BRPI0610549A2 - treatment of severe community-acquired pneumonia by administration of tissue factor pathway inhibitor (tfpi) - Google Patents

Abstract

Methods for prophylactically or therapeutically treating severe pneumonia involve the administration of tissue factor pathway inhibitor (TFPI) or a TFPI analogue to patients suffering from or at risk for developing this condition. The methods involve the use of continuous intravenous infusion of TFPI or a TFPI analogue, preferably at low doses to avoid adverse side effects.

Description

TREATMENT OF SEVERE PNEUMONIA PURCHASED IN THE COMMUNITY BY THE ADMINISTRATION OF TISSUE FACTOR INHIBITOR (TFPI)

Field of the invention

The present invention relates to a method for the therapeutic treatment of community-acquired severe pneumonia. More specifically, it relates to the administration of a fat-specific pathway inhibitor protein to attenuate exuberant or amplified physiological pathways associated with severe pneumonia.

Background of the invention

Pneumonia results from an acute infection of one or more functional elements of the lung, including spacesveolar and interstitial tissue. In the US, about 2 million people develop pneumonia each year and 40,000 to 70,000 of them die. Pneumonia ranks sixth among all disease categories as a cause of death and is the most common lethal nosocomial infection. Community Acquired Pneumonia (CAP) has a significant impact on public health costs in the United States, accounting for an estimated $ 14 billion per year in direct costs and $ 9 billion in lost investments. (Lynch JP, Martinez F J. Community Acquired Pneumonia. CurrentT Opin Pulm Med. 1998; 4: 162-172). In developing countries, lower respiratory tract infections are typically the leading cause of death or loss only for infectious diarrhea. (The Merck Manual, Sec. 6, Ch. 73, Pneumonia, 2000).

The condition known as "severe pneumonia" is characterized according to the guidelines presented by various organizations, including the American Thoracic Society (ATS). (Am J Respir Crit Care Med 2001; 163: 1730-1754). For example, ATS requires at least one major criterion, such as the need for mechanical ventilation or septic shock, in addition to other criteria for a diagnosis of severe pneumonia. Generally, pneumoniasevera may result from acute lung disease, inflammatory pulmonary disease, or any disturbance in pulmonary function due to factors such as inflammation or coagulation. A severe CAP diagnosis is based on a patient being admitted to an ICU specifically for pneumonia. Epidemiologically, this patient population comprises approximately 10% of all ICU admissions. ICU patients with pneumonia have the highest mortality of CAP patients (30% to 40%) compared to less than 15% for hospitalized patients with CAP.

Each year in the United States, community-acquired pneumonia (CAP) is diagnosed in approximately 4 million adults, with 600,000 requiring hospitalization. Fine et al., N. Engl. J. Med. 336, 243-50.1997. Overall, the incidence of CAP increases with age, with the highest prevalence found in those 65 and older. Marston et al., Arch Intern Med. 1997; 157: 1709-1718.The incidence also increases in patients with comorbidities such as chronic obstructive pulmonary disease, asthma, diabetesmellitus, alcoholism, immunosuppression, renal failure, chronic liver disease, and heart disease. Marrie, CurrOpin Pulm Med. 1996; 2: 192-197; Niedermann et al, Am RevRespir Dis. 1993; 148: 1418-1426.

Pneumonia is the leading cause of death from infections in the United States and the sixth leading cause of death in general. The pneumonia-related mortality rate increased by 22% from 1979 to 1992, with elderly patients (65 years and older) accounting for 89% of all pneumonia-related deaths in 1992. See, "Pneumonia andinfluenza death rates" - United States , 1979-1994 [published correction appears in MMWR. 1995; 44: 782]. MMWR. 1995; 44: 535-53 7. Fine and colleagues (1997) reported that certain coexisting diseases (neoplastic disease, congestive heart failure (CHF), cerebrovascular disease, renal disease, and liver disease) and certain physical examination findings (altered mental status, increased heart rate). increased respiratory rate, decreased systolic blood pressure, and abnormally low or elevated temperatures) are also associated with increased CAP-related mortality. In addition, CAP has a significant impact on public health costs in the United States, accounting for an estimated $ 14 billion a year in direct costs and $ 9 billion in lost investments. Lynch & Martinez, Curr Opin Pulm Med. 1998; 4: 162-172.

Tissue factor pathway inhibitor (TFPI) is a protein and serine protease inhibitor present in mammalian blood plasma. Thomas, Bull. Johns HopkinsHosp. 81, 26 (1947); Schneider, Am. J. Physiol. 149, 123 (1947); Broze & Miletich, Proc. Natl. Acad. Know. USA 84,1886 (1987). TFPI is also known as fatortecid inhibitor, tissue thromboplastin inhibitor, Factor III inhibitor, extrinsic pathway inhibitor (EPI) and lipoprotein-associated coagulation inhibitor (LACI). The name "tissue factor pathway inhibitor" (TFPI) was accepted by the "International Society on Thrombosis and Hemostasis" on 30 June 1991.

Activation of blood coagulation is the conversion of fluid blood into a solid gel or clot. In addition, the consumption of coagulation proteases leads to excessive bleeding. The main event is the conversion of soluble defibrinogen to insoluble fibrin filaments, although fibrin itself forms only 0.15% of the total blood clot. This conversion is the last step in a complex enzyme hull. The components (factors) are present as zymogens, inactive precursors of proteolytic enzymes, which are converted to active enzymes by proteolytic cleavage at specific sites. Activation of a small amount of one factor catalyzes the formation of larger amounts of the next, and so on, resulting in amplification that results in extremely rapid fibrin formation.

Coagulation is believed to be initiated by damage that exposes the factor Vila to tissue factor (TF), which is expressed in cells below the endothelium. The factorVIIa-TF complex cleaves factor X to factor Xa and cleaves factor IX aofactor IXa. TFPI binds to the factor Vila and factor Xa. The complex formed between TFPI, factor Vila (with its TF on) and factor Xa inhibits the additional formation of factors Xa and IXa, necessary for sustained homeostasis. Broze, Jr., Ann.Rev. Med. 46: 103 (1995).

Activation of the coagulation cascade by bacterial products, including endotoxins, introduced directly into the bloodstream may result in large fibrin deposition on arterial surfaces, as well as fibrinogen, prothrombin, factor V, and VIII platelet depletion. In addition, the fibrinolytic system is stimulated, resulting in further formation of fibrin degradation products.

While activation of coagulation is apparently initiated by bacterial products (eg endotoxin), counteracting mechanisms also appear to be activated by coagulation, ie activation of the fibrinolytic system. Activated factor XIII converts plasminogen proactivator to plasminogen activator which subsequently converts plasminogen to plasmin, thereby mediating clot lysis. Activation of plasma fibrinolytic systems may therefore also contribute to bleeding tendencies.

Endotoxemia is associated with an increase in circulating plasma plasminogen activator inhibitor (PAT) levels. This inhibitor rapidly inactivates tissue plasminogen activator (TPA), thereby impeding its ability to promote fibrinolysis through plasminogen activation to plasmin. Failure of fibrinolysis may cause fibrin deposition in the vasosanguines, thus contributing to IHD (disseminated intravascular coagulation) associated with septic shock.

Progressive efforts have been made to identify satisfactory interventions for the prevention or treatment of severe pneumonia and associated coagulopathies. An agent that disrupts the coagulation pathway is not necessarily effective as a therapeutic or prophylactic treatment of severe pneumonia. For example, heparin is a commonly used anticoagulant, however, controlling the use of heparin has been difficult because heparin can induce excessive bleeding and attenuate coagulation abnormalities but does not offer a survival benefit. See, for example, Aoki et al, "A Comparative Double-BLIND Randomized Trialof Activated Protein C and Unfractionated Heparin in theTreatment of Disseminated Intravascular Coagulation", Int.J. Hematol 75, 540-47 (2002). Several clinical trials, especially in meningococcal endotoxemia where fulminant IHD is a prominent feature, have failed to demonstrate a reduction in mortality due to heparin treatment. See, for example, Corrigan ecols., "Heparin Therapy in Septacemia with Disseminated Intravascular Coagulation. Effect on Mortality and on Correction of Hemostatic Defects," N. Engl. J. Med. 283: 778-782 (1970); Lasch et al, Heparin Therapy of Diffuse Intravascular Coagulation (DIC) ", Thrombos.Diathes. Haemorrh., 33: 105 (1974); Straub," A Case AgainstHeparin Therapy of Intravascular Coagulation ", Thrombos.Diathes. Haemorrh., 33: 107 (1974).

Patients prone to severe community-acquired pneumonia are those patients with community-acquired pneumonia who require admission to an Intensive Care Unit (ICU). Patients with community-acquired pneumonia are clinically identified as having a pulmonary parenchymal infection and / or confirmed by radiographic and clinical signs. Severe pneumonia includes severe community-acquired pneumonia, typically has well-defined pathogens, including S.pneumonae, legionella, H. influenae or several gram-negative bacilli. Most patients with severe community-acquired pneumonia live in the community prior to the CAP episode, with only about 20% admitted as a transfer from a hospital, nursing home or long-term hospitalization. Patients in the United States with severe CAP are about 50% men and about 50% women, but tend to be older. About 17% of severe CAP patients in the United States are under 50; about 24% are between 50 and 64 years old; About 21% are between 65 and 74 years old and about 38% are over 75 years old. Most patients with CAPsevera have one or more significant comorbidities. Typically, US patients with CAP who receive ICU treatment in 2003 have corresponding heart disease, COPD / cystic fibrosis, diabetes, disease, cancer, alcoholism, and / or are dependent on them. drugs.

Administration of human recombinant ala-TFPI (a TFPI analog) has been shown to improve survival rates in animal sepsis models. See, for example, U.S. Patent. No. 6,063,764. As an endogenous protein, TFPI is well tolerated. Administration of TFPI by intravenous infusion or subcutaneous injection has been shown to reduce the ability of coagulation, which is manifested as increased prothrombin time (PT). In animal and human studies, PT prolongations were linearly related to increased plasma TFPI. A. A. Creasey, Sepsis 3: 173 (1999).

There remains a need in the art for treatment approaches that will inhibit the lethal effects of pneumoniasevera while minimizing potentially serious side effects.

One embodiment of the present invention is a method of treating or preventing severe pneumonia comprising administering TFPI or a TFPI analog to a patient who has or is at risk of developing severe pneumonia.

In some embodiments, the patient has a demonstrated infection.

Another embodiment of the present invention is a method of treating severe pneumonia comprising administering to a patient a continuous intravenous infusion of an agent selected from the group consisting of TFPI or a TFPI analog. In some embodiments, the patient has a demonstrable infection.

Another embodiment of the present invention is a method for the treatment or prevention of severe pneumonia comprising administering TFPI or a TFPI analog to a human patient who has or is at risk of developing severe pneumonia in an amount that does not induce excessive bleeding.

Other embodiments include any of the above modalities wherein said TFPI or TFPI analog is administered by continuous intravenous infusion at a dose rate equivalent to the reference administration of ala-TFPI at a dose rate of less than about 2.0mg / kg / kg. hr. In a preferred embodiment, said dose rate is equivalent to the ala-TFPI reference administration at a dose rate of from about 0.00025 to about 2.0, or alternatively from about 0.001 to about 1.75mg / ml. kg / hr. In another preferred embodiment, said dose rate is equivalent to dealt-TFPI reference administration at a dose rate of about 0.005 to about 1.5 mg / kg / hr. In a more preferred embodiment said dose rate is equivalent to deala-TFPI reference administration at a dose rate of about 0.010 to about 0.75 mg / kg / hr. In a still more preferred embodiment, said TFPI or said TFPI analog is administered at a dose rate equivalent to the ala-TFPI reference administration at a dose rate of about 0.2mg / kg / hr to about 0.8 mg / kg / hr. In another preferred embodiment, said dose rate is administered to provide a total dose equivalent to the administration of ala-TFPI in a total dose of about 0.024 to about 4.8 mg / kg. In another preferred embodiment, said dose rate is administered to provide a dosage equivalent to the ala-TFPI reference administration at a daily dose of at least about 0.006 mg / kg and less than about 1.2 mg / kg.

Other embodiments include any of the above modalities, wherein said TFPI or TFPI analog is administered for at least 72 hours. In a preferred embodiment, said TFPI or TFPI analog is administered for at least 96 hours.

Other embodiments include any of the above modalities wherein said TFPI analog is unglycosylated ala-TFPI.

Other embodiments include any of the above embodiments wherein said TFPI analog comprises a first Kunitz domain consisting of Seq. No. 1: In one preferred embodiment, said TFPI analog also comprises a second Kunitz domain consisting of amino acids 90-160 of Id. De Seq. No.: 1.

Other embodiments include any of the above modalities wherein said TFPI analog comprises amino acids 1-160 of Id. De Seq. No. 1 or wherein said TFPI analogue comprises a second deKunitz domain consisting of amino acids 90-160 of Seq.No.:Id.

Other embodiments include any of the above modalities wherein said TFPI or TFPI analog is prepared from a lyophilized composition comprising TFPI or a TFPI analog.

Other embodiments include any of the above modalities wherein said TFPI or TFPI analog is administered as a formulation comprising arginine.

Other embodiments include any of the above modalities wherein said TFPI or TFPI analog is administered as a formulation comprising citrate.

Other embodiments include any of the above modalities wherein said TFPI or TFPI analog has a concentration of about 0.15 mg / ml in a formulation comprising about 300 mM dearginine hydrochloride and about 20 mM sodium citrate and which has an HP of about 5.5.

Other embodiments include any of the above modalities, which also comprises administration at the same time as, or within 24 hours of said TFPI or TFPI analog administration, of an additional selected agent from the group consisting of an antibiotic, an antibody, an endotoxin antagonist, a tissue defactor analogue having anticoagulant activity, an immunostimulant, a cell adhesion blocker, heparin, BPI protein, an IL-1 antagonist, pahase (PAF enzyme inhibitor), a TNF inhibitor, an IL-6 inhibitor, and an inhibitor of complement. In one preferred embodiment, said additional agent is an antibody which specifically binds to an antigen selected from the group consisting of TNF, IL-6, and M-CSF.

Additional embodiments of the present invention are apparent in view of the reference drawings below with the detailed description.

Detailed Description of the Invention

Administration of TFPI or TFPI analogs is effective in the prophylaxis and treatment of severe pneumonia. Continuous low dose administration of TFPI or TFPI analogs (herein "low dose TFPI administration") is also effective in the prophylaxis and treatment of severe pneumonia. Administration of TFPI or TFPI analog, particularly low-dose administration, inhibits or attenuates acute or chronic inflammation, particularly severe pneumonia. When low-dose TFPI administration is continued for at least three days, the risk of death from severe pneumonia is reduced, while the rate of complications of adverse side effects, particularly bleeding disorders, may be minimized. An additional advantage of low-dose TFPI administration is that it avoids the tolerance effects that at sufficiently high doses may reduce the plasma concentration of TFPI. Tolerance effects are stimulated to half the maximum at a TFPI plasma concentration of about 850 ng / ml, whereas with low dose TFPI plasma levels are usually below 500 ng / ml.

Low-dose TFPI administration is usually performed by continuous intravenous infusion of TFPI or a TFPI analog.

TFPI and TFPI Analogs

"TFPI" as used herein refers to serum glycoproteinamate having the sequence of 276 amino acid residues shown in Seq Id. No. 1 and a molecular weight of about 38,000 Daltons. It is a natural inhibitor of tissue factor activity and thus of coagulation activation. U.S. Patent No. 5,110,773 describes fatortecidual (TF), and U.S. Patent No. 5,106,833 describes TFPI. TFPI cDNA cloning is described in Wun et al., U.S. Patent. No. 4,966,852. TFPI is a protease inhibitor and has 3 Kunitz domains, two of which are known to interact with factors VII and Xa respectively. The function of the third domain remains unknown.

TFPI is believed to function in vivo to limit the initiation of coagulation by formation of an inert complex, quaternary factor X.sub.a: TFPI: factor VII.sub.a: fatortecidual. See reviews by Rapaport, Blood 73: 359-365 (1989) and Broze et al., Biochemistry 29: 7539-7546 (1990). Several of the structural characteristics of TFPI can be deduced from its homology with other well-studied deprotease inhibitors. TFPI is not an enzyme, so it probably inhibits its protease target in a stoichiometric manner, ie one of the Kunitz domains of TFPI inhibits a protease molecule. Preferably, Kunitz domains 1 and / or 2 will be present in TFPI molecules of the present invention, the function of Kunitz domain 3 is unknown.

A "TFPI analog" is a TFPI derivative modified with one or more amino acid additions or substitutions (generally conservative in nature), one or more amino acid deletions (e.g., TFPI fragments) or the addition of one or more chemical moieties to one or more amino acids, provided that the modifications do not destroy the biological activity of TFPI. Methods for making polypeptide analogs are known in the art and are further described below. A preferred analogue of TFPI is N-L-alanyl-TFPI (ala-TFPI), whose amino acid sequence is shown in Seq. No. 2. TFPI analogs have some measure of TFPI activity as determined by a bioactivity assay as described below, a preferred TFPI and analogous assay is the prothrombin time (PT) assay (see below).

TFPI and TFPI analogs may be glycosylated or unglycosylated. TFPI analogs are described in U.S. Patent. No.: 5,106,833. Ala-TFPI is a TFPI analog which is also known under the international drug name "tifacogin." Ala-TFPI includes the full-length mature human TFPI amino acid sequence plus an additional amino-terminal alanine residue. The ala-TFPI amino terminal alanine residue was engineered into the TFPI sequence to improve E. coli expression and to cleave what would otherwise be an amino terminal methionine residue. See U.S. Patent No. 5,212,091.

Particularly preferred TFPI analogs include substitutions that are conservative in nature, that is, those substitutions that occur in a family of amino acids that are related in their side chains. Specifically, amino acids are generally divided into four families: (1) acids - aspartate eglutamate; (2) basic-lysine, arginine, histidine; (3) nonpolar - alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar - glycine, asparagine, glutamine, cysteine, serine, threonine and tyrosine. Phenylalanine, tryptophan etyrosine are sometimes classified as aromatic amino acids. For example, it is reasonably foreseeable that an isolated leucine substitution with isoleucine or valine, a glutamate aspartate, a serine threonine, or a similar conservative amino acid substitution with a structurally related amino acid will not have a major effect on biological activity. For example, the opolipeptide of interest may include up to about 1-70 conservative or non-conservative amino acid substitutions, such as 1, 2, 3, 4, 5, 6-50, 15-25, 5-10, or any integer from 1 to 70 as long as the desired function of the molecule remains intact. One skilled in the art can readily determine molecule regions of interest which can be modified with a reasonable likelihood of retaining biological activity as defined herein.

"Homology" refers to the percentage similarity between two polynucleotides or two polypeptide moieties. Two polypeptide sequences are "substantially homologous" to each other when the sequences exhibit at least about 50%, preferably at least about 75%, more preferably at least about 80% -85%, preferably at least about 90% and even more. preferably at least about 95% -98% sequence homology or any percent homology within the specified ranges over a defined length of the molecules. As used herein, "substantially homologous" also refers to sequences that show complete identity to the specified polypeptide sequence.

In general, "identity" refers to an exact amino acid-to-amino acid match of two polypeptide sequences, respectively. The percent identity can be determined by a direct comparison of sequence information between two molecules by sequence alignment, exact number of matches between the two aligned sequences, division by shorter sequence length, and multiplication by 100.

Preferably, naturally occurring or non-naturally occurring TFPI analogs have amino acid sequences that are at least 70%, 80%, 85%, 90% or 95% or more homologous to TFPI derived from Seq Id. No. 1. Most preferably, the molecules are 98% or 99% homologous. Homology percentage is determined using the Smith-Waterman homology search algorithm that uses a related range search with an open range penalty of 12 and a range extension penalty of 2 and a BLOSUM matrix of 62. The homology search algorithm of Smith-Waterman is taught in Smithe Waterman, Adv. Appl. Math (1981) 2: 482-489.

The biological activity of TFPI and TFPI analogs may be determined by the prothrombin assay. Suitable deprothrombin assays are described in U.S. Patent No. 5,888,968 and WO 96/40784. Briefly, prothrombin times can be determined with the use of a coagulometer (eg Coag-AMate MTX II from OrganonTeknika). A suitable assay buffer is 100 mM NaCl, 50 mMTris adjusted to pH 7.5, containing 1 mg / ml bovine albumin. Additional reagents required are normal human plasma (eg "Verify 1" by Organon Teknika), thromboplastin reagent (eg "Simplastin Excel" by Organon Teknika), and TFPI standard solution (eg 20 mu.g of 100% ala-TFPI (or equivalent) per ml of assay buffer). A standard curve is obtained by analyzing the coagulation time of a series of deductions of the TFPI standard solution, for example, to final concentrations ranging from 1 to 5 mu.g / ml. For the determination of clotting time, the DTI sample or standard is first diluted in the assay buffer. Then normal human plasma is added. The coagulation reaction is initiated by the addition of thromboplastin reagent. The instrument then records the clotting time. A linear TFPI standard curve is obtained from a coagulation time vs. plot graph. TFPI concentration log. Standard curve is adjusted based on the purity of the TFPI standard to match the equivalent TFPI concentration of a 100% pure standard. For example, if the standard is a ala-TFPI preparation that is 97% biochemically pure (ie, it contains 3% by weight of molecular species without biological DTI activity), then the concentration of each standard dilution is multiplied by 0.97 to give the actual concentration of TFPI. Thus a TFPI standard that is 1.0 .mu. g / ml based on actual weight per ml of a preparation that is 97% pure will be equivalent to, and treated as, a concentration of 1.0 times 0.07, or 0.97 .mu.g / ml. Obtaining TFPI and TFPI Analogs

TFPI and TFPI analogs used in the methods of the invention may be isolated and purified from cells or tissues, chemically synthesized or produced to form matching in prokaryotic or eukaryotic cells.

TFPI can be isolated by various methods. For example, cells that secrete TFPI include endothelial cells, young endothelial cells that have been treated with

TNF for about 3 to 4 days, hepatocytes, and hepatoma cells. TFPI may be by conventional methods, including the chromatographic methods of Pedersen et al., 1990, J. Biol. Chem. 265, 16786-93, Novotny et al., 1989, J. Biol. Chem. 264, 18832-37, Novotny et al., 1991, Blood78, 394-400, Wun et al., 1990, J. Biol. Chem. 265, 16096-101, and Broze et al., 1987, Proc. Natl. Acad. Know. USA 84,1886-90. TFPI appears in the bloodstream and can be purified from the blood, see Pedersen et al 1990.

A TFPI or TFPI variant can be produced using chemical methods to synthesize its amino acid sequence, such as by direct peptide synthesis using solid phase techniques (Merrifield, J. ". Am. Chem. Soc. 85,2149-2154, 1963; Roberge et al. cols., Science 269, 202-204,1995) Protein synthesis can be performed using manual techniques or by automation Automated synthesis can be obtained, for example, using "Applied Biosystems" 431A Peptide Synthesizer (Optionally, fragments. TFPI or TFPI variants can be synthesized separately and combined using chemical methods to produce a full length molecule.

TFPI and TFPI analogs can be produced to form matching as shown in U.S. Pat. No. 4,966,852. For example, cDNA for the desired protein may be incorporated into a plasmid for expression in prokaryotes or eukaryotes. Pat. No. 4,847,201 provides details for the transformation of microorganisms with specific DNA sequences and to express them. There are many other references known to those of common technical skill which provide details on protein expression using microorganisms. Many of these are cited in U.S. Pat. No. 4,847,201, such as Maniatas et al., 1982, Molecular Cloning, Cold Spring Harbor Press.

A variety of techniques are available for microorganism transformation and for using them to express TFPI and TFPI analogs. The following are examples of possible approaches. TFPI DNA sequences must be isolated and connected to the appropriate control sequences. TFPI DNA sequences are shown in U.S. Pat. No. 4,966,852 and may be incorporated into a plasmid, such as pUNC13 or pBR3822, which are commercially available from companies such as Boehringer-Mannheim. Once TFPI DNA is inserted into a vector, it can be cloned into a suitable host. DNA can be amplified by techniques such as those shown in U.S. Pat. No. 4,683,202 to Mullis ePat. No. 4,683,195 to Mullis et al. TFPI cDNA can be obtained by inducing cells such as hepatoma cells (such as HepG2 and SKHep) to make TFPI mRNA, then identifying and isolating the mRNA and reverse-transcribing to obtain cTNA for TFPI. After the expression vector is transformed into a host such as E. coli, the bacteria can be fermented and the protein expressed. Bacteria are preferred prokaryotic microorganisms and E. coli is especially preferred. A preferred microorganism useful in the present invention is E. coli K-12 strain MM294 filed with the ATCC on February 14, 1984 (Accession No. 39607) under the provisions of the Budapest Treaty.

Of course, it is also possible to express genes that encode polypeptides in host-eukaryotic host cell cultures derived from multicellular organisms. See, for example, Tissue Culture, 1973, Cruz and Patterson, eds. Academic Press. Useful mammalian cell lines include murine myelomas N51, VERO, HeLa cells, Chinese hamster ovary (CHO) cells, COS, C127, Hep G2, and SKHep. TFPI and TFPI analogs may also be expressed on baculovirus infected insect cells (see also Pat. U.S. Nos. 4,847,201, above). See, also Pedersen et al., 1990, J. "of Biological Chemistry, 265: 16786-16793. Expression vectors for eukaryotic cells normally include promoter and control sequences compatible with mammalian cells such as, for example, early and late promoters commonly used. Simian Virus 40 (SV40) (Fiers, et al., 1978, Nature, 273: 113), or other viral promoters such as those derived from polyoma, Adenovirus 2, papillomabovine virus, or avian sarcoma virus, or deimmunoglobulin promoters and promoters. Shock aspects General aspects of mammalian cell host system transformations have been described by Axel, US Pat. No. 4,399,216.

Now it also seems that "enhancer" regions are important in optimizing expression; These are usually sequences found above the promoter region. Sources of replication may be obtained, if necessary, from viral sources. However, chromosome integration is a common mechanism for DNA replication in eukaryotes. Plant cells are also available as host, and plant cell compatible control sequences such as nopaline synthase polyadenylation promoter and deletion sequences (Depicker, A., et al., 1982, J. Mol. Appl. Gen., 1: 561) are available. Methods and vectors for transforming plant cells have been disclosed in WO 85/04899.

Methods that can be used for purification of TFPI and TFPI analogs expressed in mammalian cells include sequential application of faserverse, heparin Sepharose, MonoQ, MonoS HPLC chromatography. See, Pedersen ecols., Supra; Novotny et al., 1989, J. "Biol. Chem. 264: 18832-18837; Novotny et al., 1991, Blood, 78: 394-400; Wune et al., 1990, J. Biol. Chem. 265: 16096 -16101; Broze ecols., 1987, PNAS (USA), 84: 1886-1890; US Patent No. 5,106,833; and US Patent No. 5,466,783. These references describe various methods for the purification of mammalian-produced TFPI. TFPI can also be expressed as a recombinant proteinaglycosylated using mammalian cell hosts, including C127 mouse cells (Day ecols., Blood 76, 1538-45, 1990), hamster pup kidney cells (Pedersen et al., 1990), Chinese dehamster ovary cells and human hepatoma cells SK TFPI deC127 has been used in animal studies and has been shown effective in inhibiting fatortecidual intravascular coagulation in rabbits (Day et al., supra), in preventing arterial occlusion after thrombolysis. in dogs (Haskel ecols., Circulation 84: 821-827 (1991)), and in demortality reduction in a model of E. coli sepsis in baboons (Creasey et al., J. Clin. Invest. 91: 2850 (1993)). Ala-TFPI can be expressed as a non-glycosylated recombinant protein using E. coli host cells. Methods have been described that produce a highly active alaTFPI by in vitro reformatting of the E. coli produced recombinant protein. See, for example, WO96 / 40784.

TFPI and TFPI analogs can also be produced embacteria or yeast and subsequently purified. Generally, the procedures shown in U.S. Pat. No. 5,212,091; 6,063,764; and 6,103,500 or WO 96/40784 may be preached. Ala-TFPI and other TFPI analogs may be purified, solubilized, and reformatted according to WO96 / 40784 and Gustafson et al., Prot. Express Pur. 5: 233 (1994), which are incorporated herein by reference. For example, when prepared according to Example 9 of WO96 / 40784, ala-TFPI preparations can be obtained which contain from about 85% to 90% of the total protein per biologically active mature, appropriately formatted, mature ala-TFPI. 10% to 15% of which has one or more oxidized methionine residues. These oxidase forms have biological activity that is equivalent to the biological activity of non-derived ala-TFPI, as determined by prothrombin assay and are expected to be active in the invention disclosed herein. The remaining material comprises various modified ala-TFPI forms, including dimerized, aggregated and acetylated forms.

TFPI and TFPI analogs may have a significant number of cysteine residues and the procedure shown in U.S. Pat. No. 4,929,700 is relevant for TFPI formatting. TFPI and the like may be purified from the buffer solution by various chromatographic methods, such as those mentioned above. If desired, the methods shown in U.S. Pat. No. 4.92 9,700. Any method can be employed to purify TFPI and TFPI analogs that result in a high purity and adequate activity level for administration to humans.

Therapeutic Methods and Compositions

Generally, TFPI and TFPI analogs are useful for treating or preventing those diseases that occur due to over-regulation of tissue factor and thus TF activity caused by TNF, IL-1 or other cytokines. TFPI administration, and particularly low dose TFPI administration, may decrease the concentration of decytocins, such as IL-6 in a patient. Low dose TFPI administration is useful for the treatment of inflammation and coagulation abnormalities, usually including acute as well as chronic inflammatory conditions such as severe apnumonia.

"Severe pneumonia" is defined according to the guidelines stipulated by the American Thoracic Society.

Specifically, severe pneumonia requires a diagnosis of pneumonia and the existence of a) one or two main criteria, b) two or three minor criteria, or c) two of the four British Thoracic Society criteria (Thorax2001; 56 [suppl IV]: l -64). The main criteria are 1) need for mechanical ventilation and 2) septic shock or need for pressures for> 4 hours. The lowest criteria are 1) systolic blood pressure <90 mmHg, 2) multilobar pneumonia, and 3) hypoxemia criteria (Pa02 / Fi02) <250. The "British ThoracicSociety" criteria are 1) respiratory rate 3 0 breaths / minute, 2 ) diastolic blood pressure.ltoreq.60 mmHg, 3) blood urea nitrogen (BUN)> 7.0 mM (> 19.6 mg / dL) and 4) confusion. As is understood in the art, the hypoxemia criterion (Pa02 / Fi02) refers to arterial oxygen partial pressure for the inspired oxygen fraction and indicates the level of gas exchange failure.

Preferably, patients with severe pneumonia have a demonstrable infection by any means known in the art. Such methods include, but are not limited to, detecting a pathogenic organism in a blood culture or other normally sterile fluid or body tissue, for example by GRAM staining, culture, histochemical staining, immunochemical assay, or nucleic acid assays. A demonstrable infection can also be evidenced by a chest X-ray consistent with a diagnosis of pneumonia which is the reason for anti-infectious systemic therapies, as well as any clinical symptoms of infection, including, without limitation, an increase in respiratory rate> = 30 / min or PaCO2 / FiO2. <250, a decrease in blood pressure and an increase in body temperature. TFPI Formulations and TFPI Analogs

TFPI formulations and TFPI analogs are preferably administered by intravenous infusions. Therefore, continuous intravenous infusion is preferred.

Methods for achieving this administration are known to those of ordinary skill in the art. The infusion may be performed by means of a centerline or a peripheral line. Although large fluctuations in dose rate should be avoided, short-term deviations of the dose rates of the invention are acceptable as long as the plasma level resulting from administered TFPI is within 20% of that expected from continuous infusion at a constant dose rate accordingly. with the preferred embodiments of the invention.

Prior to administration to patients, formulator may be added to TFPI and TFPI analogs. A liquid formulation is preferred. TFPI and TFPI analogs may be formulated at different concentrations using different formulators, and at any physiologically appropriate pH compatible with the route of administration, solubility, and stability of the TFPI protein. A preferred formulation for intravenous infusion includes ala-TFPI of up to about 0.6 mg / mL, 300 mM ematin arginine hydrochloride, and sodium citrate buffer at pH 5.0-6.0.

Certain solutes such as arginine, NaCl, sucrose and mannitols serve to solubilize and / or stabilize ala-TFPI. See, WO96 / 40784. An especially preferred intravenous infusion formulation contains about 0.15 mg / ml ala-TFPI, 300 mM arginine hydrochloride, and 20 mM desodium citrate at pH 5.5. TFPI and TFPI analogs may also be formulated at concentrations of up to about 0.15 mg / mL in 150 mM NaCl and 20 mM sodium phosphate or other buffer at pH 5.5-7.2, optionally 0.005% or 0. .01% (w / v) polysorbate 80 (Tween 80). Other formulations contain up to about 0.5 mg / mL TFPI, or TFPI analog in 10 mM sodium deacetate at pH 5.5 containing 150 mM NaCl, 8% (w / v) sucrose, or 4.5% (w / v) mannitol. TFPI and TFPI analogues can also be formulated at higher concentrations up to several mg / mL using high salt.

For example, one formulation contains up to about 6.7 mg / ml ala-TFPI in 500 mM NaCl and 20 mM sodium phosphate at pH 7.0. In addition, the TFPI formulation may contain methionine, preferably in a range of about 1 to about 10 mM methione.

A preferred embodiment of a TFPI formulation isala-TFPI in about 0.1 to about 0.7 mg / ml, 200 to 500 mML-arginine, 1 to 7 mM methionine, 5 to 50 mM sodium decitrate buffer in pH 5.0-6.0. A preferred embodiment of a TFPI formulation is ala-TFPI in about 0.1 to about 0.5 mg / ml, 250 to 400 mM L-arginine, 3 to 6.5 mMmethionine, 15 to 30 mM citrate buffer. sodium at pH 5.0-6.0. In a preferred embodiment of a TFPI formulation contains ala-TFPI at about 0.15 mg / ml, L-arginine hydrochloride at about 300 mM, 5 mM methionine, 20 mM sodium citrate buffer at pH 5.5. . Another preferred embodiment of a TFPI formulation contains about 0.5 mg / ml aliphate TFPI, about 300 mM L-arginine hydrochloride, 5 mM methionine, 20 mM sodium citrate buffer at pH 5.5.

Additional examples of formulator for TFPI and TFPI analogs include oils, polymers, vitamins, carbohydrates, amino acids, salts, buffers, albumin, surfactants, or bulking agents. Preferably carbohydrates include sugar or sugar alcohols such as mono, di, or polysaccharides, or water soluble glycans. Saccharides or glycans may include fructose, dextrose, lactose, glucose, mannose, sorbose, xylose, maltose, sucrose, dextran, pullulan, dextrin, alpha and beta cyclodextrin, soluble starch, hydroxyethyl starch and carboxymethylcellulose or mixtures thereof. Sucrose is most preferred. Sugar alcohol is defined as a C4 to C8 hydrocarbon that has a -OH group and includes galactitol, inositol, mannitol, xylitol, sorbitol, glycerol, and arabitol. Mannitol is more preferred. These sugars or sugar alcohols mentioned above may be used individually or in combination.

There is no fixed limit on the amount used as long as sugar or sugar alcohol is soluble in aqueous preparation. Preferably, the sugar or sugar alcohol concentration is between 1.0 w / v% and 7.0 w / v%, more preferably between 2.0 and 6.0 w / v%.

Preferably amino acids include carnotine, arginine, and betaine levorotatory forms (L); however, other amino acids may be added.

Preferred polymers include common polyvinylpyrrolidone (PVP) average molecular weight between 2,000 and 3,000, or polyethylene glycol (PEG) with an average molecular weight between 3,000 and 5,000. It is also preferable to use a buffer in the composition to minimize pH changes in the solution before freeze drying or after reconstitution. Most physiological buffers can be used, but buffers of citrate, phosphate, succinate and glutamate or mixtures thereof are preferred. Preferably, the buffer concentration is from 0.01 to 0.3 molar. Surfactants that can be added to the formulation are shown in EP Nos. 270,799 and 268,110.

Additionally, TFPI and TFPI analogs may be chemically modified, for example, by covalently conjugating to a polymer to increase its half-life. Preferred polymers and methods for adhering to peptides are taught in U.S. Pat. U.S. Nos. 4,766,106,4,179,337, 4,495,285, and 4,609,546. Preferred polymers are oxyethylated polyols and polyethylene glycol (PEG). PEG is water soluble at room temperature and has the formula: R (O - CH2 - CH2) n - 0 - R where R may be hydrogen, or a protecting group such as an alkyl or alkanol group.

Preferably the protecting group is 1 to 8 carbons, more preferably it is methyl. N is a positive integer, preferably between 1 and 1,000, more preferably between 2 and 500. PEG has a preferred average molecular weight between 1,000 and 40,000, more preferably between 2,000 and 20,000, more preferably between 3,000 and 12,000. Preferably, PEG has at least one hydroxy group, more preferably it is a terminal hydroxy group. It is a hydroxy group which is preferably activated to react with an amino acid group on the inhibitor. However, it will be understood that the type and amount of the reactive groups may be varied to achieve a covalently conjugated PEG / TFPI of the present invention.

Water-soluble polyoxyethylated polyols are also useful in the present invention. They include polyoxyethylated sorbitol, polyoxyethylated glucose, polyoxyethylated glycerol (POG) etc. POG is preferred. One reason is that the central structure of polyoxyethylated glycerol glycerol is the same naturally occurring central structure in, for example, animals and humans in mono-, di-, triglycerides.

Therefore, this branch would not necessarily be observed as a foreign agent in the body. POG has a preferred molecular weight in the same range as PEG. The structure for POG is shown in Knauf et al., 1988, J. Bio. Chem. 263: 15064-15070, and a discussion of POG-protein conjugates is found in U.S. Pat. No. 4,766,106.

After the TFPI liquid pharmaceutical composition or TFPI analog is prepared, it can be lyophilized to prevent degradation and to preserve sterility.

Methods for the lyophilization of liquid compositions are known to those of ordinary skill in the art.

Just prior to use, the composition may be reconstituted with a sterile diluent (Ringer's solution, distilled water, or sterile saline, for example) which may include additional ingredients. By contrast, the composition is preferably administered to individuals by continuous intravenous infusion. Doses of TFPI and TFPI analogs

TFPI or TFPI analogs are administered in a concentration that is therapeutically effective for treating and preventing severe pneumonia. Such doses are also effective for other acute or chronic inflammation, and generally diseases in which cytokines over regulate tissue defactor expression. To accomplish this, TFPI or TFPI analogs are preferably administered intravenously. Methods for performing such administration are known to those skilled in the art. Generally, TFPI or TFPI analogs are given at a dose of 1 µg / kg to 30 mg / kg, more preferably 20 µg / kg to 25 mg / kg, more preferably 1 to 15 mg / kg.

The above dosages are generally administered for a period of at least about 150 hours, and preferably for a period of at least about 100 hours. In one embodiment, TFPI administration is continued for about 99 hours to about 90 hours, preferably for about 97 hours to about 94 hours and more preferably for about 96 hours. The total daily dose administered to a host in single or divided doses may be in amounts, for example, from about 2 to about 20 mg / kg body weight and preferably from about 2 to about 15 mg / kg body weight daily. from about 4 to about 10 mg / kg. Unit dosage compositions may contain such quantities or sub-multiples thereof to construct the dosage form. Smaller daily dosage amounts may be useful for prophylactic or other purposes, for example from 1 µg / kg to 2 mg / kg. The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form will vary depending on the patient treated and the particular mode of administration. The dosage regimen is selected according to various factors including type, age, weight, gender, The patient's diet and medical condition, the severity of the condition, the route of administration, pharmacological considerations such as activity, efficacy, pharmacokinetic profiles and detoxicology, whether a drug delivery system is used and whether the compound is administered as part of a drug combination. Therefore, the actual dosage regimen employed may vary widely and thus may deviate from the preferred dosage regimen set forth above. Preferably, doses of TFPI or TFPI analogs should not exceed a dose rate equivalent to the ala-TFPI dose rate of about 0.66 mg / kg / hr.

Low dose administration

When TFPI or a TFPI analog is given at a dose rate equivalent to the administration of ala-TFPI at a dosage rate of at least about 0.00025 mg / kg / hr (0.00417ug / kg / min) and less than about of 2.00 mg / kg / hr (0.833ug / kg / min) the effectiveness in treating severe eroded pneumonia and adverse side effects such as bleeding are minimized. In a preferred embodiment, ala-TFPI is administered at a dose rate of at least about 0.02 mg / kg / hr to about 1.0 mg / kg / hr, more preferably about 0.24 mg / kg / hr. hr to about 0.8mg / kg / hr. For best efficacy and safety combined, the dosage rate is preferably equivalent to a reference ala-TFPI dose rate of at least about 0.010mg / kg / hr (0.167} Kj / kg / min) and less than about 0.045mg. / kg / hr (0.833 æg / kg / min), or equivalent to a ala-TFPI fingering rate of at least about 0.020 mg / kg / hr and less than about 0.040 mg / kg / hr, and more preferably equivalent to an ala-TFPI dosage rate of at least about 0.025 mg / kg / hr (0.417 µg / kg / min). The route of administration is generally by intravenous administration with preferred continuous intravenous infusion. The infusion may be administered for at least 72, 96, 120, or 240 hours. Preferably, the continuous infusion is administered for 3 to 8 days, more preferably 3 to 6 days, and most preferably for about 4 days.

To administer by "continuous infusion" means that the infusion is maintained at approximately the prescribed rate without substantial interruption for most of the prescribed duration. Alternatively, intermittent intravenous infusion may be used. If intermittent infusion is used, then a time-weighted tapping rate should be used, which is equivalent to the tapping rates described above for continuous infusion. In addition, the intermittent infusion schedule should result in a maximum serum concentration of no more than about 20% above the maximum concentration obtained using continuous infusion. To avoid adverse patient reactions, particularly side effects involving bleeding, the dose rate should be less than the dose rate which is equivalent to continuous ala-TFPI intravenous infusion at about 0.050 mg / kg / hr.

All doses described herein, including dose rates and total doses, are subject to a variation of up to 10% in practice due to errors in determination of protein concentration and biological activity with the deprothrombin assay. Thus, any dose actually administered up to 10% higher or 10% lower than a dose set forth herein is considered to be equivalent to the set dose. For this reason, all doses were set to "about" a specific dose. For example, a dose described as "about 0.025 mg / kg / hr" is considered equivalent to any actual dose ranging from 0.0225 to 0.0275 mg / kg / hr.

A bolus injection or a slightly higher infusion rate of TFPI or a TFPI analogue may also be employed in the practice of the present invention followed by low dose administration of TFPI. For example, a bolus injection or higher infusion rate may be used to reduce the equilibration time of TFPI or TFPI analog administered to a patient's circulation.

By doing so, the eventual steady state plasma level of TFPI can be reached faster and TFPI receptors can be saturated more quickly. Administering ala-TFPI to humans at about 0.025mg / kg / hr for 2 hours increases plasma TFPI (plus ala-TFPI) levels from about 80 ng / mL to about 125 ng / mL, or an increase of approximately 50%. The same level will be reached faster if the infusion rate is increased, or a bolus injection is used. Higher infusion rates will result in higher levels if infusion is continued until steady state is achieved. The steady state level for administration of ala-TFPI at about 0.050 mg / kg / hr was found to be about 300 ng / ml, and for administration of ala-TFPI at about 0.33 or about 0.66. mg / kg / hr is about at least 2 µg / ml in patients suffering from sepsis. The total daily dose administered to a host in a single continuous infusion or divided infusion doses may be in amounts, for example, equivalent. at least about 0.006 mg / kg / day less than about 1.2 mg / kg / day ala-TFPI, more usually equivalent to about 0.24 mg / kg / day less than about 1 2 mg / kg / day ala-TFPI and preferably equivalent to about 0.6 mg / kg / day deala-TFPI. Smaller amounts within this range may be useful for prophylactic or other purposes.

Larger doses above this range may be useful for severe CAP treatment. Invention dosing protocols may also be expressed as the total dose administered to the patient. Total dose is the thematic product of infusion rate and total infusion time.

For example, at the preferred dose rate of about 0.025mg / kg / hr for ala-TFPI and the preferred infusion time of 96 hours, the total dose is about 2.4 mg ala-TFPI per kg body weight. In one embodiment, the preferred dosage rate of about 0.25 mg / kg / hr for ala-TFPI and the preferred infusion time of 96 hours, the total dose is about 24 mg ala-TFPI per kg body weight. In one embodiment, the preferred dosage rate of about 0.75 mg / kg / hr for ala-TFPI and the preferred infusion time of 96 hours, the total dose is about 72 mg of ala-TEPI per kg body weight. In another embodiment, the total dose of TFPI administered according to the invention is equivalent to at least about 0.75 µg / kg and less than about 4.8mg / kg ala-TFPI. Preferably the total dose is equivalent to at least about 1 mg / kg and less than about 4.8 mg / kg ala-TFPI. More preferably the total dose is equivalent to about 2.4 mg / kg ala-TFPI.

One factor that can be used to adjust the fingering regimen is the patient's individual coagulation function, which is typically measured using a thrombothrombin time test (PT), or the International Normalized Ratio (INR). INR is the standardization of the PT assay in which the assay is calibrated against an international reference thromboplastin reagent. See, for example, R. S.Riley et al. , J. Clin. Anal Lab. 14: 101-114 (2000). The response of INR to ala-TFPI in healthy human volunteers is approximately linear over the range of plasma concentrations observed (Fig. 3). The overall change in INR is 1.2 units per 1 µg / mL increase plasma ala-TFPI concentration.

In a pharmacodynamic model, the INR response to ala-TFPI was best described by a log-linear model in which quallog INR was linearly related to plasma ala-TFPI concentration. The log-linear nature of the responses means that individuals with high baseline INR are likely to experience higher anticoagulant responses than individuals with low baseline values who have similar levels of circulating ala-TFPI.

The dosage regimens described above, including dosage rate in terms of mg / kg / hr and total daily dose, are expressed as "equivalent to the administration of ala-TFPI deferral." This means that they are quantitatively determined by normalization to a dose of "reference ala-TFPI", which is defined as mature, 100% pure (in protein), properly formatted, biologically active, unglycosylated ala-TFPI. Ala-TFPI is a TFPI analog whose amino acid sequence is revealed in Id. From Seq. No. 2. Other forms of TFPI may also be used in the invention, including full length mature TFPI and the like. To determine the appropriate dosage range for practicing the invention with other forms of TFPI other than ala-TFPI and with ala-TFPI or other TFPI analog preparations that are less than 100% pure, the dosage ranges described herein for reference ala-TFPI may be adjusted based on the intrinsic biological activity of the particular form of TFPI and further adjusted based on the biochemical purity of the preparation.

In a preferred embodiment, the patient did not receive anticoagulant 10 days after receiving the first TFPI administration. In a preferred embodiment, the patient did not receive an anticoagulant 7 days after the first TFPI administration. Preferably, the patient has not received a form of heparin 24 hours after receiving the first administration of TFPI. In one embodiment, the patient did not receive unfractionated heparin 10 hours, preferably 12 hours after receiving the first TFPI administration. In one embodiment, the patient did not receive low molecular weight heparin 20 hours, preferably 24 hours after receiving the first TFPI administration. In one embodiment, the patient did not receive drotrecogin-alpha 10 hours, preferably 12 hours after receiving the first administration of TFPI.

The intrinsic biological activity of TFPI or a TFPI analog refers to the specific activity, as defined by the prothrombin assay, of the 100% pure, appropriately formatted mature TFPI or TFPI analogue. Thus, the equivalent dose is calculated as (dose- Reference TFPI) / ((relative intrinsic activity) x (biochemical purity)), where relative intrinsic activity refers to (analog intrinsic activity) / (reference ala-TFPI intrinsic activity). For example, if a particular TFPI analog has an intrinsic biological activity that is 80% of that of the reference ala-TFPI, then the equivalent dose for the particular TFPI analog is obtained by dividing the dose values for reference ala-TFPI by 0, 8 In addition, if the formulation administered to a patient is, for example, only 90% biochemically pure, ie comprising 10% molecular specimens that are devoid of TFPI biological activity, then an additional correction of the dose-reference values for ala-TFPI is performed by dividing the dose values by 0.9. Thus, for a hypothetical TFPI analogue that has 80% of the intrinsic ala-TFPI activity and is 90% biochemically pure as administered, the dose rate equivalent to the ala-TFPI administration of 0.025 mg / kg / hr would be 0.0347. mg / kg / hr (i.e. 0.025 / (0.8 x 0.9)).

Equivalent doses may also be determined without knowledge of intrinsic activity or purity by determining biological activity. Relative biological activity can be determined by comparing a particular TFPI analog with a standard TFPI biological activity using the prothrombin time assay. For example, ala-TFPI produced according to the method of Example 9 of WO 96/40784, which contains about 85% of the biologically active molecular species of deala-TFPI, can be used as a standard bio-activity of TFPI. Ala-TFPI produced according to the method Example 9 of WO 96/4 0784 has about 85% of the reference ala-TFPI activity in the prothrombin assay. A standard prothrombin time curve is plotted on the Clot formation is tabulated against the log concentration of TFPI. If the standard biological activity of TFPI has 85-90% of the reference ala-TFPI activity, then a standard curve that is equivalent to that for reference ala-TFPI can be prepared if the concentrations of the standard TFPI biological activity are multiplied by 0, 85 before tabulation, so that tabulated activity is equivalent to 100% pure deferral ala-TFPI activity. When the clot formation time for a particular TFPI analog is compared to the standard curve, the equivalent concentration of reference ala-TFPI can be deduced from the curve. Alternatively, if the inclination of the linear portion of the standard curve is obtained by linear regression analysis, then the slope may be corrected based on the activity of the TFPI standard biological activity relative to the reference ala-TFPI. The relative biological activity of a particular TFPI analog is thus equal to the ratio of the reference deala-TFPI activity to the analog activity. For example, if a particular analogue requires 1.43 ng to produce the same prothrombin time activity as 1.00ug of reference ala-TFPI, then the biological activity of the analog is 1.00 / 1.43, or 0.7. For that analogue, the dose equivalent to a dose of ala-TFPI reference is obtained by dividing the dose of ala-TFPI reference by the relative biological activity of the analog.

For example, a 0.025 mg / kg / hr dose for reference ala-TFPI would be equivalent to 0.0357 mg / kg / hr of the analogue (ie 0.025 / 0.7).

Although TFPI or a TFPI analog may be administered as the sole active pharmaceutical-anticoagulation agent, such molecules may also be used in combination with one or more additional therapeutic agents to provide combination therapy for the treatment of severe pneumonia. Such additional therapeutic agents include useful antibodies such as, for example, anti-endotoxin, monoclonal antibodies (for example, endotoxin-binding mabs) and anti-TNF products such as an anti-TNF murine Mab. TFPI and TFPI analogs may also be combined with deinterleukin-1 receptor antagonists, bactericidal / enhancing protein (BPI) immunostimulant compounds which have anti-inflammatory activity such as PAF antagonists and cell adhesion blockers (eg anti-platelet agents such as as GPIIb / lIIa inhibitors). When administered as a combination, the therapeutic agents may be formulated as separate compositions which are administered at the same time or at different times.

Preferably, the additional therapeutic agents are given at the same time (i.e. during the period of administration of TFPI or TFPI analogs) or within 24 hours of the period of administration of TFPI or TFPI analogs (ie 24 hours before onset or 24 hours after the end of the administration period of TFPI or TFPI analogs). Additional therapeutic agents may also be given as a single composition together with TFPI or TFPI analogs. TFPI and a TFPI analog may also be given in combination with other agents that would be effective. to treat severe pneumonia. For example, the following may be administered in combination with TFPI or a TFPI analog: antibiotics that can treat underlying bacterial infection, monoclonal antibodies that are directed against bacterial cell wall components, receptors that can form complexes with cytokines that are involved in the pathway of severe pneumonia and generally any agent or protein that may interact with amplified pathophysiological cytokines or pitras that include complement protein to attenuate severe pneumonia and / or its symptoms.

Useful antibiotics include those in the general category of beta-lactam (penicillin) rings, amino glycosidic linkage sugars (aminoglycosides), lactone macrolytic rings (macrolides), polycyclic denaftacenecarboxanide derivatives (tetracyclines), dichloroacetic acid nitrobenzene derivatives, peptides, bactridine (bacillid), polymyxin), large-rings common double bond conjugate system (polyenes), sulfanamide-derived desulfa drugs (sulfonamides), 5-nitro-2-furanyl groups (nitrofurans), dequinolone carboxylic acids (nalidixic acid) and many others. Other antibiotics and further versions of the above specific antibiotics can be found in "Encyclopedia of Chemical Technology", 3rd Edition, Kirk-Othymer (ed.), Vol. 2, pages 782-1036 (1978) and Vol. 3, pages 1-78, Zinsser, MicroBiology, 17th Edition W. Joldik et al. (Eds.) Pages 235-277 (1980), or "Dorland's Illustrated Medical Dictionary", 27th Edition, W. B. Saunders Company (1988).

Other agents that may be combined with TFPI or with a TFPI analog include endotoxin antagonists such as E5531 (a Lipid A analog, see Asai ecols., Biol. Pharm. Buli. 22: 432 (1999)), TF analogs with anticoagulant activity. (see, for example, Kelley ecols., Blood 89: 3219 (1997) and Lee & Kelley, J. Biol. Chem. 277: 4149 (1998)), paracytokine directed monoclonal antibodies, such as those monoclonal antibodies directed to IL-6 or M-CSF, see US Ser. No. Ser. 7 / 451,218, filed December 15, 1989, monoclonal antibodies directed to TNF, see Ceramics., Pat. No. 4,603,106, protein inhibitors that cleave the mature TNF prohormone of the cell in which it was produced, (see US Ser. No. 07 / 395,253, filed August 16, 1989), IL-1 antagonists, (see, US Ser. No. 07 / 517,276, filed May 1, 1990), IL-6 cytokine expression inhibitors such as inhibin, (see US Patent 5,942,220) and receptor-based inhibitors of various cytokines. such as IL-1. Complement antibodies or complement protein inhibitors such as CRi, DAF, and MCP may also be used.

All patents, patent applications, and references cited in this disclosure are incorporated herein by reference in their entirety.

The present invention will now be illustrated by reference to the following examples which have particularly advantageous embodiments. However, it should be understood that such embodiments are illustrative and are not to be construed as limiting the invention in any way.

EXAMPLES

Example 1

Ala-TFPI Treatment of Patients with Severe Pneumonia

Patients with severe pneumonia were evaluated to explore the potential effect of ala-TFPI treatment in a relatively homogeneous group. Compneumonia patients were identified if a source of sepsis documented by the investigator was coded as pneumonia. Other sites of infection could also be present. Due to the difficulty in differentiating infectious chemical sequences, aspiration pneumonia patients were not included. Patients identified as having pneumonia were then classified as positive culture (any evidence of infection such as culture or Gram stain), or negative culture (negative culture or non-culture). Patients were treated by continuous intravenous infusion with a preparation of unglycosylated ala-TFPI expressed in E. coli at a dose of 0.025 mg / kg / h formulated in a buffer containing 300 mM L-arginine, 20 mM sodium decitrate, pH 5.5, osmolarity of 560 +/- 110 mOsm. Placebo consisted of the same buffer without ala-TFPI and was infused at the same rate as the study drug. The results of these analyzes demonstrated a positive effect of ala-TFPI treatment in those patients with positive culture pneumonia (Table 1). Those patients without evidence of an infectious source demonstrated a negative effect.

Table 1. Mortality by Pneumonia Status

<table> table see original document page 43 </column> </row> <table>

Table 2. Mortality by INR Low Pneumonia Status

<table> table see original document page 43 </column> </row> <table>

Increased mortality in the high INR negative culture group appeared to be present in patient populations with or without added heparin administration, although it should be noted that the number of individuals in the non-heparin negative culture pneumonia group is relatively small (Table 3). A strong positive detrimental effect was observed in the culture-positive / heparin-free group. Table 3. Mortality by Pneumonia and Heparin Status

<table> table see original document page 44 </column> </row> <table>

Example 2

Investigation of baseline severity of disease variables

Numerous baseline severities of disease variables were evaluated to determine if there was any group imbalance that could explain the outcome. These data indicate that the difference in results associated with culture status is not due to base imbalances.

Therefore, the results appear to represent a different effect of IPTp treatment due to biological differences between patients with and without infection. Despite the fact that the severity indicators (eg, APACHE II score or organ dysfunction score) were equal to placebo or lower in the TFPI-negative culture-negative group the negative culture group showed the highest overall mortality (Table 4 ). Table 4. Baseline severity of disease by Pneumonia Status

<table> table see original document page 45 </column> </row> <table>

IL-6 is an inflammatory cytokine that is early in sepsis, reflects the intensity of inflammatory response, and is associated with the outcome, and, as a result, IL-6 levels are lower in patients clinically identified as having pneumonia but no infection (Table 5). . This suggests that there is a biological difference between patients with a documented infectious source of pneumonia versus those without an apparent infectious source. Paradoxically, the negative culture TFPI group has the lowest IL-6 levels at the highest mortality rate. In a population with sepsis IL-6 levels fall over time. The rate of IL-6 kedana is reduced in individuals with culture-negative pneumonia treated with TFPI (Table 5). This suggests that the biological effect of IPTF may differ in those with and without infection.

Table 5. IL-6 by Pneumonia Status

<table> table see original document page 46 </column> </row> <table>

Example 3

Analysis of Patients with Severe Pneumonia by Type of Infection Documentation

As discussed above, a general benefit of ala-TFPI treatment was observed in those patients with the highest certainty of infection, ie those with a positive blood culture. In an analysis of patients with severe pneumonia by type of infection documentation, a benefit of ala-TFPI treatment was observed in subjects with a positive blood culture and those with other evidence (Table 6). The effect was stronger in the bacteremia group, ie the group with the highest probability of infection or with the most demonstrable source of infection.

Table 6. Mortality by Culture Status and Pneumonia Status

<table> table see original document page 47 </column> </row> <table>

As previously shown, patients with documented infection (blood + "other") benefited from treatment with IPTF in the absence of heparin. This result is mainly due to the benefit derived from the pneumonia group (Table 7). This finding seems to indicate that the benefit of endogenous anticoagulants is highest in those patients with severe pulmonary infections.

Table 7. Mortality by Infection Status, Pneumonia Status and Heparin Use

<table> table see original document page 47 </column> </row> <table> <table> table see original document page 48 </column> </row> <table>

To also limit heterogeneity, future experiments may be focused on community-acquired pneumonia (CAP). Patients who develop pneumonia while in hospital (nosocomial pneumonia) are more likely to be colonized with pathogenic organisms and have other lung disorders making the diagnosis of infectious pneumonia more difficult. In addition, patients with CAP are less likely to be exposed to heparin than patients with pneumonia. When data were analyzed by length of hospital stay prior to treatment, a similar benefit was noted for positive culture patients hospitalized for <2 days (community acquired) versus those hospitalized for more than 2 days (nosocomial).

The negative effect in the culture negative patients was primarily in the nosocomial group (Table 8). Table 8. Mortality by pneumonia status and time to hospital

<table> table see original document page 49 </column> </row> <table>

Example 4

Alveolar fibrin deposition and alteration in fibrinolytic activity is a distinguishing feature of pneumonia and acute lung damage. Tissue factor was considered as an important coagulation initiator in this environment and inhibitor of tissue factor pathway (IPTT) has been shown to avoid reducing lung damage in animal models.

Recombinant tissue factor (tifacogin) pathway inhibitor was investigated in severe sepsis and a randomized, double-blind, placebo-controlled phase III experiment was completed (TFP007). The study failed to show a significant reduction in mortality by 28 days for any cause. A post-hoc analysis was performed to assess whether patients who develop severe sepsis due to CAP can benefit from tifacogin. In addition, the possible interaction of heparin and the importance of microbiological documentation were investigated.Material and Methods

Patients (pts) listed on TFP007 with CAP as the primary cause of severe sepsis were maintained for post hoc analysis. 28-day mortality from any cause and safety were determined for the general population of CAP. The influence of concomitant administration of heparin for DVT prophylaxis and the importance of microbiological documentation were also analyzed.

Results

TFP007 included 1,955 patients of whom 435 had CAP (TFPI 217, placebo 218). The baseline characteristics were similar in both groups (mean age 62.3 vs 62; mean APACHE II 24.9 vs 24; mean OD 3 vs 3) in TFPI eplacebo respectively (NS). 28-day mortality data for any cause are given below in Table 9. Mortality tended to be reduced in patients treated with tifacogin compared to placebo in particular when no concomitant heparin was administered and in the presence of microbiological documentation.

Serious bleeding events occurred in 12 treated patients (5.5%) vs 5 patients (2.2%) in placebo (NS). CNS bleeding was observed in 6 patients treated with tifacogin (2.7%) vs 2 in placebo ((1%) (NS) for the first 28-day period.

Table 9: 28-day mortality from any cause in CAP with concomitant heparin and microbiological evidence.

<table> table see original document page 50 </column> </row> <table> <table> table see original document page 51 </column> </row> <table>

Hep: Heparin ME: Microbiological Evidence

Conclusion

From this post hoc analysis, tifacogin appears to reduce 28-day mortality from any cause in severe CAP patients. A greater benefit seems to be obtained in patients who have a documented PAC and / or lack of concomitant heparin administration. Security operfil is acceptable. The potential benefit detifacogin is currently evaluated in a phase III clinical study that includes patients with severe CAP.

Occurrences of bleeding and serious bleeding for all patients listed on TFP007, as well as for various subpopulations, are provided in tables 9A-9F.

Tables 9A-9F

Table 9A: TFP007 bleeding for TFP007 population (all individuals)

<table> table see original document page 51 </column> </row> <table>

* Chisq test; + Hospital- acquired pneumonia # N = number of individuals; n (%) = number of bleeds (rate) Table 9B: TFP007 serious bleeding for TFP007 population (all individuals)

<table> table see original document page 52 </column> </row> <table>

Table 9C. TFP007 bleeding for CAP / PAH subpopulations (all subjects)

<table> table see original document page 52 </column> </row> <table>

* Chisq test; + Hospital- Acquired Pneumonia

# N = number of individuals; n (%) = number of bleeds (rate)

Table 9D: TFP007 bleeding for CAP / PAH subpopulations (all individuals)

<table> table see original document page 52 </column> </row> <table> <table> table see original document page 53 </column> </row> <table>

* Chisq test; + Hospital- acquired pneumonia # N = number of individuals; n (%) = number of bleeds

serious (fee)

Table 9E. TFP007 bleeding for subpopulations of

<table> table see original document page 53 </column> </row> <table> Table 9F. TFP007 serious bleeding for subpopulations (all individuals)

<table> table see original document page 54 </column> </row> <table>

* Fisher's exact test; + Hospital- acquired pneumonia # N = number of individuals; n (%) = number of serious bleeds (rate) Table 10

SNC TFP007 ischemic adverse event listing <table> table see original document page 55 </column> </row> <table> <table> table see original document page 56 </column> </row> <table> <table > table see original document page 57 </column> </row> <table> <table> table see original document page 58 </column> </row> <table> <table> table see original document page 59 </ column > </row> <table> <table> table see original document page 60 </column> </row> <table> <table> table see original document page 61 </column> </row> <table> <table > table see original document page 62 </column> </row> <table> <table> table see original document page 63 </column> </row> <table> <table> table see original document page 64 </ column > </row> <table> <table> table see original document page 65 </column> </row> <table> <table> table see original document page 66 </column> </row> <table> <table > table see original document page 67 </column> </row> <table> <table> table see original document page 68 </column> </row> <table> table see original document page 69 </ column > </row> <table> <table> table see original document page 70 </column> </row> <table> <table> table see original document page 71 </column> </row> <table> <table > table see original document page 72 </column> </row> <table> <table> table see original document page 73 </column> </row> <table> <table> table see original document page 74 </ column > </row> <table> <table> table see original document page 77 </column> </row> <table> <table> table see original document page 76 </column> </row> <table> <table > table see original document page 79 </column> </row> <table> <table> table see original document page 78 </column> </row> <table> table see original document page 81 </ column > </row> <table> Table 12

<table> table see original document page 80 </column> </row> <table>

The present invention has been described with reference to specific embodiments. However, this application is intended to cover those changes and substitutions that may be made by those skilled in the art without departing from the spirit and scope of the appended claims.

2 1 276 PRT Homo sapiens 1 Asp Be Glu Glu Asp Glu Glu His Thr From He Thr Asp Thr Glu Leu1 5 10 15 Pro Pro Leu Lys Leu Met His Ser Phe Cys Ala Phe Lys Ala Asp 20 25 30 Gly ProCys Lys Ala lie Met Lys Arg Phe Phe Phe Asn From Phe Thr 35 40 45 Arg Gln Cys Glu Glu PheDe Tyr Gly Gly Cys Glu Gly Asn Gln Asn 50 55 60 Arg Phe Glu Be Leu Glu Cys Lys LysMet Cys Thr Arg Asp Asn 65 70 75 80 Ala Asn Arg He Ilc Lys Thr Thr Leu Gln Gln Glu LysPro Asp Phe 85 90 95 Cys Phe Leu Glu Glu Asp Pro Gly He Cys Arg Gly Tyr From Thr Arg 100105 110 Tyr Phe Tyr Asn Gln Cys Glu Cys Glu Arg Phe Lys Tyr Gly 115 120 125 GlyCys Read Gly Asn Met Asn Asn Phe Glu Thr Read Le Glu Glu Cys Lys 130 135 140 Asn From Cys GluAsp Gly Pro Asn Gly Phe Gln Go Asp Asn Tyr Gly 145 150 155 160 Thr Gln Leu Asn Ala VaiAsn Asn Ser Read Thr Pro Gln Be Thr Lys 165 170 175 Go Pro Be Read Le Phe Glu Phe His GlyPro Be Trp Cys Read Le Thr Pro 180 185 190 Wing Asp Arg Gly Leu Cys Arg Wing Asn Glu Asn ArgPhe Tyr Tyr Asn 195 200 205 Be Go He Gly Lys Cys Arg Pro Phe Lys Tyr Be Gly Cys GlyGly 210 215 220 Asn Glu Asn Asn Phe Thr Be Lys Gln Glu Cys Leu Arg Wing Cys Lys 225 230235 240 Lys Gly Phe He Gln Arg To Be Lys Gly Gly Leu He Lys Thr Lys 245 250 255 Arg LysArg Lys Lys Gln Arg Go Wing Lys Tyr Glu Glu From Phe 260 265 270 Go Lys Asn Met 275 2277 PRT Homo sapiens 2 Allah Asp Ser Glu Asp Glu Glu His Thr From Thr Asp Thr Glu 1 510 15 Leu Pro Pro Read Lys Read Met His Be Phe Cys Wing Phe Lys Wing Asp 20 25 30 Asp GlyPro Cys Lys Wing He Met Lys Arg Phe Phe Phe Asn From Phe 35 40 45 Thr Arg Gln Cys GluPhe From Tyr Gly Gly Cys Glu Gly Asn Gln 50 55 60 Asn Arg Phe Glu Be Leu Glu Glu Cys LysLys Met Cys Thr Arg Asp 65 70 75 80 Asn Wing Asn Arg Lie Of Lys Thr Thr Leu Gln Gln GluLys Pro Asp 85 90 95 Phe Cys Phe Leu Glu Glu Asp Pro Gly Be Cys Arg Gly Tyr From Thr 100105 110 Arg Tyr Phe Tyr Asii Asn Gln Thr Lys Gln Cys Glu Arg Phe Lys Tyr 115 120 125 GlyGly Cys Read Gly Asn Met Asn Phe Glu Thr Read Glu Glu Cys 130 135 140 Lys Asn He Cys Glu Asp Gly Pro Asn Gly Phe Gln Go Asp Asn Tyr 145 150 155 160 Gly Thr Gln Read Asn AlaVai Asn Asn Be Read Thr Pro Gln Be Thr 165 170 175 Lys Go Pro Be Read Phe Glu Phe HisGly Pro Be Trp Cys Leu Thr 1S0 185 190 Pro Wing Asp Arg Gly Leu Cys Arg Wing Asn Glu Asn

Arg Phe Tyr Tyr 195 200 205 Asn Gonna Go Gly Lys Cys Arg Pro Phe Lys Tyr Be Gly CysGly 210 215 220 Gly Asn Glu Asn Asn Phe Thr Be Lys Gln Glu Cys Read Arg Wing Cys 225 230235 240 Lys Lys Gly Phe Of Gln Arg He Ser Lys Gly Gly Leu From Lys Thr 245 250 255 Lys ArgLys Arg Lys Lys Gln Arg Goes Wing Lys Tyr Glu Glu 260 265 270 Phe Goes Lys Asn Met275

Claims (43)

A method of treating or preventing pneumonias should comprise: administering TFPI or a TFPI analogue to a patient who is at risk or is at risk of severe continuous intravenous infusion pneumonia at a dose rate equivalent to the reference administration of ala-TFPI. at a dose rate of less than about 1.0 mg / kg / hr and the patient did not receive an anticoagulant within 24 hours after TFPI or TFPI analog administration. Method according to claim 1, characterized in that said TFPI analog is unglycosylated TFPI. Method according to claim 1, characterized in that said patient has a demonstrable infection. A method according to claim 1, wherein said TFPI or TFPI analog is administered by continuous intravenous infusion at a dose rate equivalent to the ala-TFPI reference administration at a dose rate of less than about 0.80mg / kg / hr. A method according to claim 4, wherein said dose rate is equivalent to the ala-TFPI reference administration at a dose rate of about 0.025 to about 0.10 mg / kg / hr and wherein said TFPI or TFPI analog is administered for at least about 72 hours. A method according to claim 5, wherein said dose rate is equivalent to the ala-TFPI reference administration at a dose rate of from about 0.010 to about 0.10 mg / kg / hr. Method according to claim 6, characterized in that said TFPI analogue is unglycosylated TFPI. A method according to claim 6, wherein said dose rate is equivalent to the ala-TFPI reference administration at a dose rate of from about 0.020 mg / kg / hr to about 0.08mg / kg. / hr. Method according to claim 8, characterized in that said TFPI analog is unglycosylated TFPI. Method according to claim 1, characterized in that said TFPI or said TFPI analog is administered for about 96 hours. Method according to claim 10, characterized in that said TFPI analog isala-unglycosylated TFPI. The method of claim 10, wherein said TFPI or TFPI analog is administered by continuous intravenous infusion to provide a total dose equivalent to the administration of ala-TFPI in a total dose of about 0.025 to about 2.5 mg / kg. Method according to claim 12, characterized in that said TFPI analog isala-unglycosylated TFPI. A method according to claim 10, characterized in that said TFPI or TFPI analog is administered by continuous intravenous infusion at a dose rate equivalent to the ala-TFPI reference administration at a dose rate of about 0 ° C. 02 mg / kg / hr about 0.09 mg / kg / hr. Method according to claim 14, characterized in that said TFPI analogue is unglycosylated TFPI. A method according to claim 1, wherein said TFPI or TFPI analog is administered by continuous intravenous infusion to provide a daily dose equivalent to the ala-TFPI reference administration at a daily dose of about 0.06 mg / kg to about 4 mg / kg. Method according to claim 16, characterized in that said TFPI analogue is unglycosylated TFPI. Method according to claim 1, characterized in that said TFPI analog comprises a first Kunitz domain consisting of amino acids 19-89 of Id. De Seq. No.: 1. The method of claim 18 wherein said TFPI analog also comprises a second Kunitz domain consisting of amino acids 90-160 of Id. De Seq. No.: 1. A method according to claim 1, wherein said TFPI analog comprises amino acids 1-160 of Id. De Seq. No.: 1. Method according to claim 1, characterized in that said TFPI analog comprises a second Kunitz domain consisting of amino acids 90-160 of Id. De Seq. No.: 1. Method according to claim 21, characterized in that said TFPI analog isala-unglycosylated TFPI. A method according to claim 1, wherein said TFPI or TFPI analog is prepared from a lyophilized composition comprising TFPI or a TFPI analog. A method according to claim 23, characterized in that said TFPI analogue is unglycosylated TFPI. A method according to claim 1, wherein said TFPI or TFPI analog is administered as a formulation comprising arginine. Method according to claim 25, characterized in that said TFPI analogue is unglycosylated TFPI. A method according to claim 1, wherein said TFPI or TFPI analog is administered as a formulation comprising nitrate. A method according to claim 27, characterized in that said TFPI analogue is unglycosylated TFPI. A method according to claim 1, wherein said TFPI or TFPI analog has a concentration of about 0.15 mg / ml in a formulation comprising about 300 mM dearginine hydrochloride and about 20 mM citrate. sodium and which has a pH of about 5.5. A method according to claim 29, characterized in that said TFPI analog is unglycosylated TFPI. The method of claim 1 further comprising administering at the same time as, or up to 24 hours after the administration of said TFPI or TFPI analog, an additional selected agent from the group consisting of an antibiotic, an antibody. , an endotoxin antagonist, a tissue defactor analogue that has anticoagulant activity, an immunostimulant, a cell adhesion blocker, heparin, BPI protein, an IL-1 antagonist, pahase (PAF enzyme inhibitor), a TNF inhibitor, an inhibitor IL-6 is a complement inhibitor. A method according to claim 31, characterized in that said TFPI analog is non-glycosylated TFPI. A method according to claim 31 wherein said additional agent is an antibody, wherein said antibody specifically binds to an antigen selected from the group consisting of TNF, IL-6 and M-CSF. A method according to claim 33, characterized in that said TFPI analog isala-unglycosylated TFPI. 35. A method for the treatment of severe pneumonia comprising: administering to a patient (i) TFPI or a TFPI analogue and (ii) an additional agent selected from the group consisting of an antibiotic, a monoclonal antibody, a decitocin inhibitor and a complement inhibitor. The method of claim 35, wherein said TFPI analog isala-unglycosylated TFPI. A method according to claim 35, characterized in that said patient has a demonstrable infection. A method according to claim 35, wherein said TFPI or TFPI analog is administered by continuous intravenous infusion at a dose rate equivalent to the ala-TFPI reference administration at a dose rate of less than about 1.0mg / kg / hr. The method of claim 38, wherein said dose rate is equivalent to the reference administration of ala-TFPI at a dose rate of from about 0.001 to about 0.090mg / kg / hr. The method of claim 39, wherein said dose rate is equivalent to the reference administration of ala-TFPI at a dose rate of from about 0.002 to about 0.050mg / kg / hr. The method of claim 40, wherein said dose rate is equivalent to the reference administration of ala-TFPI at a dose rate of from about 0.002 to about 0.010mg / kg / hr. The method of claim 41, wherein said dose rate is equivalent to the reference administration of ala-TFPI at a dose rate of from about 0.0025 to about 0.075mg / kg / hr. 43. A method of treating or preventing pneumoniasevera comprising administering to a human patient who has or is at risk of having pneumoniasevera, TFPI or a TFPI analog in an amount that does not induce excessive bleeding.