WO2009129164A1 - Pharmaceutical combinations of tfpi-fragments and an antibiotic - Google Patents

Abstract

The present disclosure describes combinations (e.g., pharmaceutical combinations) of anti-bacterial therapeutic agents for the treatment of Gram-positive bacterial infections, e.g., bacterial infections leading to severe pneumonia and/or sepsis. Novel combinations (e.g., pharmaceutical combinations, e.g., pharmaceutical compositions) disclosed herein comprise: 1) C-terminal polypeptides of TFPI in combination with an antibiotic (e.g., vancomycin); and 2) C-terminal polypeptides of TFPI in combination with TFPI or ala-TFPI and an antibiotic. Disclosed herein are methods of using such combinations and compositions for treating Gram-positive bacterial infections, in the manufacture of medicaments for treating such infections, and for reducing cytokine induction by Gram-positive bacteria.

Description

PHARMACKUTICAL COMBINATIONS OF TFPI-FRAGMENTS AND AN ANTIBIOTIC

This application claims priority to US Provisional Application No. 61/045011, filed April 15, 2008, which is incorporated herein by reference in its entirety.

All patents, patent applications, online information and references cited in this disclosure are incorporated herein by reference in their entireties,

BACKGROUND During severe bacterial infection, blood clotting and inflammation become activated to pathological levels, leading to multiple organ failure and death, despite large doses of antibiotics. 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.

BRIEF SUMMARY

Disclosed herein are pharmaceutical 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. In one embodiment, 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 another embodiment, said C-terminal polypeptide of TFPI consists of the amino acid sequence set forth in SEQ ID NO:3, 4, 5 or 8. In another embodiment, said antibiotic is vancomycin or erythromycin. In another embodiment, the pharmaceutical composition further comprises TFPI or ala-TFPI. In another embodiment, said ala-TFPI consists of the amino acid sequence set forth in SEQ ID NO:2. In another embodiment, 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 are 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. Disclosed herein is the use of 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. Disclosed herein are pharmaceutical combinations 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. In one embodiment, said C-terminal polypeptide of TFPI consists essentially of the amino acid sequence set forth in SEQ ID N0:3, 4, 5 or 8. In another embodiment, said C-terminal polypeptide of TFPI consists of the amino acid sequence set forth in SEQ ID N0:3, 4, 5 or 8. In another embodiment, said antibiotic is vancomycin or erythromycin. In another embodiment, the pharmaceutical combination further comprises TFPI or ala-TFPI. In another embodiment, said ala-TFPI consists of the amino acid sequence set forth in SEQ ID N0:2. In another embodiment, 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. Disclosed herein are 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.

Disclosed herein are methods of reducing 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 In one embodiment, 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, 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, said antibiotic is vancomycin or erythromycin. In one embodiment, the method further comprises administering TFPI or ala-TFPI to the plurality of cells. In one embodiment, 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 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.

Disclosed herein is 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.

Disclosed herein is a C-terminal polypeptide of TFPI and an antibiotic for use in a method of treating a Gram- positive bacterial infection in an animal.

Disclosed herein is 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.

Disclosed herein is 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.

Disclosed herein is the use of 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.

Disclosed herein is the use of 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.

Disclosed herein is 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. Disclosed herein is 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.

Disclosed herein is 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.

Disclosed herein is 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.

Disclosed herein is 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.

Disclosed herein is 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.

Disclosed herein is the use of 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.

Disclosed herein is the use of 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. Disclosed herein is the use of 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.

Disclosed herein is the use of 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.

Disclosed herein is the use of 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.

Disclosed herein is the use of 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 TFPI₁ and wherein the animal has previously been treated with or will subsequently be treated with an antibiotic.

In one embodiment of any of the above uses, 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.

Disclosed herein is 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. In one embodiment of the commercial package, said C-terminal polypeptide of TFPI consists essentially of the amino acid sequence set forth in SEQ ID N0:3, 4, 5 or 8. In one embodiment of the commercial package, said C-terminal polypeptide of TFPI consists of the amino acid sequence set forth in SEQ ID N0:3, 4, 5 or 8. In one embodiment of the commercial package, said antibiotic is vancomycin or erythromycin. In one embodiment of the commercial package, 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 commercial package, 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.

DETAILED DESCRIPTION

The present application describes combinations (e.g., pharmaceutical combinations, e.g., pharmaceutical compositions) of anti-microbial therapeutic agents for the treatment of Gram-positive bacterial infections, e.g., bacterial infections leading to 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.

TFPI and TFPI Analogs

Tissue Factor Pathway Inhibitor (TFPI) 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). The K1 domain inhibits clotting factor Vila- tissue factor (TF) complex. The K2 domain inhibits factor Xa. Thus far no serine protease has been associated with K3, but recent experiments suggest that K3 functions in binding TFPI to a GPI anchored receptor on cell surfaces [3]. The CTD is also involved in celi association, heparin binding, and optimal Xa inhibition.

"TFPI" as used herein 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.

A "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.

Preferably, TFPI analogs comprise all three Kunitz domains. TFPI and TFPI analogs can be either glycosylated or non-glycosylated.

To maintain anti-bacterial activity, it is preferred that a TFPI analog should retain its C-terminal domain (CTD), as this region is where the anti-bacterial activity has been localized. Typically, it is preferred to retain substantially all of the amino acids downstream of the most-downstream thrombin cleavage site in TFPI (e.g., downstream of amino acid 254 of SEQ ID NO:1, in which thrombin cleaves between residues 254 & 255). 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.

Provided herein are: (1) a TFPI analog, wherein the analog lacks the thrombin cleavage site found near the

C-terminus of natural TFPI; (2) a TFPI analog, wherein the analog lacks the thrombin cleavage site present between amino acids Lys-254 and Thr-255 of natural TFPI; (3) 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; (4) 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; (5) a TFPI analog, wherein the analog contains fewer than two (i.e., one or none) Lys-Thr dipeptides. The natural cleavage site (Lys-Thr) can be removed in various ways. For instance, the lysine and/or the threonine can be substituted with different amino acids to give a dipeptide that is not recognized by thrombin. As an alternative, the lysine and/or the threonine can be deleted. As a further alternative, one or more amino acids can be inserted between the lysine and the threonine. After the modification has been made, the TFPI analog can be incubated with thrombin in a test digestion to confirm that the natural C-terminus cleavage no longer takes place. Provided herein are: (1) a TFPI analog, wherein the analog includes Kunitz domain 3, but lacks the C-terminus domain; (2) a 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.

C-Terminal Polypeptides of TFPI

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). Cleavage at this site liberates a 22 amino acid peptide (SEQ ID NO:3) which has been shown to have anti-bacterial activity and may bind to bacterial LPS. 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. Disclosed herein is the synergistic activity of such C-terminal polypeptides of TFPI in combination with antibiotics, e.g., vancomycin and erythromycin, as well as the synergistic activity of such peptides in combination with TFPI or ala-TFPI and antibiotics, e.g., vancomycin and erythromycin.

Provided herein are the following 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 NCW₁ 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 or 8 is a D-amino acid; (6) a polypeptide comprising a fragment of at least 3 (e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21) consecutive amino acids of amino acid sequence SEQ ID N0:3, 4, 5 or 8, provided that said polypeptide is not TFPI; (7) a polypeptide comprising at least 3 {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, 55, 60, 65, 70, 75 or more) amino acids from the C-terminus of amino acid sequence SEQ ID NO:1, provided that said polypeptide is not TFPI or a TFPI analog.

Antimicrobial activity has also been seen in peptides derived from the CTD, but not including the most C-terminal residues of TFPI. Provided herein are 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.

Also provided are 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. Also provided are 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, 10O₁ 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). Provided herein are 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,

21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500 etc.). 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,

20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500 efc). The value of 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,

21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500 efc.}. It is preferred that the value of 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 π% sequence identity to the c amino acids which are C-terminal of sequence -B- in the sequence. In general, 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.

In some embodiments, 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 disclosed herein may comprise amino xid sequences that have sequence identity to SEQ ID NO:3, 4, 5, 8 or 9. These polypeptides include homologs, orthologs, allelic variants and mutants. Identity between polypeptides is preferably determined by the Smith-Waterman homology search algorithm as implemented in the MPSRCH program (Oxford Molecular), using an affine gap search with parameters gap open penalty=12 and gap extension penalty=1. These 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. Genetically encoded 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. In general, substitution of single amino acids within these families does not have a major effect on the biological activity. Moreover, 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. Furthermore, 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. As an alternative to chemical synthesis, biological synthesis may be used, e.g., the 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.).

C-terminal polypeptides of TFPI disclosed herein are preferably provided in purified or substantially purified form i.e., substantially free from other polypeptides (e.g., free from naturally-occurring polypeptides), and are generally at least about 50% pure (by weight), and usually at least about 90% pure, i.e., less than about 50%, and more preferably less than about 10% (e.g., 5% or less) of a composition ;= made up of other expressed polypeptides.

The term "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. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, efc), as well as other modifications known in the art. 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. For example, a contemplated sequence includes -X₁-Y₁- X2-Y2- , or X1-X2-Y1 or -X1-X2- etc. In one embodiment, Y is an N-terminal leader sequence as seen for example in SEQ ID N0:11 or 12. In a further embodiment, Y is a C-terminat T-helper sequence as seen for example in SEQ ID NO:13 or 14.

Antibiotics 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. Such 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, ampicilϋn, ticarcillin, ticarcillin with clavulanate. In some embodiments of the invention, vancomycin or erythromycin are used as part of the pharmaceutical combination. In other embodiments, combinations of 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.

Pharmaceutical Combinations of C-terminal Polypeptides of TFPi and Uses Thereof

TFPI and ala-TFPI have an anti-coagulant effect and interrupt potentially harmful endotoxin signaling. In addition, 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.

Provided herein are: (1) 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 TFPI₁ 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 bacterial infection in a patient or reducing cytokine induction by Gram-positive bacteria comprising simultaneous, separate or sequential administration of

TFPI or a TFPI analog, a C-terminal polypeptide of TFPI and an antibiotic; (8) 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; (9) a method for reducing cytokine induction by Gram-positive bacteria using TFPI or a TFPI analog, 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.

In some embodiments, the TFPI or TFPI analog, or the C-terminai polypeptide of TFPI, or the antibiotic may be administered without a pharmaceutically acceptable carrier.

In some embodiments, 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. In other embodiments, the pharmaceutical combination or composition contains ala-TFPI (SEQ ID NO:2).

As used herein, "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 TFPI₁ 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). Thus, the phrase "pharmaceutical combination" includes a pharmaceutical composition. The term "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. In one embodiment, the present disclosure provides to a pharmaceutical combination which comprises:

(a) one or more unit dosage forms of a TFPI or a TFPI analog (e.g., ala-TFPI as set forth in SEQ ID NO:2);

(b) one or more unit dosage forms of a C-terminal polypeptide of TFPI (e.g., SEQ ID NO:4); and

(c) one or more unit dosage forms of an antibiotic (e.g., vancomycin or erythromycin).

In one embodiment, 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).

In some embodiments, the subject (e.g., animal, e.g., mammal, e.g., human) for treatment will have previously been treated with or will subsequently be treated with a component of the combination {e.g., a C-terminal polypeptide of TFPI, TFPI or ala-TFPI, or an antibiotic). For example, a subject may first be administered an antibiotic, and later wilt be administered, e.g., a C-terminal polypeptide of TFPI, by a physician. In such a case, the subject has previously been administered an antibiotic and subsequently administered a C-terminal polypeptide of TFPI. In the case of the novel combinations disclosed herein, e.g., a combination comprising an antibiotic, a C- terminal polypeptide of TFPI and TFPI or a TFPI analog (e.g., ala-TFPI of SEQ ID N0:2), 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. Thus 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. Such additional components include anticoagulants, antiinflammatories, vasopressors, etc.

It will be understood that references to the components are meant to also include the pharmaceutically acceptable salts of any of the active substances. !f 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.

Also combinations of two or more of sequential, separate and simultaneous administration are possible, preferably such that the pharmaceutical combination shows a joint therapeutic effect that exceeds the effect found when the components are used independently at time intervals so large that no mutual effect on their therapeutic efficiency can be found. Pharmaceutical combinations as disclosed herein may be provided in the form of a commercial package or kit. The term "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. Moreover, 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. In one embodiment, 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.

In the case of the use of the combination of the components and of the commercial package, 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.

Pharmaceutical preparations 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 lyophiϋzing 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.

In preparing pharmaceutical compositions for oral administration, 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. The pharmaceutical 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. For example, the peptide backbone may be replaced by a non-peptide backbone while retaining important amino acid side chains. The peptidomimetic molecule may compπse sugar amino acids [24]. Peptoids may be used.

To assist in trie design of peptidomimetic molecules, a pharmacophore (i.e., a collection of chemical features and 3D constraints that expresses specific characteristics responsible for activity) can be defined for the peptides. 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), CERlUS², 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. Whilst linking of functional groups in this way can be done manually, perhaps with the help of software such as QUANTA or SYBYL₁ automated or semi-automated de novo design approaches are also available, such as: - MCSS/HOOK [26, 27], which links multiple functional groups with molecular templates taken from a database. - LUD! [28], which computes the points of interaction that would ideally be fulfilled by a ligand, places fragments in the binding site based on their ability to interact with the receptor, and then connects them to produce a ligand.

- MCDLNG [29], which fills a receptor binding site with a close-packed array of generic atoms and uses a Monte Carlo procedure to randomly vary atom types, positions, bonding arrangements and other properties.

- GROW [30], which starts with an initial 'seed' fragment (placed manually or automatically) and grows the ligand outwards.

- 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.

- LEAPFROG [33], which evaluates ligands by making small stepwise structural changes and rapidly evaluating the binding energy of the new compound. Changes are kept or discarded based on the altered binding energy, and structures evolve to increase the interaction energy with the receptor.

- GROUPBUILD [34], which uses a library of common organic templates and a complete empirical force field description of the non-bonding interactions between a ligand and receptor to construct ligands that have chemically reasonable structure and have steric and electrostatic properties complimentary to the receptor binding site.

- RA3SE [35]

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.).

As well as being useful compounds individually, 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. Provided herein are: (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.

Therapeutic Methods and Compositions

Provided herein 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. 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. When a therapeutically effective amount of a component of the pharmaceutical combination is administered orally, the binding agent may be in the form of a tablet, capsule, powder, solution or elixir. When administered in tablet form, the pharmaceutical combination may additionally contain a solid carrier such as a gelatin or an adjuvant. When administered in liquid form, 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.

When a therapeutically effective amount of a component of the pharmaceutical combination is administered by intravenous, cutaneous or subcutaneous injection, the component of the combination will be in the form of a pyrogen-free, parenterafiy acceptable aqueous solution. In one embodiment, 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. In one embodiment, ala-TFPI is formulated at 0.45 mgtøiL C-termina! poly peptides may be provided at, e.g., 3 μM -300 nM, or 30 nM or 3 nM or

0.3 nM, and may be formulated with tifacogin at, e.g., 0.45 mg/ml or 0.15 mg/ml. Antibiotics may be provided at doses well known to physicians and clinicians, In another embodiment, ala-TFPI is formulated at 0.15 mg/mL In another embodiment, 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. In another embodiment, 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. In one embodiment, ala-TFPI is administered as a continuous infusion for 96 hours. Also contemplated is subcutaneous (s.c.) therapy using a component of the pharmaceutical combinations disclosed herein. The attending physician will decide on the appropriate duration of i.v. or s.c. therapy, and the timing of administration of the therapy, using the pharmaceutical combination.

Dosages and routes of administration can be determined by routine experimentation. 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). Other nonlimiting examples of routes of administration include 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.

As used herein, 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. When applied to an individual active ingredient (component), administered alone, the term refers to that ingredient alone. When applied to a combination, 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.

Gram-positive Bacterial Infections

The bacterial infection may be with a single Gram-positive bacterial species, or with several Gram-positive bacterial species. Thus "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. Typical, non-limiting examples of Gram-positive bacteria involved in severe infections, and which may be treated with a pharmaceutical combination as presently disclosed, 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. (See, e.g., Ostapchuk et al. (2004) Am. Family Physician 70:5:899-908; Arancibia et al. (2002) Arch. Intern. Med. 162:1849-58). In one embodiment, the Gram-positive bacterial infection results from infection by Staphylococcus aureus or Streptococcus pneumonia. In one embodiment, 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.

Provided herein is also the use of the disclosed combinations in the manufacture of a medicament for treating a patient at risk of developing or diagnosed as having a Gram-positive bacterial infection.

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.

General

The term "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.

The term "about" in relation to a numerical value x means, for example, x+10%. Where necessary, the term "about" can be omitted.

The word "substantially" does not exclude "completely," e.g., a composition which is "substantially free" from Y may be completely free from 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],

As indicated in the above text, nucleic acids and polypeptides disclosed herein may include sequences that:

(a) are identical (/.©., 100% identical) to the sequences disclosed in the sequence listing;

(b) share sequence identity with the sequences disclosed in the sequence listing;

(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

(a) or (b); and

(d) when aligned with a particular sequence from the sequence listing using a pairwise alignment algorithm, a moving window of x monomers (amino acids or nucleotides) moving from start (N-terminus or 5') to end (C-terminus or 3'), such that for an alignment that extends to p monomers {where p>x) there are p-x+1 such windows, 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

Needleman-Wunsch global alignment algorithm [37], using default parameters (e.g., with Gap opening penalty = 10.0, and with Gap extension penalty = 0.5, using the EBLOSUM62 scoring matrix). This algorithm is conveniently implemented in the needle tool in the EMBOSS package [38].

The nucleic acids and polypeptides herein may additionally have further sequences to the N-terminus/5¹ and/or C-terminus/3' of these sequences (a) to (d).

The term "animal" refers to any member of the animal kingdom including human beings.

The term "treat" or "treating" 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.

The term "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.

The methods herein will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, molecular biology, immunology and pharmacology, within the skill of the art. Such techniques are explained fully in the literature, e.g., see references [39-46], etc.

BRIEF DESCRIPTION OF DRAWINGS

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 tifacogiπ 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.

EXAMPLES

The following Examples provide illustrative embodiments and do not in any way limit the scope of the disclosed subject matter. One of ordinary skill in the art will recognize that numerous other embodiments are encompassed within the scope of the disclosed subject matter.

In the examples that follow, all exogenous TFPI is the TFPI analog, ata-TFPI.

EXAMPLE 1. METHODS

Antibacterial effect of tifacogin and C-terminal polypeptides of TFPI To characterize response to infection in a whole blood culture system, a non-pathogenic, Gram-positive bacteria, Staphylococcus epidermidis (ATCC 700583), which is efficiently removed from blood by the innate immune system of humans, is used. Under conditions of 10% fresh donor blood diluted within 5 minutes with RPMI (without phenol red) and distributed into 96 well tissue culture dishes (round bottom, Coming) slow clotting over the course of an hour is observed (Johnson et al., Blood. 1996 Jun 15;87(12):5051-60). In the standard experiment, cultures are inoculated with 10,000 colony forming units (CFU) of S. epidermidis, treated with or without hirudin (12 units/ml) or tifacogin (100 nM), incubated overnight in a tissue culture incubator (37⁰C, 5% CO2), and bacterial titer measured by plating on growth agar (trypticase soy agar). Some samples contain 3 μM of 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). Activity of CSS-3 is compared to cultures containing 3 μM 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 37⁰C and then diluting into RPMI to a final concentration of 10 units/ml.

Anti-inflammatory activity

Anti-inflammatory activity is assessed by measuring induced levels of IL-6, IL-1β, IL-8 and TNF-α 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 μg/ml) or erythromycin (50 μg/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, TNFα and IL-1 β) were measured after 20 to 24 hours.

Example 2 RESULTS

As shown in Figure 1, growth of S. epidermidis is inhibited in the presence of tifacogin (10OnM). As shown in Table 1, 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.

Table 1 - Growth of S. epidermidis

As shown in Figure 2, 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-α 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

In light of the above, 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).

As shown in Figure 3, either tifacogin or a C-terminal polypeptide of TFPI (CSS-3, SEQ ID NO:4) 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-α (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.

Table 3 - Cytokine levels (10000 CFU/well S. epidermidis)

Since complement is required for SEQ ID N0:4 (CSS-3 - a C-terminal polypeptide of TFPI) to kill E. coli, the effect of complement inactivation using cobra venom factor (CVF) is tested on S. epidermidis in whole blood cultures (Table 4). CVF added to coagulating blood or tifacogin anti-coagulated blood produces a major increase in bacterial growth, showing the complement sensitivity of $. epidermidis. CVF has no effect on the CSS-3 activity killing S. epidermidis. Given that the bacteria are the likely trigger for inflammatory cytokines, it is highly surprising to see that CVF disconnects the positive relationship between bacterial titer and cytokine induction (Table 5). CVF prevents clearance of S, epidermidis in tifacogin containing blood and there is a large increase in cytokines in those samples. However, while CSS-3 kills S. epidermidis in the presence of CVF₁ 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. These data indicate that although CSS-3 can directly kill S. epidermidis, CSS-3 works through complement to lower inflammatory cytokines induced by S. epidermidis bacteria. "Peptide 2" of Table 4 corresponds to the scrambled peptide set forth in SEQ ID N0:7.

Table 4 - effect of complement inactivation using cobra venom factor (CVF) on S. epidermidis growth

Table 5 - Cytokine levels (10000 CFU/well S. epidermidis)

The induction of inflammatory cytokines in blood containing CSS-3 (SEQ ID NO:4) by S. epidermidis suggests that killing bacteria via antibiotics may not sufficiently remove the bacteria from acting as inflammatory triggers. As shown in Figure 4, adding vancomycin to a whole blood cultures inoculated with S. epidermidis minimally reduces the induction of IL-6. However, vancomycin in combination with tifacogin (or other anticoagulants, e.g., hirudin) significantly reduces the cytokine induction by S. epidermidis. In spite of this reduction, there is still an inflammatory signal in the presence of tifacogin and vancomycin in combination. Reductions in IM β, IL-8 and TNFα all show a similar pattern (Table 6).

Table 6 - Cytokine Levels (10000 CFU/well S. epidermidis)

Attempts to clear the diverse organisms S. aureus and S. pneumoniae from whole blood cultures show that the antibacterial activity of blood is not sufficient to control these pathogens with or without anti-coagulants (data not shown). These strains are well documented to be resistant to the combined effects of complement and leukocytes. As shown in Figures 5 and 6 (IL-6 data) and Tables 3 and 4 (IL-6, IL-1 β, IL-8 and TNFα data), the combination of tifacogin and CSS-3 (SEQ ID NO:4) with vancomycin reduces cytokines IL-6, IL-1β, IL-8 and TNFα induced by S. aureus (Table 7) or S. pneumoniae (Table 8) to near background levels. Similar results are obtained with erythromycin instead of vancomycin (data not shown).

Table 7 - Cytokine levels (10000 CFU/well S. aureus)

Table 8 - Cytokine levels (300 CFU/well S. pneumonia]

IL-1β IL-6 IL-8 TNF-α

Blood+Vanc 57 56 1800 1840 12700 12600 170 150 Blood+Vanc+tifacogin 12 14 250 245 4800 3300 31 35

Blood+vanc+CSS-3 9 8 187 192 1810 2600 18 18

Blood+vanc+tff+CSS-3 19 23 32 35 256 260 31 33

Example 3

Discussion and Conclusion

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. These patients suffer disseminated intravascular coagulopathy (DIC) and excessive inflammation, leading to multiple organ failure. A retrospective analysis of a large clinical trial suggests that tifacogin may be effective in sCAP patients at decreasing mortality, however, the tifacogin mechanisms are unclear.

TFPI inhibits the initiation of blood clotting by binding to the tissue factor, Vila and Xa complex. Thus, part of the tifacogin mechanism is likely that it restores the haemostatic balance resolving DIC. However, in 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). Thus, 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

CVF.

In parallel, herein it is shown that 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. Interestingly, the data provided herein shows that inflammatory cytokines are induced whether bacteria are alive or dead - what matters more is active complement and an ability to enhance complement interaction with the bacteria were critical to controlling cytokines. These data show the inflammatory activity of S. aureus, S. epidermidis and S. pneumoniae in cultures with either vancomycin or erythromycin. Tifacogin and C-terminal polypeptides of TFPI in combination with antibiotics in reducing cytokine induction and treating Gram-positive bacterial infection re expected to be clinically relevant, Bacterial strategies to evade opsonization and phagocytosis are properties of the bacterial capsule and likely persist after death, potentially extending bacteria's pathological effects, even though infection has been controlled.

Disclosed herein is the finding that C-terminal polypeptides of TFPI directly kill S. epidermidis. This may be through the same mechanism as cationic bactericidal peptides. In whole blood CSS-3 (SEQ ID N0:4) remains active. 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.

In retrospective analysis of clinical trial data (OPTIMIST) efficacy of the recombinant anticoagulant tifacogin was unexpectedly correlated with bacterial infections and not other causes of sepsis, Although these data suggest that Tifacogin in some way affects the pathologies associated with a bacterial infection, this was not anticipated from the documented activities of TFPI, In an investigation to understand this, it was discovered that fragments of tifacogin or synthetic peptides based on the C-terminal TFPI amino acids (243 to 276) killed E. coli in a mechanism dependent on serum complement. Gram-negative £ coli is not a frequent cause of pneumonia. The same fragments were tested for the ability to kill S. aureus or S. pneumoniae, Gram-positive bacteria associated with the majority of community acquired pneumonia cases. However, growth of both pathogens was unaffected by tifacogin, tifacogin fragments and C-terminal peptides in serum containing assays, which suggested an absence of activity against Gram-positive bacteria.

Conversely, disclosed herein is a whole blood culture system inoculated with Gram-positive bacteria as a means of measuring the complicated interactions between tifacogin and its derivatives, and bacteria, antibiotics, blood clotting, complement, leukocytes and inflammatory cytokines. Disclosed herein are the findings that tifacogin (ala- TFPI of SEQ ID N0:2) and a C-terminal polypeptide of TFPI (SEQ ID N0:4) increase clearance of a Gram-positive bacteria and decrease the induction of inflammatory cytokines. Either tifacogin or a C-terminal polypeptide of TFPI has similar effects. Since the C-terminal polypeptide of TFPI is wholly contained within tifacogin, it is surprising to see that the combination of these two agents synergize. Further, it is determined that either agent combined with antibiotics is superior to antibiotics alone in decreasing induced cytokines, and the combination of antibiotics with tifacogin plus C-terminal peptides is unexpectedly powerful.

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Claims

WHAT IS CLAIMED IS:

I. A pharmaceutical composition 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. 2. The pharmaceutical composition as set forth in claim 1, wherein said C-terminal polypeptide of TFPI consists essentially of the amino acid sequence set forth in SEQ ID N0:3, 4, 5 or 8.

3. The pharmaceutical composition as set forth in any one of claims 1-2, wherein said C-terminal polypeptide of TFPI consists of the amino acid sequence set forth in SEQ ID N0:3, 4, 5 or 8.

4. The pharmaceutical composition as set forth in any one of claims 1-3, wherein said antibiotic is vancomycin or erythromycin.

5. The pharmaceutical composition as set forth in any one of claims 1-4, further comprising TFPI or ala-TFPI.

6. The pharmaceutical composition as set forth in claim 5, wherein said ala-TFPI consists of the amino acid sequence set forth in SEQ ID N0:2.

7. The pharmaceutical composition as set forth in any one of claims 1-6, wherein said Gram-positive bacteria! infection results from infection by Staphylococcus aureus or Streptococcus pneumonia.

8. A pharmaceutical combination 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.

9. The pharmaceutical combination as set forth in claim 8, wherein said C-terminal polypeptide of TFPI consists essentially of the amino acid sequence set forth in SEQ ID NO:3, .4, 5 or 8. 10. The pharmaceutical combination as set forth in claim 9, wherein said TFPI C-terminal polypeptide consists of the amino acid sequence set forth in SEQ ID NO:3, 4, 5 or 8.

II. The pharmaceutical combination as set forth in any one of claims 8-10, wherein said antibiotic is vancomycin or erythromycin,

12. The pharmaceutical combination as set forth in any one of claims 8-11, further comprising TFPI or ala-TFPI. 13. The pharmaceutical combination as set forth in claim 12, wherein said ala-TFPI consists of the amino acid sequence set forth in SEQ ID NO:2.

14. The pharmaceutical combination as set forth in any one of claims 8-13, wherein said Gram-positive bacterial infection results from infection by Staphylococcus aureus or Streptococcus pneumonia.

15. A method of treating a Gram-positive bacterial infection in an animal comprising administering a therapeutically effective amount of the pharmaceutical composition set forth in any one of claims 1-7 to a subject in need thereof.

16. A method of treating a Gram-positive bacterial infection in an animal comprising administering a therapeutically effective amount of the pharmaceutical combination set forth in any one of claims 8-14 to a subject in need thereof.

17. The method of claim 15, wherein said Gram-positive bacterial infection results in severe community acquired pneumonia or sepsis.

18. The method of claim 16, wherein said Gram-positive bacterial infection results in severe community acquired pneumonia or sepsis.

19. A method of reducing 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.

20. The method as set forth in claim 19, wherein said C-terrrtinal polypeptide of TFPI consists essentially of the amino acid sequence set forth in SEQ ID NO:3, 4, 5 or 8.

21. The method as set forth in claim 20, wherein said C-terminal polypeptide of TFPI consists of the amino acid sequence set forth in SEQ ID NO:3, 4, 5 or 8.

22. The method as set forth in any one of claims 19-21, wherein said antibiotic is vancomycin or erythromycin.

23. The method as set forth in any one of claims 19-22, further comprising administering TFPI or ala-TFPI to said plurality of cells.

24. The method as set forth in claim 23, wherein said ala-TFPI consists of the amino acid sequence set forth in SEQ ID NO:2.

25. The method as set forth in any one of claim 19-24, wherein said Gram-positive bacterial infection results from infection by Staphylococcus aureus or Streptococcus pneumonia. 26. The method as set forth in any one of claim 19-25, wherein said Gram-positive bacterial infection results in severe community acquired pneumonia or sepsis.

27. 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.

28. A C-terminal polypeptide of TFPI and an antibiotic for combined use in treating a Gram-positive bacterial infection in an animal.

29. A combination of a C-terminal polypeptide of TFPI and an antibiotic for use in treating a Gram-positive bacterial infection in an animal. 30. A C-terminal polypeptide of TFPI and an antibiotic for use in a method of treating a Gram-positive bacterial infection in an animal.

31. 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. 32. 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.

33. Use of a therapeutically effective amount of a pharmaceutical composition as claimed in any one of claims 1-7 in the manufacture of a medicament for the treatment of a Gram-positive bacterial infection in an animal.

34. Use of 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.

35. 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.

36. Use of 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.

37. 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.

38. 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.

39. 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.

40. 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.

41. 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.

42. 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. 43. 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 TFPf and TFPI or ala-TFPI.

44. 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.

45. Use of a therapeutically effective amount of a pharmaceutical combination as claimed in any one of claims 8- 14 in the manufacture of a medicament for the treatment of a Gram-positive bacterial infection in an animal.

46. Use of 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.

47. 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.

48. Use of 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. 49. Use of 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.

50. 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-TFPI.

51. Use of 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.

52. Use of 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. 53. 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-TFPl.

54. Use of 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 wherein the animal has previously been treated with or will subsequently be treated with an antibiotic.

55. The use set forth in any of the above claims, wherein said C-terminal polypeptide of TFP! consists essentially of the amino acid sequence set forth in SEQ ID NO:3, 4, 5 or 8. 56. The use set forth in claim 55, wherein said C-terminal polypeptide of TFPI consists of the amino acid sequence set forth in SEQ ID NO:3, 4, 5 or 8.

57. The use set forth in any of the above claims, wherein said antibiotic is vancomycin or erythromycin.

58. The use set forth in any of the above claims, wherein said TFPI or ala-TFPI is ala-TFPI, and wherein said ala- TFPI consists of the amino acid sequence set forth in SEQ ID NO:2. 59. The use set forth in any of the above claims, wherein said Gram-positive bacterial infection results from infection by Staphylococcus aureus or Streptococcus pneumonia.

60. The use set forth in any of the above claims, wherein said Gram-positive bacterial infection results in severe community acquired pneumonia or sepsis.

61. 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.

62. The commercial package set forth in claim 61 , wherein said C-terminal polypeptide of TFPI consists essentially of the amino acid sequence set forth in SEQ ID NO:3, 4, 5 or 8.

63. The commercial package set forth in claim 62, wherein said C-terminal polypeptide of TFPI consists of the amino acid sequence set forth in SEQ ID NO:3, 4, 5 or 8.

64. The commercial package set forth in any of claims 61-63, wherein said antibiotic is vancomycin or erythromycin.

65. The commercial package set forth in any of ciaims 61-64, wherein 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.

66. The commercial package set forth in any of claims 61-65, wherein said Gram-positive bacterial infection results from infection by Staphylococcus aureus or Streptococcus pneumonia.

67. The commercial package set forth in any of claims 61-66, wherein said Gram-positive bacterial infection results in severe community acquired pneumonia or sepsis.