MX2007016372A - Airway administration of activated protein c in inflammatory conditions affecting the respiratory tract - Google Patents

Abstract

The present invention provides methods for the local treatment of acute and chronic extravascular pulmonary fibrin deposition and/or reducing unwanted effects associated with systemic administration of anticoagulants to a subject via airway administration to the subject by intratracheal, intrabronchial or intraalveolar routes of human activated protein C or biologically active derivatives thereof .

Description

ADMINISTRATION TO THE RESPIRATORY ROUTES OF THE ACTIVATED C? INFLAMMATORY CONDITIONS QUK AFECTAM EL, TRACTO Field of the invention The present invention provides methods for stopping the deposition of acute, recurrent and chronic fibrin in the alveoli of the respiratory tract, in particular the alveoli and / or bronchioles, in any disease associated with sedimentation in children and adults. These diseases include inflammatory diseases of the lungs such as acute lung injury (ALI), which can be directly or indirectly related to pulmonary trauma, for example, after ventilator therapy (ventilator-induced lung injury (VILI, its acronym in English), inflammatory conditions such as autoimmune diseases, pancreatitis, aspiration pneumonitis, inhalation of toxic fumes, acute respiratory distress syndrome (ARDS), < Thu is a more severe manifestation of ALI, infections such as sepsis, severe sepsis and septic shock; pneumonia of any cause; Acute and chronic bronchoalveolar diseases, fibrous alveolitis, bronchiolitis, cystic fibrosis and also diseases with severe respiratory tract hyperactivity, for example, bronchial asthma and pulmonary insufficiency REF. s 188890 drug-induced, for example, after chemotherapy such as bleomycin. In the methods of the present invention, anticoagulants such as activated protein C, whether these agents are derived from the plasma or prepared by recombinant DNA technology, are administered intratracheally, intrabronchially, or the alveolar space by means of administration to the respiratory tract. These methods are useful in clinical medicine, especially critical or intensive care medicine and respiratory medicine.

Background of the Invention The deposition of fibrin in the alveoli of the respiratory tract, particularly the alveolar and bronchial alveoli, is a frequent complication of systemic inflammatory conditions such as those resulting from trauma, sepsis, severe sepsis and septic shock, ALI induced by drug, ARDS or pneu onitis (for example, due to methotrexate, bleomycin or sirolimus). { Amigues L, Klouche K, Massanet P, Gaillard N, Garrigue V, Beraud JJ, Mourad G. Sirolimus-associated acute respiratory distress syndrome in a renal transplant recipient. Transplant Proc. 2005; 37: 2830-1.) And ventilated-induced lung injury (VILI) (Maclntyre NR, Current issues in mechanical ventilation for respiratory failure, Chest, 2005; 128: 561S-567S) and lung injury following mechanical ventilation ( VILI (The Acute Respiratory Distress Syndrome Network: Ventilation with lower tidal volumes as compared with traditional tidal volumes for lung injury and respiratory distress syndrome N Engl J Med. 2000; 342: 1301-1308. These conditions lead to an activation systemic coagulation, finally resulting in fibrin sedimentation in the intravascular and extravascular spaces, that is, in the alveolar and bronchial compartments.At the same time, the inflammatory activation causes the release of pro-inflammatory cytokines such as necrosis factor alpha of tumor (TNF-α), interleukin 1-beta (IL-1β), interleukin 6 (IL-6) and interleukin 8 (IL-8) from activated inflammatory cells. n intravascular fibrin and the release of pro-inflammatory cytokines in the alveoli of the lungs causes a tissue injury characterized by increased permeability of the alveolar-capillary membrane with diffuse alveolar damage and the accumulation in the alveoli of edema fluid rich in plasma proteins, including the components of the blood coagulation system, and a reduction in the production of surfactants. As a result, the fibrin-rich hyaline membrane is formed in the alveolar ducts and alveoli. In the last phase, massive infiltration of neutrophils and other inflammatory cells occurs, after the organization of exudates and fibrosis. This pathological sequence has been described and reviewed by Bellingan GJ, 2002: "The pathogenesis of ALI / ARDS", Thorax 57: 540-546. The clinical conditions that correspond to their pathology are called ALI or ARDS, they differ only in that ARDS is more severe and is characterized by greater hypoxemia in such a way that the ratio of arterial P02 to a fraction of inspired oxygen (Pa02 / Flo2) = 200 mmHg. ALI and ARDS is presented as part of the systemic inflammatory response syndrome (SIRS), which may be due to infectious or noninfectious causes such as pancreatitis or direct or indirect lung trauma.; When the cause of SIRS is infectious, it is called sepsis, which, when associated with organ dysfunction, is defined as severe sepsis, and when it is associated with significant hypotension, such as septic shock. A similar sequence of pathological events occurs in pneumonias due to a variety of causes, including viral, bacterial and fungal agents, for example Pneumocystis carinii pneumonia (PCP), bronchiolitis (for example following viral pneumonitis and / or graft-versus-disease). pulmonary host (GVHD)), which leads to alveolar exudates and fibrin sedimentation in those regions of the lung affected by the inflammatory process. It has been pointed out (for example by Levi M et al., 20 3: "Bronchoalveolar coagulation and fibrinolysis in endotoxemia and pneumonia", Crit Care Med 31: S238-S242) that the lung is particularly susceptible to fibrin sedimentation in sepsis, which shows this phenomenon to a greater degree than other organs. Sedimentation of extensive local fibrin suggests that local activation of coagulation or disturbance of local physiological regulatory systems may be involved. In the bronchoal eolar compartment, tissue factor (TF), expressed locally in alveolar cells and in the epithelium, is seen as having an essential role in the initiation of coagulation, while physiological anticoagulation is due to antithrombin and C protein system is dysfunctional. It has been documented that the protein C system breaks down markedly in patients with ALI / ARDS of both septic and non-septic causes and there is evidence of both circulatory and intra-alveolar alterations in the path of protein C in ALI / ARDS (Ware LB et al., 2003: "Protein C and thrombomodulin in human lung injury", Am J Physiol Lung Cell Mol Physiol 285: L514-L521). At the same time, there is a marked depression of local fibrinolysis, that is, coagulation is locally overregulated in the injured lung, whereas fibrinolytic activity is markedly depressed.

Brief Description of the Invention The purpose of the present invention is to improve the treatment of ALI, ARDS, pneumonia and inflammatory lung diseases by directing the local pulmonary activation of the coagulation system and the local deficiency of anticoagulant mechanisms, by applying the relevant anticoagulants, or agents capable of blocking the local initiation of coagulation, by local administration in the respiratory tract. In this way a high local concentration of these acids can be reached in the affected airways, so that the deposition of extravascular fibrin can be inhibited more efficiently than by the systemic (intravenous) administration of the same agents, but the effects are avoided or reduced adverse systemic. Administration to the respiratory tract of these agents can occur either alone or as a complement to the intravenous administration of the same or other agents. Due to the "interference" between coagulation and inflammation, administration to the respiratory tract of these agents is also expected to modulate local lung inflammation, by reducing the activation of local thrombin and in certain cases also by the direct anti-inflammatory action . One aspect of the present invention relates to a method for reducing the deposition of extravascular fibrin in the respiratory tract, especially in the alveolar or bronchoalveolar spaces, in human subjects with inflammatory and / or infectious lung conditions leading to such fibrin settling, The method comprises the administration by the respiratory tract of anticoagulants, either purified from the plasma or obtained by recombinant DNA technology.

Detailed Description of the Invention The present invention relates to administration to the respiratory tract, by any suitable method including, but not limited to, intratracheal, intrabronchial or intraalveolar administration, to a human subject including both adults and children, of purified or concentrated human activated protein C (APC), or derivatives thereof, however prepared, to prevent or reduce the deposition of extravascular fibrin in the respiratory tract, especially the alveolar or bronchoalveolar alveoli. Such fibrin deposition can result from an acute condition, a recurring condition or a chronic condition and can be due to a variety of causes, including but not limited to trauma, direct or indirect, inflammation or infection, due to fibrin sedimentation induced by drug in the respiratory tract and pulmonary interstitium, congenital diseases type cystic fibrosis, or due to a combination of such possible causes. For example, it is considered that the methods of the present invention will be useful in the treatment of alveolar fibrin sedimentation characteristic of ALI or ARDS that occurs in a large proportion of patients with sepsis of varying degrees, in patients with severe pneumonias, bronchiolitis. obstructive and in fibrous alveolitis.

Definitions Affinity: The resistance of the bond between the receptors and their ligands, for example between an antibody and its antigen. Amino acid residue: That part of the amino acid that occurs in the polypeptide chain in which the amino acid is linked to other amino acids by peptide (amide) bonds. The amino acid residues described herein are preferably in the "L" isomeric form. However, the amino acid encompasses each amino acid such as amino acid L, amino acid D, alpha amino acid, beta amino acid, gamma amino acid, natural amino acid and synthetic amino acid or the like, so that the desired functional property is maintained by the polypeptide. Also included are natural or synthetic amino acids that have been modified. NH2 refers to the free amino group present at the amino termini of a polypeptide. COOH refers to the free carboxy group present at the carboxy terminus of the polypeptide. Standard polypeptide abbreviations for amino acid residues are used herein. It should be noted that all sequences of amino acid residues represented herein by the formulas have a left to right orientation in the conventional direction from the amino terminal to the carboxy terminus. Additionally, it will be noted that a point at the start or end of an amino acid residue sequence indicates a peptide bond to an additional sequence of one or more amino acid residues or a covalent bond to an amino terminal group or a covalent bond to a group of amino terminal such as NH2 or acetyl or a carboxy terminal group such as COOH.

Modified amino acid: An amino acid where an arbitrary group of it is chemically modified. In particular, a chemically modified amino acid that is modified at the alpha carbon atom to an alpha amino acid is preferred. Polypeptide: The phrase "polypeptide" refers to a molecule that comprises amino acid residues that do not contain ligatures other than the amide linkers between adjacent amino acid residues.

APC Molecule The present invention relates to the use of APC molecules in the manufacture of a medicament for the local treatment of sedimentation of acute and extravascular pulmonary fibrin. The term "APC molecule" is used herein to refer to any molecule capable of binding to, and proteolytically destroying the Va and Villa coagulation factors and which can bind and neutralize PAI-1 and / or specifically bind to the protein receptor. C (EPCR protein C endothelial receptor). Methods for evaluating the functional activity of APC molecules for use in the present invention include those described by Gruber A, Griffin JH. Direct detection of activated protein C in the blood of human subjects. Blood. 1992 May 1; 79 (9): 2340-8, and Strandberg K, Kjellberg M, Knebel R, Lilja H, Stenflo J. A sensitive immunochemical assay for measuring the concentration of the activated protein C protein C inhibitor complex in plasma: use of a catcher antibody specific for the complexed / cleaved form of the inhibitor. Thromb Haemost. 2001; 86 (2): 604-10. It is understood that the activity of the APC molecules for use in the present invention may be less potent or more potent than the native APC. APC is a serine proteinase dependent on vitamin K and is the activated form of protein C. APC has both anticoagulant and fibrinolytic properties. Human protein C is a 461 amino acid polypeptide, which undergoes various post-translational modifications to create activated protein C. These modifications include 1) the unfolding of a signal sequence of 42 amino acids; 2) the cleavage of lysine and arginine residues (positions 156 and 157) to make an inactive zymogen of 2 chains (a light chain of a 155 amino acid residue linked by means of a bisulfide bridge to a heavy chain of residue of 262 amino acids); 3) vitamin K-dependent carboxylation of nine glutamic acid residues located within 45 residues of the amino terminal (gla domain); and the 4-carbohydrate bond in four sites (one in the light chain and three in the heavy chain). Finally, the 2-chain zymogen can be activated by the removal of a dodecapeptide at the N-terminus of the heavy chain. The present invention further includes the use of human APC polypeptides produced recombinantly or synthetically or transgenically. Thus in one embodiment, the APC molecule is an APC homolog. Human activated protein C which is intended to be administered according to the present invention comprises naturally occurring human activated protein C, or biologically active analogues thereof, if they are prepared from plasma or are produced recombinantly or transgenically or synthetically. Recombinant human activated protein C can incorporate modifications (e.g., amino acid substitutions and / or deletions and / or additions of heterologous amino acid sequences), which can result in analogues with increased biological activity. For example, APC can be produced using eukaryotic cell culture systems (eg, human kidney 293, HEPG-2, LLC-MK2, CHO or AV12 cells), transgenic animals, transgenic plants, or in vitro systems. In these systems, the protein can be produced as an inactive precursor, which, after purification, is activated by unfolding of thrombin and formulated for administration. Alternatively, APC can be produced by the direct secretion of the activated form of protein C. Details of production, purification, activation, and formulation of APCs are known in the art and are described, for example, in US Pat. No. 6,156,734, which is incorporated herein by reference in its entirety. Also, APC genes and plasmids that can be used in these methods are described in the U.S. Patent. Nos. 4,981,952; 4,775,624; and 4,992,373, which are also incorporated herein by reference. Activated protein C can also be obtained from commercial sources. For example, a specific example of an APC that can be used in the invention is produced by Eli Lilly and Company, under the name XIGRIS (TM) (recombinant human activated protein C). In a preferred embodiment of the present invention, the APC molecule is an APC analog. An "APC analog" is defined as a molecule having one or more (such as 20 or less, for example 17 or less, such as 15 or less, for example 13 or less, such as 11 or less, for example 9 or less, such as 7 or less, for example 5 or less, such as 3 or less, for example 2 or less, such as 1 or less) amino acid substitutions, deletions, inversions, or additions relative to APC and may include forms of amino acid D. APC analogs have also been described in WO 01/57193 and include APC analogs which are created from protein C analogs containing at least two of the following amino acid substitutions in SEQ ID NO 1; His in position 10 is replaced with Gln; Being in position 11 is replaced with Gly; Being in position 12 is replaced with Lys; Gln at position 32 is replaced with Glu; Asn in position 33 is replaced with Asp or Phe; and the amino acid at position 194, 195, 228, 249, 254, 302 or 306 is substituted with an amino acid selected from Ser, Ala, Thr, His, Lys, Leu, Arg, Asn, Asp, Glu, Gly, and Gln , or the amide form thereof, and pharmaceutically acceptable salts thereof. Preferred APC molecules used in the present invention also include APC analogs in which one or more amino acids that do not occur in the original sequence are added or deleted, and derivatives thereof. Such analogs are described, for example, in EP 0 946 715, where the Gla domain of protein C is replaced by the Gla domains of other vitamin K dependent polypeptides, such as factor VII, factor X and prothrombin. In one embodiment of the present invention the APC analog exhibits an increasing anticoagulant activity compared to the wild-type protein. In a preferred embodiment the analog has a higher binding affinity for its binding partners, such as for example factor FVa, and FVIIIa, than the wild-type protein. In yet another embodiment, the APC analog has a lower binding affinity for its inhibitors, such as, for example, the C protein inhibitor, a-anti ripsin or plasminogen activator inhibitor 1, than the wild-type protein. In one embodiment of the present invention, the APC analog shows improved profibrinolytic activity compared to the wild-type protein. In a preferred embodiment, the analog has a higher binding affinity to PAI-1 than the wild-type protein. In another embodiment, the APC analog can inhibit PAI-1. In an additional embodiment the modifications result in a stabilization of the analog APC. Such analogs are described, for example, in patent applications WO 99/20767 and WO 03/073980, which are hereby incorporated by reference in their entirety. The APC analogs can be used according to the present invention alone or in combination with other APC analogues and / or homologs and / or derivatives and / or conjugates. For example, an APC analog with a higher binding affinity for FVa and / or FIBA can be used in combination with an APC homolog that can selectively bind and inhibit PAI-1. In another preferred embodiment of the present invention, the APC molecule is an APC derivative. An "APC derivative" is defined as a molecule having the amino acid sequence of APC or an APC analog, but additionally comprises chemical modification of one or more of its amino acid side groups, alpha carbon atoms, terminal amino group, or terminal carboxylic acid group. A chemical modification includes, but is not limited to, chemical portions added, creations of new links, and elimination of chemical portions. Modifications in amino acid side groups include, without limitation, acylation of epsilon amino groups lysine, N alkylation of arginine, histidine, or lysine, alkylation of carboxylic, aspartic or glutamic acid groups, and deamidation of glutamine or asparagine. Modifications of the amino terminus include, without limitation, des-amino, lower alkyl N, lower alkyl di N, and modifications of acyl N. Modifications of the carboxy terminal group include, without limitation, amide, lower alkyl amide, amide of dialkyl, and modifications of lower alkyl ester. Lower alkyl is C1-C4 alkyl. In addition, one or more side groups, or terminal groups, can be protected by protecting groups known to the ordinarily skilled protein chemist. The alpha carbon of an amino acid can be mono or dimethylated.

Homologues of APC molecules A homolog of one or more of the sequences specified herein may vary in one or more amino acids as compared to the defined sequences, but is capable of performing the same function, that is, a homolog can be contemplated as a functional equivalent of a predetermined sequence. Thus, in a preferred embodiment of the present invention, the APC molecule is a homolog of any of the molecules described herein, such as a homolog of any of the molecules of the group consisting of: o APC ° XIGRIS. Alpha Activated Drotrecogin Thus, in a preferred embodiment of the present invention, the APC molecule is a peptide that contains one or more amino acid substitutions, reversals, additions and / or deletions, compared to any of the molecules described herein, such as a molecule selected from the group consisting of: o APC or XIGRIS • Drotrecogin alfa In one embodiment, the number of substitutions, deletions, or additions is 20 amino acids or less, such as 15 amino acids or less, for example 10 amino acids or less, such as 9 amino acids or less, for example 8 amino acids or less, such as 7 amino acids or less, for example 6 amino acids or less, such as 5 amino acids or less, for example 4 amino acids or less, such as 3 amino acids or less, for example 2 amino acids or less (such as 1), or any whole between these amounts. In one aspect of the invention, the substitutions include one or more conservative substitutions, such as 20 or fewer conservative substitutions, for example 18 or less, such as 16 or less, for example 14 or less, such as 12 or less, for example 10 or less, such as 8 or less, for example 6 or less, such as 4 or less, for example 3 or less, such as 2 or less conservative substitutions. A "conservative" substitution denotes the replacement of one amino acid residue by another, related amino acid residue belongs to the same group of amino acids, such as those with a hydrophobic side chain, those with an aromatic side chain, those with a basic side chain, those with a an acid side chain, those with a hydroxyl side chain and those with a non-ionized polar side chain. Examples of conservative substitution include the substitution of a hydrophobic residue, such as isoleucine, valine, leucine or methionine by another, or the substitution of a basic residue by another, such as the substitution of lysine by arginine, or the substitution of a residue. acid by another, such as glutamic acid by aspartic acid, or the substitution of a non-ionized polar waste by another, such as the substitution of glutamine by asparagine, and the like. The following table lists illustratively, but does not limit, conservative amino acid substitutions.

Other APC homologs suitable for the uses and methods of the present invention are peptide sequences having greater than 50% sequence identity, and preferably greater than 90% sequence identity (such as greater than 91% sequence identity, example greater than 92% sequence identity, such as greater than 93% sequence identity, for example greater than 94% sequence identity, such as greater than 95% sequence identity, eg greater than 96% sequence identity, such as greater than 97% sequence identity, for example greater than 98% sequence identity, such as greater than 99% sequence identity, eg greater than 99.5% sequence identity), to any of the molecules described herein, such as a molecule selected from the group consisting of: o APC or XIGRIS. Drotrecogin alfa As used herein, "sequence identity" refers to a comparison made between two molecules using standard algorithms well known in the art. The preferred algorithm for calculating sequence identity by the present invention is the Smith-Waterman algorithm, where the reference sequence is used to define the percentage identity of polypeptide homologs over its length. The choice of parameter values for matches, no matches, and inserts or deletions are arbitrary, through some parameter values have been found to produce more biologically realistic results than others. A preferred set of parameter values for the Smith-Waterman algorithm is set in the approximate "maximum similarity segments", which use values of 1 for a matching residue and -1/3 for a mismatched residue (a residue that is already be a single nucleotide or single amino acid) (Waterman, Bull, Math, Biol. 46, 473-500 (1984)). Insertions and deletions (indels), x, are weighted as xk = 1 + k / 3, where k is the number of residues in a given insert or elimination (Id.). For example, a sequence that is identical to a sequence of 42 amino acid residues, except for 18 amino acid substitutions and an insertion of 3 amino acids, should have an identical percentage given by: [(1x42 matches) - (1/3 x 18 no matches) - 1 + 3/3 indels)] / 42 = 81% identity In a preferred embodiment of the present invention, the truncations at the end of the molecule are not taken into account when the sequence identity is calculated (this is, if one molecule is larger than another, only the overlapping lengths of the molecules are used in the sequence identity analysis); in another preferred embodiment of the present invention, the truncations are counted as eliminations. An APC analog can include amino acid D forms and can be a molecule having one or more amino acid substitutions, deletions, inversions, or additions relative to any of the molecules described herein, such as a molecule selected from the group consisting of from: • APC • XIGRIS. Drotrecogin alfa In a preferred embodiment of the present invention, the APC molecule is a peptide that contains one or more amino acid substitutions, inversion, additions or deletions, compared to APC. In one embodiment, the number of substitutions, deletions or additions is 20 amino acids or less, such as 15 amino acids or less, for example 10 amino acids or less, such as 9 amino acids or less, for example, 8 amino acids or less such as 7 amino acids or less. amino acid or less, for example, 6 amino acids or less, such as 5 amino acids or less, for example, 4 amino acids or less, such as 3 amino acids or less, for example, 2 amino acids or less (such as 1), or any integer between these amounts. In one aspect of the invention, the substitutions include one or more conservative substitutions. Examples of suitable conservative substitutions are given above.

Other APC homologs suitable for the uses and methods of the present invention are peptide sequences derived from protein C sequences that have more than 50% sequence identity and preferably more than about 90% sequence identity (such as more than about 91% sequence identity, for example, more than about 92% sequence identity, such as more than about 93% sequence identity, for example, more than about 94% identity of sequence, such as more than about 95% sequence identity, for example, more than about 96% sequence identity, such as more than about 97% sequence identity, for example, more than about 98 % sequence identity, such as more than about 99% sequence identity, eg, more than about 99.5% sequence identity) up to (1) SEQ ID NO: l / o (2) for truncated sequences thereof. As used herein, sequence identity refers to a comparison made between two molecules using standard algorithms well known in the art. The preferred algorithm for calculating the sequence identity by the present invention is the Smith-Waterman algorithm, as described above. An APC homolog may also be a molecule having one or more amino acid substitutions, deletions, inversions, or additions relative to human APC and may include amino acid forms D.

In another embodiment of the present invention, the homolog of any of the predetermined sequences herein, such as SEQ ID NO: 1 can be defined as: i) homologs comprising an amino acid sequence capable of selectively binding to the coagulation factors. and Villa, and / or ii) homologs that have substantially similar or higher binding affinity to the Va and Villa coagulation factors, than the human APC and / or üi) homologs that comprise an amino acid sequence that can selectively bind to and inhibit PAI -1 and / or iv) homologs having a binding affinity higher or substantially similar to PAI-1 and / or v) homologs having a binding affinity higher or substantially similar to the protein C endothelial receptor (ECPCR) and / or vi) homologues that have a substantially similar, higher or lower half-life after sedimentation in the respiratory tract.Chemically Derived APC Molecules It will further be understood that APC molecules suitable for use in the present invention can be chemically derived or altered, for example, peptides with unnatural amino acid residues (eg, taurine residue, beta and gamma amino acid residues and amino acid residues D), modifications of the C-terminal functional group, such as amides, esters and modifications of C-terminal ketone and modifications of the N-terminal functional group, such as acylated amines, Schiff bases, or cyclization, such as are found , for example, in the pyroglutamic acid of the amino acid.

Conjugates of the APC molecule The APC molecules of the present invention can also be modified with non-polypeptide portions, such as PEG or sugar portions, to produce APC compounds having increased inactivation resistance by, for example, human plasma and al-antitrypsin , therefore they display an increased in vivo half-life. Preferred examples include protein C conjugates, wherein at least one additional site of N-glycosylation in vivo has been introduced. Suitable examples are described in the U.S. patent. No. 6,933,367 ("Protein C or activated molecules of the C-protein type"), the contents of which are incorporated herein by reference. Thus, in one embodiment of the present invention, the APC molecule used is an N-glycosylated APC polypeptide or an analog thereof. In another embodiment of the present invention, the APC molecule is a glycosylated APC polypeptide or an analog thereof. In a further embodiment, the APC molecule, wherein at least one additional N-glycosylation site has been introduced. In another embodiment of the present invention, the APC molecule used is an APC polypeptide or an analog thereof also contains N-linked or O-linked fatty acids.

APC fragments In one embodiment the APC molecule can be an APC fragment. A fragment is a portion of APC, APC homolog or APC derivative. Examples of the fragments include domains of the type EGF 1, or 2 or the domain of serine protease, or deletions of the N-terminal or the C-terminal or both. The APC fragments comprise at least 20 consecutive amino acids of SEQ ID NO: 1. For example, a fragment may be 20 amino acids or more, such as 25 amino acids or more, for example, 30 amino acids or more, such as 50 amino acids or more, for example, 100 amino acids or more, such as 150 amino acids or more, example, 200 amino acids or more, such as 250 amino acids or more, for example, 300 amino acids in length or more, such as 350 amino acids or more, for example 400 amino acids or more, such as 450 amino acids or more, for example 460 amino acids of length or any integer between these quantities.

Intrathecal, intrabronchial or intraalveolar administration Methods of administration include, but are not limited to, spraying, washing, inhaling, jet washing or installation, using as a fluid a physiologically acceptable composition in which the blood coagulation factor (s) also They dissolve. When used herein the terms "intratracheal, intrabronchial or intraalveolar administration" include all forms of such administration therefore the coagulation factor is applied in the trachea, the bronchus or the alveolus, respectively, either by the installation of a Factor solution, by applying the factor in a powder form, or by allowing the factor to extend the relevant part of the respiratory tract by inhaling the factor as an aerosol or nebulizer solution or powder or gel, with or without adding stabilizers or other excipients. In another embodiment, intratracheal, intrabronchial, intraalveolar administration does not include inhalation of the product but the instillation or application of a factor solution or a powder or gel containing the factor in the lower respiratory tract or in the trachea.

Methods of intrabronchial / alveolar administration include, but are not limited to the administration (BAL) of bronchoalveolar lavage according to methods well known to those skilled in the art, using as a wash fluid a physiologically acceptable composition in which the APC and / or homologous APC and / or derivative and / or conjugate are dissolved or actually by any other effective form of intrabronchial administration including the use of nebulizer powders containing the anticoagulant in dry form, with or without excipients, or direct application of the anticoagulant in the form of a solution or powder or gel during bronchoscopy. Methods of intratracheal administration include, but are not limited to, blind tracheal lavage with a similar solution of dissolved activated protein C, or inhalation of nebulized aerosol fluid droplets containing dissolved activated protein C obtained by the use of any nebulizer device suitable for this purpose. The present invention provides a new useful addition for methods for treating ALI, ARDS, pneumonia and other conditions associated with bronchial-alveolar fibrin sedimentation. In addition, the administration of anticoagulants via the respiratory tract is expected to avoid the undesirable haemorrhagic adverse effects of the systemic administration of anticoagulants such as APC, whose intravenous use is associated with a significant incidence of internal bleeding including cerebral hemorrhage. At the same time, the application of anticoagulants via the respiratory tract is expected to enhance their effect on the deposition of extravascular fibrin in the lungs when compared to its systemic administration. It is expected that the total dose of an anticoagulant and anti-inflammatory agent such as APC can only be used crazily within the respiratory tract or can be divided between the conventional intravenous route and the airway route of the present invention to obtain the optimum balance between the local systemic and pulmonary effects of the treatment and reduce the incidence of the adverse effect of the drug, for example, in patient with severe sepsis, septic shock and ARDS. In addition, the time interval ("window of opportunity") during which the intravenous use of the APC is probably beneficial is limited. A long interval time of the drug response can be expected when the agent is used in the post-septic phase or even in the ARDS phase later by the alveolar fibrin sedimentation, for example, as observed in ALI and ARDS. A preferred embodiment of the present invention comprises local intrabronchial administration to human patients with APC ARDS by means of bronchoalveolar lavage with wash fluid (eg, 25 ml to 100 ml isotonic saline) in which a suitable dose (e.g. , 2 mg to 5 mg or more) of APC also dissolve. This administration was repeated at intervals one or more days dependent on the duration of either earlier or later phases of A i or ARDS. As supplementary or combination treatments in patients who meet the indications for intravenous administration of APC, APC can also be given by intravenous infusion. Other preferred methods of administration may include using the following devices: 1. Using pressurized nebulizers comprising an air / oxygen mixture. 2. Ultrasonic nebulizers. 3. Electronic micropump nebulizers (for example, Aeroneb professional nebulizer) 4. Measured dose inhaler (MDI) 5. Dry powder inhaler (DPI) systems, The aerosol can be released by means of a) facial masks or b) by means of of endotracheal tubes in patients intubated during mechanical ventilation (device 1, 2 and 3). Devices 4 and 5 can also be used by the patient without assistance with the condition that the patient can by himself activate the aerosol device. The preferred concentrations for a solution comprising APC and / or homologs and / or APC derivatives are in the range of 0.1 μg to 10000 μg of active ingredient per ml of the solution. Using monomeric forms of the compounds, suitable concentrations are frequently in the range from about 0.1 μg to 5000 μg per ml of the solution, such as in the range from about 0.1 μg to 3000 μg per ml of the solution and especially in the range from about 0.1 μg up to 1000 μg per ml of the solution, such as in the range from about 0.1 μg to 250 μg per ml of the solution. A preferred concentration should be from about 0.1 to about 5.0 mg, preferably from about 0.3 mg to about 3.0 mg, such as from about 0.5 to about 1.5 mg and especially in the range from .8 to 1.0 mg per my of the solution. Using multimeric forms of the compounds, suitable concentrations are frequently in the range from 0.1 μg to 1000 μg per ml of the solution, such as in the range from about 0.1 μg to 750 μg per ml of the solution and especially in the range from about 0.1 μg to 500 μg per ml of the solution, such as in the range from about 0.1 μg to 250 μg per ml of the solution. A preferred concentration should be from about 0.1 to about 5.0 mg, preferably from about 0.3 mg to about 3.0 mg, such as from about 0.5 to 1.5 mg and especially in the range from 0.8 to 1.0 mg per ml of the solution.

Indications An aspect of the present invention relates to a method for treating or preventing sedimentation of extravascular fibrin in the respiratory tract. Thus, the present invention relates to the treatment of individuals suffering from or at risk of suffering from extravascular fibrin depositions caused by an inflammatory lung disease. In a preferred aspect inflammatory lung disease is selected from the group consisting of: ALI ARDS Pneumonia Acute bronchial-alveolar disease Chronic bronchial-alveolar disease Fibrous alveoliths or bronchial asthma Alveolitis Bronchiolitis Organized pneumonia due to bronchiolitis obliterans (BOPA) Graft-versus-host disease (C-HDV) Pneumocystis carinii pneumonia (PCP) Pneumonitis, for example, aspiration pneumonitis Drug-induced pneumonitis (for example, due to methotrexate, bleomycin, or sirolimus) Fibrous, acute, or chronic alveolitis Cystic fibrosis Idiopathic pulmonary fibrosis In another modality, the inflammatory disease of the lung is related to a condition selected from the group consisting of: Direct or indirect pulmonary trauma Pancreatitis Pneumonitis by aspiration sepsis severe sepsis and / or septic shock Pneumocystis carinii pneumonia (PCP) as a prophylactic adjuvant or therapy preventive or as a treatment of ARDS manifest by PCP, induced by PCP, that is, before or early in the PCP phase or in ARDS manifestation concomitant with antibiotic therapy anti-Pneumocystis with for example, Sulfamethoxazole with Trimethoprim.

Pharmaceutical Compositions Pharmaceutical compositions of formulations for use in the present invention include an APC preparation in combination with, preferably dissolved in, a pharmaceutically acceptable carrier, preferably an aqueous carrier or diluent. The pharmaceutical composition can be a solid, liquid, a gel or an aerosol. A variety of aqueous carriers can be used, such as 0.9% saline, buffered saline, physiologically compatible buffer solutions and the like, the compositions can be sterilized by conventional techniques well known to those skilled in the art. The resulting aqueous solutions can be packaged for use or filtered under aseptic conditions and frozen-dried, the frozen-dried preparation is dissolved in a sterile aqueous solution before administration. The compositions may contain pharmaceutically acceptable adjuvants or auxiliaries, including, without limitation, pH adjusting agents and buffering agents and / or tonicity adjusting agents, such as, for example, sodium acetate, sodium lactate, Sodium chloride, potassium chloride, calcium chloride, etc. The formulations may contain pharmaceutically acceptable carriers and excipients including microspheres, liposomes, microcapsules, nanoparticles or the like. Conventional liposomes are typically composed of phospholipids (neutrally or negatively charged) and / or cholesterol. Liposomes are vesicular structures based on lipid bilayers wrapped in aqueous compartments. They can vary in their physicochemical properties such as size, lipid composition, surface charge and number and fluidity of the phospholipid bilayers. The most frequently used lipid for liposome formation are: 1, 2-Dilauriol-sn-Glycero-3-phosphocholine (DLPC), 1,2-Dimyristoyl-sn-Glycero-3-phosphocholine (DMPC), 1,2-Dipalmitoil -sn-Glycero-3-phosphocholine (DPPC), 1,2-Distearoyl-sn-Glycero-3-phosphocholine (DSPC), 1,2-Dioleoyl-sn-Glycero-3-phosphocholine < DOPC), 1, 2-Dimiristoil-sn-Glycero-3-phosphoethanolamine (DMPE), 1,2-Dipalmitoyl-sn-Glycero-3-phosphoethanolamine (DPPE), 1,2-Dioleoyl-sn-Glycero-3-phosphoethanolamine (DOPE), 1, 2-Dimiristoil-sn-Glycero-3-phosphate (Monosodium Salt) (DMPA), 1,2-Dipalmitoyl-sn-Glycero-3-phosphate (Monosodium Salt) (DPPA), 1,2-Dioleoyl-sn-Glycero-3-Phosphate (Monosodium Salt) (DOPA), 1,2-Dimyristoyl-sn-Glycero-3- [phospho-rac- (l-glycerol)] (Sodium salt) ) (DMPG), 1,2-Dipalmitoyl-sn-Glycero-3- [Phospho- rac- (1-glycerol)] (Sodium salt) (DPPG), 1,2-Dioleoyl-sn-Glycero-3- [ phospho-rac- (1-glycerol)] (Sodium salt) (DOPG), 1,2-Dimyristoyl-sn-Glycero-3- [phospho-L-Serine] (Sodium salt) (DMPS), 1, 2 -Dipalmitoil-sn-Glycero-3- [phospho-L-Serine). { Sodium Salt) (DPPS), 1,2-Dioleoyl-sn-Glycero-3- [phospho-L-Serine] (Sodium Salt) (DOPS), 1,2-Dioleoyl-sn-Glycero-3-phosphoethanolamine- N- (glutaryl) (Sodium salt) and 1, 1 ', 2, 2' -Tetramiristoil Cardiolipin (Ammonium salt). Composite formulations of DPPC in combination with other lipids or liposome modifiers are preferred, for example, in combination with cholesterol and / or phosphatidylcholine. The long-circulation liposomes are characterized by their ability to extravasate sites in the body where the permeability of the vascular wall increases. The most popular means of producing long-circulation liposomes is to bind polyethylene glycol hydrophilic polymer (PEG) covalently to the outer surface of the liposome. Some of the preferred lipids are: 1, 2-Dipalmitoyl-sn-Glycero-3-Phosphoethanolamine-N- [Methoxy (Polyethylene glycol) -2000] (Ammonium salt), 1,2-Dipalmitoyl-sn-Glycero-3- Phosphoethanolamine-N- [Methoxy (Polyethylene glycol) -5000] (Ammonium salt), 1,2-Dioleoyl-3-trimethylammonium-Propane (Salt Chloride) (DOTAP). Possible lipids applicable for liposomes are supplied by Avanti, Polar Lipids, Inc., Alaba ter, AL. Additionally, the liposome suspension can include lipid protection agents which protect the lipids against free radicals and damage by lipid peroxidation in storage. Lipophilic free radical quenchers, such as alpha-tocopherol and specific water-soluble iron chelators, such as ferrioxianin, are preferred. A variety of methods are available for preparing liposomes, as described in, for example, Szoka et al., Ann. Rev. Biophys. Bioeng. 9: 467 (1980), Pat. E.U. Nos. 4,235,871, 4,501,728 and 4,837,028, all of which are incorporated herein by reference. Another method produces multilamellar vesicles of heterogeneous sizes. In this method, the vesicle forming lipids are dissolved in a suitable organic solvent or solvent system and dried under vacuum or in an inert gas to form a thin lipid film. If desired, the film can be again dissolved in a suitable solvent, such as tertiary butanol, and then lyophilized to form a more homogeneous lipid mixture which is in a more easily hydrated form as a powder. This film is covered with an aqueous solution of the target drug and the target component and allowed to hydrate, typically over a period of 15-60 minutes with agitation. The size distribution of the resultant multilamellar vesicle can be changed to smaller sizes by hydrating the lipids under more vigorous agitation conditions or by adding solubilizing detergents such as deoxycholate. The micelles are formed by surfactants (molecules that contain a hydrophobic portion and one or more ionic or otherwise strongly hydrophilic groups) in aqueous solution. Common surfactants well known to one of skill in the art may be used in the micelles of the present invention. Suitable surfactants include sodium laureate, sodium oleate, sodium lauryl sulfate, octaoxyethylene glycol monododecyl ether, octoxynol 9 and PLURONIC F-127 (Wyandotte Chemicals Corp.). Preferred surfactants are nonionic polyoxyethylene and polyoxypropylene detergents compatible with IV injection such as, TWEEN-80, PLURONIC F-68, n-octyl-beta-D-glucopyranoside, and the like, In addition, phospholipids, such as those described for use in the production of liposomes, can also be used for of micelos. In some cases, it will be advantageous to include a compound, which promotes the delivery of the substance to its objective Dosage regimens The preparations are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective. The amount to be administered depends on the subject to be treated, including, for example, the weight and age of the subject, the disease to be treated and the stage of the disease. Suitable dosage ranges are per kilogram of body weight usually in the order of several hundred μg of active ingredient per administration with a preferred range of about O.lμg to 100OOμg per kilogram of body weight. By using monomeric forms of the compounds, the appropriate dosage is frequently in the range of O.lμg to 5000μg per kilo of body weight, such as in the range of about O.lμg to 3000μg per kilo of body weight, and especially in the range from around O.lμg to lOOOμg per kilo of body weight. Using multimeric forms of the compounds, suitable dosage is obtained in the range of about 0.1 μg to 750 μg per kilo of body weight, and especially in the range of about 0.1 μg to 500 μg per kilo of body weight as in the range of about 0.1 μg to 250 μg per kilo of body weight. A preferred dosage would be from about 0.1 to about 5.0 mg, preferably from about 0.3 mg to about 3.0 mg, such as from about 0.5 to about 1.5 mg and especially in the range from 0.8 to 1.0 per administration. Administration will be performed once or may be followed by subsequent administrations. The dosage also depends on the route of administration and will vary according to the age, sex and weight of the subject to be treated. A preferred dosage of multimeric forms would be in the range of 1 mg to 70 mg per 70 kg of body weight. The proper daily dosage ranges are per kilogram of weight per day, usually in the order of several hundred μg of active ingredient per day with a preferred range of about 0.1 μg to 10000 μg per kilo of body weight per day. Using monomeric forms of the compounds, the appropriate dosage is obtained in the range of about 0.1 μg to 5000 μg per kilo of body weight per day, such as in the range of about 0.1 μg to 3000 μg per kilo of body weight, and especially in the range of 0.1 μg to 1000 μg per kilo of body weight per day. Using multimeric forms of the compounds, the appropriate dosage is obtained in the range of about 0.1 μg to 1000 μg per kilo of body weight per day, such as in the range of about 0.1 μg to 750 μg per kilo of body weight per day, and especially in the range of about 0.1 μg to 500 μg per kilo of body weight per day, such as in the range of about 0.1 μg to 250 μg per kilo of body weight per day. A preferred dosage would be from about 0.1 to about 100 μg, preferably from about 0.1 to about 50 μg, such as from about 0.3 μg to about 30 μg and especially in the range from 1.0 to 10 μg per kilogram. body weight per day. Administration will be performed once or may be followed by subsequent administrations. The dosage also depends on the route of administration and will vary with the age, sex and weight of the subject to be treated. A preferred dosage of multimeric forms would be in the range of 1 mg per 70 kilograms of body weight per day.

Medical packaging The compounds used in the invention can be administered alone or in combination with pharmaceutically acceptable carriers or excipients, in either single or multiple doses. The formulations can be conveniently presented in unit dosage form by methods known to those skilled in the art. It is preferred that the compounds according to the invention are provided in a kit. Such a kit typically contains an active compound in dosage forms for administration. A dosage form contains a sufficient amount of active compound such that a desired effect can be obtained when administered to the subject. A) YesIt is preferred that the medical package comprises a quantity of dosage units corresponding to the relevant dosage regimen. Thus, in one embodiment, the medical package comprises a pharmaceutical composition comprising a compound as defined above or a pharmaceutically acceptable salt thereof and pharmaceutically acceptable carriers, vehicles and / or excipients, the package comprising from 1 to 7 units of dosage, therefore it has dosage units for one or more days, or from 7 to 21 dosage units, or multiple thereof, therefore it has dosage units for a week of administration or several weeks of administration. The dosage units can be as defined above. Medical packages can be in any form suitable for intratracheal, intrabronchial or intraalveolar administration. In a preferred embodiment the package is in the form of a vial, ampoule, tube, bubble pack, cartridge or capsule. When the medical package comprises more than one dosage unit, it is preferred that the medical package be provided with a mechanism for adjusting each administration for one dosage only. Preferably, a kit contains instructions indicating the use of the dosage form to achieve a desired affect and the amount of dosage form to be taken during a specific period of time. Accordingly, in one embodiment the medical package comprises instructions for administering the pharmaceutical composition.

EXAMPLES Example 1 A 60-year-old patient develops severe ARDS with opacities mainly located in the right lung as judged from the radiograph. The bromo-saclar lavage (BAL) was performed in the bronchus of the right lower lobe by means of a bronchoscope with a simple dose of 5 mg activated drotrecogin-alpha (Xigris) dissolved in 20 ml of normal saline. There were no apparent adverse effects and no bleeding of the respiratory tract was observed. The next day there is some clearing of the opacities in the right lower lobe that had been treated with BAL. On the other hand, the single dose of APC did not improve gas exchange as monitored by the PaO2 / Fi02 ratio.

Example 2 An 11-year-old boy with Mb. Hodgkin 2003 was initially treated with chemotherapy. A relapse was documented in 2005, where it was treated with radiation therapy and bone marrow transplantation (BMT). The lung biopsy is performed before the ICU admission shown. No sign of disease graft against pulmonary host (GvHD). There are no signs of lymphoma infiltration admitted on days 9 ICU after BMT for acute pulmonary insufficiency (ALI) and continuous high fever with sepsis. Initially it is treated with empirical broad-spectrum antibiotics. On day 4 the oxygenation capacity was further reduced with a Pa02 / Fi02 < 200 despite the high PEEP. A film of the chest shows diffuse bilateral infiltrations located in the 4 quadrants of the lung field. Echocardiography shows no cardiac bypass or valvular insufficiency with normal myocardial function. No other organ dysfunction was detected. The patient has mono organ dysfunction with severe ARDS. Bronchoalveolar lavage (BAL) was without Candida spp. Aspergillus, virus or bacteria. Based on the decision of the Institutional Review Board (IRB), inhalation therapy with activated protein C (activated alpha Drotrecogin) in a dose of 5 mg x 3 by means of a nebulizer (Aeroneb®) was initiated. Already after 3 doses after 12 hours later, the infiltrates in the breast film were clearly reduced; concomitantly with the arterial oxygen saturation increased from 85% to 95% while in Fi02 = 1.0. There were no serious adverse effects at any time, for example, bleeding of the respiratory tract during or after the inhalation of APC. 24 hours after the initiation of inhalation therapy with APC, the patient died due to circulatory failure due to the lack of a right ventricle of the heart, that is, acute or pulmonary.

It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims (13)

1. Claim When the invention has been described as above, the content of the following claims is claimed as property: 1. The use of a human activated protein C or a biologically active derivative thereof for the manufacture of a medicament for use as the sole drug for treat or prevent the sedimentation of extravascular fibrin in the alveolar or bronchioalveolar spaces, in a human by administration through the respiratory tract by means of intratracheal, intrabronchial or intraalveolar administration.
2. 2. The use according to claim 1, wherein the fibrin sedimentation is caused by an inflammatory lung disease.
3. 3. The use according to claim 2, wherein the inflammatory lung disease is selected from the group consisting of: ALI, ARDS, pneumonia, acute bronchial-alveolar diseases, chronic bronchial-alveolar diseases, fibrous alveolitis or bronchial asthma.
4. 4. The use according to any of the preceding claims, wherein the inflammatory lung disease is related to a condition selected from the group consisting of: direct or indirect lung trauma, pancreatitis, aspiration pneumonitis, sepsis, severe sepsis and / or septic shock.
5. 5. The use according to any of the preceding claims, wherein the activated protein C is administered by bronchoalveolar lavage with an activated protein C solution.
6. 6. The use according to any of the preceding claims, wherein the activated protein C is administered by washing the blind trachea with a solution of the activated protein C.
7. The use according to any of the preceding claims, wherein activated protein C is administered by inhalation of a nebulized solution of activated protein C.
8. 8. The use according to any of the preceding claims, wherein activated protein C is administered by inhalation of activated protein C in an inhaled powder form.
9. 9. The use according to any of the preceding claims, wherein the activated protein C is administered by the direct application of activated protein C during bronchoscopy.
10. 10. The use according to any of claims 1 to 9, wherein the human is an adult.
11. 11. The use according to any of claims 1 to 9, wherein the human is a child.
12. 12. The use according to any of the preceding claims, wherein the APC is administered in an amount from 0.1 μg / kg to about 10 mg / kg of body weight per day.
13. 13. The use according to any of the preceding claims, wherein the inflammatory lung disease is selected from ALI and ARDS.