used in part or in full.
  • polypeptides may be produced by translation. This may be carried out in vitro or in vivo.
  • Biological methods are in general restricted to the production of polypeptides based on L-amino acids, but manipulation of translation machinery (e.g., of aminoacyl tRNA molecules) can be used to allow the introduction of D-amino acids (or of other non natural amino acids, such as iodotyrosine or methylphenylalanine, azidohomoalanine, etc.) [15]. Where D- amino acids are included, however, it is preferred to use chemical synthesis. Polypeptides may have covalent modifications at the C-terminus and/or N-terminus.
  • C-terminal polypeptides of TFPI disclosed herein can take various forms (e.g., native, fusions, glycosylated, non-glycosylated, lipidated, non-lipidated, phosphorylated, non-phosphorylated, myristoylated, non-myristoylated, monomeric, multimeric, particulate, denatured, efc.).
  • polypeptide refers to amino acid polymers of any length.
  • the polymer may be linear, branched or circular, it may comprise modified amino acids, and it may be interrupted by non-amino acids.
  • the terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylate, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component.
  • polypeptides containing one or more analogs of an amino acid including, for example, unnatural amino acids, efc
  • Polypeptides can occur as single chains or associated chains.
  • C-terminal polypeptides of TFPI disclosed herein may comprise one or more sequences -X-Y- or -Y-X- or -X-X-, wherein: -X- is an amino acid sequence as defined above and -Y- is not a sequence as defined above, i.e., C- terminal polypeptides of TFPI that are fusion proteins.
  • a contemplated sequence includes -X 1 -Y 1 - X2-Y2- , or X1-X2-Y1 or -X1-X2- etc.
  • Y is an N-terminal leader sequence as seen for example in SEQ ID N0:11 or 12.
  • Y is a C-terminat T-helper sequence as seen for example in SEQ ID NO:13 or 14.
  • Antibiotics for use in the disclosed combinations may be selected from antibiotics know for use in treatment of Gram-positive bacteria, and will be selected based on the particular species of bacteria responsible for the bacterial infection to be treated.
  • antibiotics include penicillins, cephalosporins, macrolides, fluoroquinolones, sulfonamides, tetracyclines, and aminoglycosides.
  • Non-limiting examples of antibiotics include, e.g., azithromycin, clarithromycin, erythromycin, doxycycline, gemifloxacin, levofloxacin, moxifloxacin, gatifloxacin, ceftriaxone, cefotaximine, ceftazidime, cefepime, vancomycin, amoxicillin, amoxicillin with clavulanate, ampicil ⁇ n, ticarcillin, ticarcillin with clavulanate.
  • vancomycin or erythromycin are used as part of the pharmaceutical combination.
  • antibiotics are used, e.g., a betalactam plus macrolide ⁇ e.g., ciarithromycin or azithromycin) or a respiratory quinoline (e.g., levofloxacin or moxifloxacin), e.g., a betalactam with antipsuedomonal activity plus ciprofloxacin or levofloxacin, a macrolide plus amoxicillin, etc.
  • macrolide e.g., ciarithromycin or azithromycin
  • a respiratory quinoline e.g., levofloxacin or moxifloxacin
  • betalactam with antipsuedomonal activity plus ciprofloxacin or levofloxacin e.g., a betalactam with antipsuedomonal activity plus ciprofloxacin or levofloxacin, a macrolide plus amoxicillin, etc.
  • TFPI and ala-TFPI have an anti-coagulant effect and interrupt potentially harmful endotoxin signaling.
  • TFPI and ala-TFP! have an anti-bacterial mediated by its C-terminal domain, It is shown herein that C-terminal polypeptides of TFPI, as defined above, unexpectedly enhance the anti-bacterial effect of TFPI and TFPI analogs in the presence of antibiotics during treatment of Gram-positive bacterial infections. It is also shown herein that C- terminal polypeptides of TFPI in conjunction with antibiotics unexpectedly enhance the anti-bacterial effect of antibiotics in treating Gram-positive bacteria! infections.
  • a pharmaceutical combination (e.g., as a set combination of corresponding components, or as a combination of individual components or individual components in a time-staggered sequence) comprising or consisting of TFPI or a TFPI analog, a C-terminal polypeptide of TFPI 1 and an antibiotic; (2) a pharmaceutical combination comprising or consisting of a C-terminal polypeptide of TFPI and an antibiotic; (3) a pharmaceutical composition comprising or consisting of TFPI or a TFPI analog, a C-terminal polypeptide of TFPI, and an antibiotic; (4) a pharmaceutical composition comprising or consisting of a C-terminal polypeptide of TFPI and an antibiotic (5) TFPI or a TFPI analog, a C-terminal polypeptide of TFPI, and an antibiotic for simultaneous, separate or sequential administration; (6) a C-terminal polypeptide of TFPI and an antibiotic for simultaneous, separate or sequential administration; (7) a method for treating a Gram-positive
  • TFPI or a TFPI analog a C-terminal polypeptide of TFPI and an antibiotic
  • a method for treating a Gram- positive bacterial infection in a patient or reducing cytokine induction by Gram-positive bacteria comprising simultaneous, separate or sequential administration of a C-terminal polypeptide of TFPI and an antibiotic
  • (10) a method for reducing cytokine induction by Gram-positive bacteria using a C-terminal polypeptide of TFPI and an antibiotic.
  • the TFPI or TFPI analog, or the C-terminai polypeptide of TFPI, or the antibiotic may be administered without a pharmaceutically acceptable carrier.
  • the C-terminal polypeptide of TFPI is the polypeptide set forth in SEQ ID N0:3, 4, 5 or 8, e.g., the polypeptide set forth in SEQ ID N0:4.
  • the pharmaceutical combination or composition contains ala-TFPI (SEQ ID NO:2).
  • pharmaceutical combination refers to simultaneously, separately or sequentially administered C- terminal polypeptides of TFPI and antibiotics, or simultaneously, separately or sequentially administered C- terminal polypeptides of TFPI 1 TFPI or ala-TFPI and antibiotics, e.g., as part of a single pharmaceutical composition (e.g., for simultaneous administration) or in separate pharmaceutical compositions (e.g., for separate administration).
  • pharmaceutical combination includes a pharmaceutical composition.
  • component refers to an active ingredient in the pharmaceutical combinations disclosed herein (e.g., TFPI or a TFPI analog, a C-terminal polypeptide of TFPI, or antibiotics).
  • the components of a pharmaceutical combination can be dosed independently or by use of different fixed combinations with distinguished amounts of the components, i.e., simultaneously or at different time points.
  • the parts of the pharmaceutical combination can then, e.g., be administered simultaneously or chronologically staggered, that is, at different time points and with equal or different time intervals for any component.
  • the ratio of the total amounts of the components to be administered in the pharmaceutical combination can be varied, e.g., in crder to cope with the needs of a patient sub-population to be treated or the needs of the single patient based on the severity of any side effects that the patient experiences.
  • the present disclosure provides to a pharmaceutical combination which comprises:
  • the present disclosure provides a pharmaceutical combination which comprises: (a) one or more unit dosage forms of a C-terminal polypeptide of TFPI (e.g., SEQ ID NO:4); and (b) one or more unit dosage forms of an antibiotic (e.g., vancomycin or erythromycin).
  • a pharmaceutical combination which comprises: (a) one or more unit dosage forms of a C-terminal polypeptide of TFPI (e.g., SEQ ID NO:4); and (b) one or more unit dosage forms of an antibiotic (e.g., vancomycin or erythromycin).
  • the subject e.g., animal, e.g., mammal, e.g., human
  • a component of the combination e.g., a C-terminal polypeptide of TFPI, TFPI or ala-TFPI, or an antibiotic.
  • a subject may first be administered an antibiotic, and later wilt be administered, e.g., a C-terminal polypeptide of TFPI, by a physician.
  • the subject has previously been administered an antibiotic and subsequently administered a C-terminal polypeptide of TFPI.
  • any sequential administration may occur, e.g., variation 1, order of components administered: a) antibiotic, b) a C-terminal polypeptide of TFPI, and c) TFPI or a TFPI analog; variation 2, order of components administered: a) antibiotic, b) TFPI or a TFPI analog, and c) a C-terminal polypeptide of TFPI; variation 3, order of components administered: a) TFPI or a TFPI analog; b) antibiotic; c) a C-terminal polypeptide of TFPI, etc.
  • the physician will determine the appropriate sequence of administration, and the appropriate time between administration of the components of the novel combinations.
  • the TFPI or TFPI analog used in these combinations may include, or alternatively may lack, the C-terminal polypeptide of TFPI.
  • the TFPI or TFPI analog may lack up to q C-terminus amino acids, as described above.
  • Additional components may be found in the pharmaceutical combinations disclosed herein, e.g., an additional antibiotic, an additional C-terminal polypeptide of TFPI, an additional TFPI analog, or, e.g., a different component belonging to a similar or separate class of molecules useful for treating a Gram-positive bacterial infection or useful for reducing cytokine induction by Gram-positive bacteria.
  • additional components include anticoagulants, antiinflammatories, vasopressors, etc.
  • references to the components are meant to also include the pharmaceutically acceptable salts of any of the active substances.
  • active substances comprised by the components have, for example, at least one basic center, they can form acid addition salts, Corresponding acid addition salts can also be formed having, if desired, an additionally present basic center.
  • Active substances having an acid group e.g., COOH, can form salts with bases.
  • the active substances comprised in the components or a pharmaceutically acceptable salts thereof may also be used in form of a hydrate or include other solvents used for crystallization.
  • Simultaneous administration may, e.g., take place in the form of one fixed combination with two or more active ingredients, or by simultaneously administering two or more active ingredients that are formulated independently.
  • Sequential use (administration) preferably means administration of one (or more) components of a combination at one time point, other components at a different time point, that is, in a chronically staggered manner, preferably such that the combination shows more efficiency than the single compounds administered independently (especially showing synergism). If the pharmaceutical combination is provided sequentially, at the onset of administration of the second (or third) component of the combination, the first (or second) of the two components may still be detectable at effective concentrations at the site of treatment.
  • Separate use (administration) means that administration of the components of the pharmaceutical combination act independently of each other at different time points.
  • compositions as disclosed herein may be provided in the form of a commercial package or kit.
  • a commercial package as used herein defines a "kit” in the sense that the components can be dosed independently or by use of different fixed combinations with distinguished amounts of the components, i.e., simultaneously or at different time points.
  • these terms comprise a commercial package comprising (e.g., combining) as active ingredients the components, together with instructions for simultaneous, sequential (chronically staggered, in time-specific sequence) or separate use thereof in the delay of progression or treatment of a Gram-positive bacterial infection.
  • the parts of the kit of parts can then, e.g., be administered simultaneously or chronologically staggered, that is at different time points and with equal or different time intervals for any part of the kit of parts.
  • the time intervals may be chosen such that the effect on the treated disease in the combined use of the parts is larger than the effect which would be obtained by use of only any one of the components (as can be determined according to standard methods).
  • the ratio of the total amounts of the components to be administered in the combined preparation can be varied, e.g., in order to cope with the needs of a patient sub-population to be treated or the needs of the single patient which different needs can be due to the particular disease, age, sex, body weight, etc. of the patients.
  • there is at least one beneficial effect e.g., a mutual enhancing of the effect of the components, in particular a more than additive effect, which hence could be achieved with lower doses of each of the components, respectively, than tolerable in the case of treatment with the individual components only without combination, producing additional advantageous effects, e.g., less side effects or a combined therapeutic effect in a non-effective dosage of one or more of the components, e.g., a strong synergism of the components.
  • beneficial effect e.g., a mutual enhancing of the effect of the components, in particular a more than additive effect, which hence could be achieved with lower doses of each of the components, respectively, than tolerable in the case of treatment with the individual components only without combination, producing additional advantageous effects, e.g., less side effects or a combined therapeutic effect in a non-effective dosage of one or more of the components, e.g., a strong synergism of the components.
  • any combination of simultaneous, sequential and separate use is also possible, meaning that the components may be administered at one time point simultaneously, followed by administration of only one component with lower host toxicity either chronically, e.g., more than 3-4 weeks of daily dosing, at a later time point and subsequently the other component or the combination of both components at a still later time point (in subsequent drug combination treatment courses for an optimal anti-tumor effect) or the like.
  • compositions for the combination therapy for enteral or parenteral administration are, e.g., those in unit dosage forms, such as sugar-coated tablets, capsules or suppositories, and furthermore ampoules. If not indicated otherwise, these formulations are prepared by conventional means, e.g., by means of conventional mixing, granulating, sugar-coating, dissolving or lyophi ⁇ zing processes. It will be appreciated that the unit content of a combination partner contained in an individual dose of each dosage form need not in itself constitute an effective amount since the necessary effective amount can be reached by administration of a plurality of dosage units. One of skill in the art has the ability to determine appropriate pharmaceutically effective amounts of the combination components.
  • Components of the pharmaceutical compositions may be administered as an oral pharmaceutical formulation in the form of a tablet, capsule or syrup; or as parenteral injections if appropriate.
  • any pharmaceutically acceptable media may be employed such as water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents.
  • Pharmaceutically acceptable carriers include starches, sugars, microcrystalline celluloses, diluents, granulating agents, lubricants, binders, disintegrating agents.
  • Solutions of an active ingredient, and also suspensions, and isotonic aqueous solutions or suspensions, are useful for parenteral administration of an active ingredient, it being possible, e.g., in the case of lyophilized compositions that comprise the active ingredient alone or together with a pharmaceutically acceptable carrier, e.g., mannitol, for such solutions or suspensions to be produced prior to use.
  • a pharmaceutically acceptable carrier e.g., mannitol
  • compositions may be sterilized and/or may comprise excipients, e.g., preservatives, stabilizers, wetting and/or emulsifying agents, solubilizers, salts for regulating the osmotic pressure and/or buffers, and are prepared in a manner known per se, e.g., by means of conventional dissolving or lyophilizing processes,
  • the solutions or suspensions may comprise viscosity- increasing substances, such as sodium carboxymethylcellulose, carboxymethylcellulose, dextran, polyvinylpyrrolidone or gelatin.
  • Suspensions in oil comprise as the oil component the vegetable, synthetic or semisynthetic oils customary for injection purposes.
  • the isotonic agent may be selected from any of those known in the art, e.g., mannitol, dextrose, glucose and sodium chloride.
  • the infusion formulation may be diluted with the aqueous medium.
  • the amount of aqueous medium employed as a diluent is chosen according to the desired concentration of active ingredient in the infusion solution.
  • Infusion solutions may contain other excipients commonly employed in formulations to be administered intravenously such as antioxidants. Drug design and peptidomimetics
  • C-termina! polypeptides of TFPI may be refined to improve anti-bacterial activity, i.e., anti-Gram-positive bacterial activity, or to improve pharmacologically important features such as bio-availability, toxicology, metabolism, pharmacokinetics, ete.
  • the C-terminal polypeptides of TFPI may therefore be used as lead compounds for further research and refinement.
  • C-terminal polypeptides of TFPI can be used for designing peptidomimetic molecules [16-21]
  • Peptidomimetic techniques have successfully been used to design thrombin inhibitors [22,23]. These will typically be isosteric with respect to the C-terminal polypeptides of TFPI but will lack one or more of their peptide bonds.
  • the peptide backbone may be replaced by a non-peptide backbone while retaining important amino acid side chains.
  • the peptidomimetic molecule may comp ⁇ se sugar amino acids [24].
  • Peptoids may be used.
  • a pharmacophore i.e., a collection of chemical features and 3D constraints that expresses specific characteristics responsible for activity
  • the pharmacophore preferably includes surface-accessible features, more preferably including hydrogen bond donors and acceptors, charged/ionizable groups, and/or hydrophobic patches. These may be weighted depending on their relative importance in conferring activity [25].
  • Pharmacophores can be determined using software such as CATALYST (including HypoGen or HipHop), CERlUS 2 , or constructed by hand from a known conformation of a polypeptide.
  • the pharmacophore can be used to screen structural libraries, using a program such as CATALYST
  • the CLIX program can also be used, which searches for orientations of candidate molecules in structural databases that yield maximum spatial coincidence with chemical groups which interact with the receptor.
  • the binding surface or pharmacophore can be used to map favorable interaction positions for functional groups ⁇ e.g., protons, hydroxyl groups, amine groups, hydrophobic groups) or small molecule fragments.
  • Compounds can then be designed de novo in which the relevant functional groups are located in substantially the same spatial relationship as in polypeptides disclosed herein.
  • Functional groups can be linked in a single compound using either bridging fragments with the correct size and geometry or frameworks which can support the functional groups at favorable orientations, thereby providing a peptidomimetic compound for use in the combinations disclosed herein.
  • - SPROUT [31] suite which includes modules to: identify favorable hydrogen bonding and hydrophobic regions within a binding pocket (HIPPO module); select functional groups and position them at target sites to form starting fragments for structure generation (EIeFAnT); generate skeletons that satisfy the steric constraints of the binding pocket by growing spacer fragments onto the start fragments and then connecting the resulting part skeletons (SPIDeR); substitute hetero atoms into the skeletons to generate molecules with the electrostatic properties that are complementary to those of the receptor site (MARABOU).
  • the solutions can be clustered and scored using the ALLigaTOR module.
  • - CAVEAT [32] which designs linking units to constrain acyclic molecules.
  • These methods identify relevant compounds. These compounds may be designed de novo, may be known compounds, or may be based on known compounds. The compounds may be useful themselves, or they may be prototypes which can be used for further pharmaceutical refinement ⁇ i.e., lead compounds) in order to improve binding affinity or other pharmacologically important features (e.g., bio-availability, toxicology, metabolism, pharmacokinetics etc.).
  • pharmacologically important features e.g., bio-availability, toxicology, metabolism, pharmacokinetics etc.
  • peptidomimetics identified in silico by the structure-based design techniques can also be used to suggest libraries of compounds for 'traditional' in vitro or in vivo screening methods. Important pharmaceutical motifs in the ligands can be identified and mimicked in compound libraries (e.g., combinatorial libraries) for screening for anti-bacterial activity.
  • a compound identified using these drug design methods (i) a compound identified using these drug design methods; (ii) a compound identified using these drug design methods, for use as a pharmaceutical; (iii) the use of a compound identified using these drug design methods in the manufacture of an anti-bacterial; and (iv) a method of treating an animal with a Gram- positive bacterial infection, comprising administering a therapeutically effective amount of a compound identified using these drug design methods.
  • compositions are: (a) pharmaceutical combinations of compounds, polypeptides, and/or peptidomimetics; and (b) a pharmaceutically acceptable carrier. These pharmaceutical combinations are useful to treat patients at risk of developing, or diagnosed as having, a Gram-positive bacterial infection, or to lower the risk of the infection developing into a severe infection for one or a group of patients.
  • Component (a) includes the active ingredients in the combination (e.g., a C-terminal polypeptide of TFPI and an antibiotic; or a C-terminal polypeptide of TFPI, TFPI or ala-TFPI and an antibiotic), which are present at a therapeutically effective amount, i.e., an amount sufficient to inhibit Gram-positive bacterial growth and/or survival in a patient, and preferably an amount sufficient to eliminate Gram-positive bacterial infection.
  • a therapeutically effective amount i.e., an amount sufficient to inhibit Gram-positive bacterial growth and/or survival in a patient, and preferably an amount sufficient to eliminate Gram-positive bacterial infection.
  • the precise effective amount of each component of the pharmaceutical combination for a given patient will depend upon the patient's size and health, the nature and extent of infection, and the composition or combination of compositions selected for administration. The effective amounts can be determined by routine experimentation and is within the judgment of the clinician.
  • An effective dose of components will generally be from about 0.01mg/kg to about 5 mg/kg, or about 0.01 mg/ kg to about 50 mg/kg or about 0.05 mg/kg to about 10 mg/kg.
  • Pharmaceutical compositions based on polypeptides are well known in the art. Polypeptides may be included in the composition in the form of salts and/or esters.
  • a 'pharmaceutically acceptable carrier' includes any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition.
  • Suitable carriers are typically large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, sucrose, trehalose, lactose, and lipid aggregates (such as oil droplets or liposomes).
  • Such carriers are well known to those of ordinary skill in the art.
  • a component of the pharmaceutical combination may be in the form of a liposome in which a C-terminal polypeptide of TFPI and/or TFPI (or ala-TFPI) is combined, in addition to other pharmaceutically acceptable carriers, with amphipathic agents such as lipids that exist in aggregated form as micelles, insoluble monolayers, liquid crystals, or lamellar layers in aqueous solution.
  • Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids, etc.
  • a carrier may be 5-CNAC.
  • the binding agent may be in the form of a tablet, capsule, powder, solution or elixir.
  • the pharmaceutical combination may additionally contain a solid carrier such as a gelatin or an adjuvant.
  • a liquid carrier such as water, petroleum, oils of animal or plant origin such as peanut oil (exercising caution in relation to peanut allergies), mineral oil, soybean oil, or sesame oil, or synthetic oils may be added.
  • the liquid form of the pharmaceutical combination may further contain physiological saline solution, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol, or polyethylene glycol.
  • a component of the pharmaceutical combination for intravenous, cutaneous, or subcutaneous injection should contain, in addition to the component, an isotonic vehicle such as sodium chloride injection, Ringer's injection, dextrose injection, dextrose and sodium chloride injection, lactated Ringer's injection, or other vehicle as known in the art.
  • Intravenous formulations for TFPI and ala-TFPI are provided in, e.g., WO01/24814 and WO06/113360.
  • ala-TFPI is formulated at 0.45 mgt ⁇ iL C-termina! poly peptides may be provided at, e.g., 3 ⁇ M -300 nM, or 30 nM or 3 nM or
  • ala-TFPI is formulated at 0.15 mg/mL
  • ala-TFPI is formulated in a solution containing 300 mM L-arginine free base, 5 mM L- methionine and 20 mM sodium citrate/citric acid buffer pH 5.5.
  • the amount of components of the pharmaceutical combination used will depend upon the nature and severity of the condition being treated, and on the nature of prior treatments that the patient has undergone. Ultimately, the attending physician will decide the amount of the components of the pharmaceutical combination with which to treat each individual patient. Initially, the attending physician may administer low doses of components and observe the patient's response. Larger doses of the components may be administered until the optima! therapeutic effect is obtained for the patient, and at that point the dosage is not generally increased further. Dosing schedules for ala-TFPI are provided in, e.g., WO06/113360. In one embodiment, ala-TFPI is administered via intravenous (i.v.) therapy at a dose of 0.075 mg/kg/hr.
  • i.v. intravenous
  • ala-TFPI is administered via i.v. therapy at a dose of 0,025 mg/kg/hr.
  • the components of the pharmaceutical combination may be administered by means known in the art. This can include, but is not limited to, topical application and intravenous, aerosol, subcutaneous, and intramuscular routes.
  • the components of the pharmaceutical combination can be given as a single dose or in multiple doses.
  • the duration of i.v. therapy using a component of the pharmaceutical combination will vary, depending on the severity of the disease being treated and the condition and potential idiosyncratic response of each individual patient.
  • ala-TFPI is administered as a continuous infusion for 96 hours.
  • a pharmaceutical composition is formulated to be compatible with its intended route of administration (e.g., oral compositions generally include an inert diluent or an edible carrier).
  • routes of administration include parenteral (e.g., intravenous), intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration.
  • parenteral e.g., intravenous
  • intradermal subcutaneous
  • oral e.g., inhalation
  • transdermal topical
  • transmucosal and rectal administration.
  • the pharmaceutical compositions compatible with each intended route are well known in the art.
  • the term "therapeutically effective amount' means the total amount of each active component of the pharmaceutical composition or method that is sufficient to show a meaningful patient benefit, e.g., amelioration of symptoms of, healing of, or increase in rate of healing of such conditions.
  • a meaningful patient benefit e.g., amelioration of symptoms of, healing of, or increase in rate of healing of such conditions.
  • the term refers to that ingredient alone.
  • the term refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously.
  • Combinations disclosed herein may include an additional antibacterial agent, e.g., antibiotic, particularly if packaged in a multiple dose format.
  • an additional antibacterial agent e.g., antibiotic
  • the bacterial infection may be with a single Gram-positive bacterial species, or with several Gram-positive bacterial species.
  • a Gram-positive bacterial infection refers to infection by at least one Gram-positive bacterial species.
  • An individual may also be infected with Gram-positive and Gram-negative bacteria (or a virus, yeast or fungi), and still be treatable with the present pharmaceutical combinations, as long as there is present an infection caused by at least one Gram-positive bacterial species.
  • Gram-positive bacteria involved in severe infections include, e.g., Haemophilus influenzae, Legionella pneumophila, Chlamydia pneumoniae, Chlamydia psittaci, Mycoplasma pneumoniae, Streptococcus pneumoniae, Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus species, Streptococcus agalactiae and Streptococcus pyogenes.
  • Haemophilus influenzae Legionella pneumophila
  • Chlamydia pneumoniae Chlamydia psittaci
  • Mycoplasma pneumoniae Streptococcus pneumoniae
  • Staphylococcus aureus Staphylococcus epidermidis
  • Enterococcus species Streptococcus agalactiae
  • Streptococcus pyogenes See, e.g., Ostapchuk et al. (2004) Am.
  • the Gram-positive bacterial infection results from infection by Staphylococcus aureus or Streptococcus pneumonia.
  • the G ram- positive bacterial infection results in severe community acquired pneumonia or sepsis.
  • Bacterial infections include, for example, respiratory infections, e.g., pneumonia (e.g., severe pneumonia, e.g., severe community acquired pneumonia (sCAP) or hospital acquired pneumonia (HAP)), bacteremia, septic shock, sepsis, ear infections, diarrhea, urinary tract infections, digestive tract infections, nervous system infections, skin disorders, topical and mucosal infections.
  • respiratory infections e.g., pneumonia (e.g., severe pneumonia, e.g., severe community acquired pneumonia (sCAP) or hospital acquired pneumonia (HAP)), bacteremia, septic shock, sepsis, ear infections, diarrhea, urinary tract infections, digestive tract infections, nervous system infections, skin disorders, topical and mucosal infections.
  • sCAP severe community acquired pneumonia
  • HAP hospital acquired pneumonia
  • Preferred patients for treatment are human, including children (e.g., a toddler or infant), teenagers and adults.
  • the pharmaceutical combination can also be applied in combination with other treatments, e.g., surgical intervention.
  • composition “comprising” encompasses “including” as well as “consisting,” e.g., a composition “comprising” X may consist exclusively of X or may include something additional, e.g., X + Y.
  • Percent sequence identity can be determined using the Smith-Waterman homology search algorithm using an affine gap search with a gap open penalty of 12 and a gap extension penalty of 2, BLOSUM matrix of 62.
  • the Smith-Waterman homology search algorithm is taught in ref. [36],
  • nucleic acids and polypeptides disclosed herein may include sequences that:
  • (c) have 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 single nucleotide or amino acid alterations (deletions, insertions, substitutions), which may be at separate locations or may be contiguous, as compared to the sequences of
  • each window has at least xy identical aligned monomers, where: x is selected from 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, / is selected from 0.50, 0.60, 0.70, 0.75, 0.80, 0.85, 0.90, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99; and if xy is not an integer then it is rounded up to the nearest integer.
  • the preferred pairwise alignment algorithm is the
  • nucleic acids and polypeptides herein may additionally have further sequences to the N-terminus/5 1 and/or C-terminus/3' of these sequences (a) to (d).
  • animal refers to any member of the animal kingdom including human beings.
  • treat refers to decreasing or ameliorating a condition before or after it has occurred. As compared with an equivalent untreated control, such reduction or degree of prevention is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, or 100% as measured by any standard technique.
  • a subject who is being treated for a Gram-positive bacterial infection is one who a medical practitioner has diagnosed as having such a condition. Diagnosis may be by any suitable means.
  • preventing refers to delaying the onset of a condition (e.g., a Gram-positive bacterial infection), or prohibiting the onset of such a condition in an animal likely to develop such a condition.
  • a condition e.g., a Gram-positive bacterial infection
  • Figure 1 shows that the growth of S. epidermidis (1000 CRU input) in blood is inhibited in the presence of tifacogin (10OnM).
  • Figure 2 shows that induction of IL-6 in blood samples containing 10,000 CFU S. epidermidis is reduced by tifacogin or hirudin, Two representative examples are depicted.
  • Figure 3 shows that either tifacogin or SEQ ID NO:4 (CSS-3 - a C-terminal polypeptide of TFPI) reduce IL-6 induction by S. epidermidis compared to clotted blood. Two representative examples are depicted.
  • Peptide 2 is the scrambled peptide of SEQ ID NO:7.
  • Figure 4 shows that induction of IL-6 by S. epidermidis is inhibited in the presence of tifacogin or hirudin in combination with vancomycin, but not by vancomycin alone. Two representative examples are depicted.
  • Figure 5 shows that tifacogi ⁇ and a C-terminal polypeptide of TFPI (SEQ ID N0:4, CSS-3) synergize to suppress IL-6 induction in vancomycin treated blood inoculated with 10,000 CFU of S. aureus. Two representative examples are depicted.
  • Figure 6 shows that tifacogin and a C-terminal polypeptide of TFPI (SEQ ID NO:4, CSS-3) synergize to suppress IL-6 induction in vancomycin treated blood inoculated with 300 CFU of S. pneumoniae. Two representative examples are depicted.
  • telomeres a C-terminal polypeptide of TFPI having the sequence set forth in SEQ ID NO:4 (CSS-3, processed TFPI amino acids 243 to 269, GFIQRISKGGLIKTKRKRKKQRVKIAY, AnaSpec, CA).
  • CSS-3 Activity of CSS-3 is compared to cultures containing 3 ⁇ M LL37 peptide control (sequence: LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES, AnaSpec, CA, SEQ ID NO:6).
  • Complement is depleted by mixing whole blood with 100 units/ml cobra venom factor (CVF) for 10 minutes at 37 0 C and then diluting into RPMI to a final concentration of 10 units/ml.
  • CVF cobra venom factor
  • Anti-inflammatory activity is assessed by measuring induced levels of IL-6, IL-1 ⁇ , IL-8 and TNF- ⁇ in the culture media after 20 to 24 hours using fluorescent antibody beads (Luminex, TX).
  • Peptide 2 is a scrambled sequence spanning amino acids 255 to 276 NFQRKEKREVIYKVKTKIKAMR (SEQ ID NO:7). Combinations of tifacogin and C-terminal polypeptides of TFPI with vancomycin
  • Effective doses of vancomycin (5 ⁇ g/ml) or erythromycin (50 ⁇ g/ml) were added to whole blood cultures containing buffer or tifacogin or CSS-3 (SEQ ID N0:4) or tifacogin and CSS-3, and inoculated with 10,000 CFU of S. epidermidis or S. aureus (ATCC 25923), or 300 CFU of Streptococcus pneumoniae (ATCC 6306) and cytokines (IL-6, IL-8, TNF ⁇ and IL-1 ⁇ ) were measured after 20 to 24 hours.
  • tifacogin 10OnM
  • tifacogin increases trie antibacterial action of blood inoculated with 10,000 CFU of S, epidermidis. The cellular fraction of blood is required eliminate the bacteria.
  • induction of IL-6 is reduced in blood samples containing 10,000 CFU S. epidermidis by tifacogin or hirudin, suggesting that the inhibitory effect is through blocking fibrin generation.
  • the lack of additivity of hirudin and tifacogin suggests that either agent produces full anticoagulation, which is supported by the lack of blood clots in the samples.
  • Induction of IL-8 and TNF- ⁇ are also inhibited in a similar pattern (data not shown). These data indicate that deposition of fibrin allows S. epidermidis to evade clearance.
  • Table 2 compares the activity of the C-terminal polypeptide of TFPI of SEQ ID NO:4 (CSS-3) to neutrophil antimicrobial peptide cathelicidin/LL37 (SEQ ID NO:6).
  • LL37 is not effective against S. epidermidis in RPMI or blood cultures, while CSS-3 is active under both conditions.
  • Table 2 - Growth of S. epidermidis
  • CSS-3 (SEQ ID NO:4) appears to act directly on S. epidermidis, as opposed to tifacogin, which requires blood cells for antibacterial action (Tables 1 and 2).
  • tifacogin or a C-terminal polypeptide of TFPI reduce IL-6 induction compared to clotted blood.
  • the combination reduces the measured cytokines to near baseline. Similar reductions are seen in IL-Ip, IL-8 and TNF- ⁇ (Table 3).
  • the same effect is seen with combinations of CSS-3 and hirudin (data not shown). Because CSS-3 retains potency against S epidermidis in whole blood, no combined effect of tifacogin and CSS-3 on S. epidermidis growth is seen (data not shown)
  • "Peptide 2" of Figure 3 and Table 3 corresponds to the scrambled peptide set forth in SEQ ID NO:7.
  • CVF prevents clearance of S, epidermidis in tifacogin containing blood and there is a large increase in cytokines in those samples.
  • CSS-3 kills S. epidermidis in the presence of CVF 1 CVF also blocks the ability of CSS-3 to suppress IL-6 and IL-8 induction.
  • Combination of tifacogin and CSS-3 fails to suppress cytokine induction in the presence of CVF, factoring out the potential effect of clotting.
  • Tifacogin produced by bacterial fermentation, purification and refolding, is non-glycosylated version of TFPI with an additional N-terminal alanine. It is currently being evaluated for ability to improve survival in patients with severe community acquired pneumonia (sCAP). sCAP is most frequently caused by Gram-positive bacterial infection and is characterized by loss of lung function due to fibrin deposition and fluid accumulation in the lungs. Underlying these conditions are activation of the clotting cascade and production of inflammatory cytokines triggered by the bacterial components. Antibiotics alone can be effective at bringing the infection under control. However, in some cases, patients continue to decline, enter septic shock and die.
  • sCAP severe community acquired pneumonia
  • DIC disseminated intravascular coagulopathy
  • TFPI inhibits the initiation of blood clotting by binding to the tissue factor, Vila and Xa complex.
  • tissue factor tissue factor
  • Xai tissue factor
  • a study of septic baboons it was found that an anticoagulant, factor Xai, normalized consumption of clotting factors - yet had no effect on survival (Taylor et al. (1991) Blood.;78(2):364-8; Randolph et al. (1998) 79(5): 1048-53).
  • anticoagulation per se, and restoration of the haemostatic balance appears to be insufficient for survival.
  • Fibrin deposition and its inhibition potentially complicate understanding effects of bacterial clearance and inflammation.
  • Staphylococcus species express proteins that trigger clotting, bind to fibrin and digest fibrin. These activities may cause a reversal in the usual relationship between clotting and infection.
  • Fibrin deposition at the site of an infection is an evolutionarily old method of trapping microbes and preventing their spread. Pathogens like
  • Staphylococcus bacteria may use fibrin bound to their surface to decoy complement binding and prevent engulfment by phagocytes.
  • the data herein is consistent with this idea. In whole blood cultures, slowly clotting blood allows S. epidermidis to persist. Addition of any anticoagulant protein increases the clearance activity of blood. This clearance depends on the cellular fraction of blood and disappears when complement is inactivated by
  • tifacogin and other protein anticoagulants lower, but do not eliminate, the bacterial induction of cytokines in whole blood. This is directly related to inhibition of fibrin generation and persistence of S. epidermidis as a source of inflammatory signals. Clotting protease activation alone does not induce inflammatory cytokine signaling, and inhibition of the clotting cascade does not diminish cytokine levels if bacteria are present, as cytokines are induced to a very high level when complement is inactivated, despite complete inactivation of the clotting cascade with anticoagulants.
  • C-terminal polypeptides of TFPI directly kill S. epidermidis. This may be through the same mechanism as cationic bactericidal peptides.
  • CSS-3 SEQ ID N0:4
  • the interpretation of this result is unclear since serum proteins inactivate cationic peptides, but serum cooperates with CSS-3 to engage complement.
  • Adding CVF has no effect on the bactericidal activity of the C- terminal polypeptide of TFPI, showing it maintained direct killing action on S. epidermidis in blood proteins.
  • a C-terminal polypeptide of TFPI (CSS-3/SEQ ID N0:4) is also effective at lowering cytokines induced by S. epidermidis. This is not the simple result of killing the bacteria - complement is required. Removal of the inflammatory trigger seems to require opsonization and phagocytosis of S. epidermidis. To obtain complete neutralization of the inflammatory signal in blood requires both intact tifacogin and CSS-3. This may allow full access of the innate immune system to the bacteria by preventing fibrin formation concurrent with enhanced binding of complement to bacteria. This combined stimulating effect may be general. Shown herein, S. aureus and S.
  • pneumoniae are potent inducers of cytokines, even when their infection is controlled with antibiotic, e.g., vancomycin.
  • Tifacogin and CSS-3 are each able to reduce the level of inflammatory cytokines and their combination virtually eliminates inflammatory cytokines from cultures. Possibly these agents work by two different mechanisms - intact tifacogin through preventing fibrin formation and C-terminal polypeptides of TFPI through enhancing recognition of opsonization resistant bacteria. In both cases, active complement is required for activity.
  • tifacogin ala- TFPI of SEQ ID N0:2
  • SEQ ID N0:4 a C-terminal polypeptide of TFPI

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Abstract

La présente invention concerne des combinaisons (par ex. des combinaisons pharmaceutiques) d'agents thérapeutiques antibactériens conçues pour traiter des infections bactériennes à Gram positif, par ex. des infections bactériennes conduisant à une pneumonie sévère et/ou une septicémie sévère. De nouvelles combinaisons (par ex. des combinaisons pharmaceutiques) comprennent 1) des polypeptides de TFPI à terminaison C combinés à un antibiotique (par ex. de la vancomycine) et 2) des polypeptides de TFPI à terminaison C combinés à TFPI ou ala-TFPI et un antibiotique. L'invention concerne aussi des procédés d'utilisation de telles combinaisons et compositions pour traiter des infections bactériennes à Gram positif, pour produire des médicaments permettant de traiter de telles infections et pour réduire l'induction de cytokines par des bactéries à Gram positif.
PCT/US2009/040322 2008-04-15 2009-04-13 Combinaisons pharmaceutiques de fragments de tfpi et d'un antibiotique WO2009129164A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003055442A2 (fr) * 2001-10-15 2003-07-10 Chiron Corporation Traitement d'une maladie infectieuse par l'administration d'une faible dose d'un inhibiteur de voie d'acces de thromboplastine tissulaire (tfpi)
WO2006113360A2 (fr) * 2005-04-15 2006-10-26 Novartis Vaccines And Diagnostics Inc. Traitement de la pneumonie aigue communautaire par l'administration d'un inhibiteur de la voie du facteur tissulaire (tfpi)
WO2006122139A2 (fr) * 2005-05-06 2006-11-16 Novartis Ag Utilisation de tfpi dans le traitement d'infections bacteriennes aigues
WO2007014199A2 (fr) * 2005-07-22 2007-02-01 Novartis Ag Agents antimicrobiens interagissant avec le systeme du complement

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003055442A2 (fr) * 2001-10-15 2003-07-10 Chiron Corporation Traitement d'une maladie infectieuse par l'administration d'une faible dose d'un inhibiteur de voie d'acces de thromboplastine tissulaire (tfpi)
WO2006113360A2 (fr) * 2005-04-15 2006-10-26 Novartis Vaccines And Diagnostics Inc. Traitement de la pneumonie aigue communautaire par l'administration d'un inhibiteur de la voie du facteur tissulaire (tfpi)
WO2006122139A2 (fr) * 2005-05-06 2006-11-16 Novartis Ag Utilisation de tfpi dans le traitement d'infections bacteriennes aigues
WO2007014199A2 (fr) * 2005-07-22 2007-02-01 Novartis Ag Agents antimicrobiens interagissant avec le systeme du complement

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CHOI GODA ET AL: "Antithrombin inhibits bronchoalveolar activation of coagulation and limits lung injury during Streptococcus pneumoniae pneumonia in rats", CRITICAL CARE MEDICINE, WILLIAMS AND WILKINGS COMPANY, BALTIMORE, MA, US, vol. 36, no. 1, 1 January 2008 (2008-01-01), pages 204 - 210, XP009119598, ISSN: 0090-3493 *
SCHIRM SABINE ET AL: "Fragmented tissue factor pathway inhibitor (TFPI) and TFPI C-terminal peptides eliminate serum-resistant Escherichia coli from blood cultures.", THE JOURNAL OF INFECTIOUS DISEASES 15 JUN 2009, vol. 199, no. 12, 15 June 2009 (2009-06-15), pages 1807 - 1815, XP009119679, ISSN: 0022-1899 *
SNE NIV ET AL: "Tifacogin (Chiron Corp/Pharmacia Corp)", IDRUGS, CURRENT DRUGS LTD, GB, vol. 5, no. 1, 15 February 2002 (2002-02-15), pages 91 - 97, XP009119597, ISSN: 1369-7056 *

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