WO2008100258A1 - Intraperitoneal administration of antithrombin iii, related compositions and methods - Google Patents

Abstract

This invention is related, in part, to the intraperitoneal administration of antithrombin III, as well as compositions and methods related thereto.

Description

INTRAPERITONEAL ADMINISTRATION OF ANTITHROMBIN III, RELATED

COMPOSITIONS AND METHODS

FIELD OF THE INVENTION This invention is related, in part, to the intraperitoneal administration of antithrombin

III, as well as compositions and methods related thereto.

BACKGROUND OF THE INVENTION

In sepsis, coagulation disorders commonly occur and correlate with multiple organ dysfunction and mortality [I]. In animal models, anticoagulants, such as antithrombin (AT), have demonstrated favorable effects on morbidity and survival, suggesting that the coagulation cascade can be used as therapeutic target in sepsis [2—4]. In a large, world-wide, double-blind, placebo-controlled, randomized phase-Ill trial (KyberSept), i.v. administration of recombinant human antithrombin (rhAT), however, was shown to lack efficacy in reducing mortality and was associated with increased risk of bleeding, especially with heparin coadministration [5]. A meta-analysis has revealed that with increasing severity, of illness — reflected by increasing mortality rate of the control group — increased survival is associated with use of anticoagulant therapy. However, in this meta-analysis, increased risk of bleeding was reconfirmed. Therefore, bleedings are a serious drawback for systemic AT therapy in sepsis [6].

The anticoagulant and anti-inflammatory effects of AT are attributed to several mechanisms. AT is a serine protease inhibitor and inhibits multiple components of the common, intrinsic and extrinsic coagulation pathways: .thrombin, factor (F) IXa, FXa and FXIa [4,9,10]. AT also functions with heparin and tissue factor (TF) pathway inhibitor to inactivate the TF- FVIIa complex [H]. AT reduces the inflammatory response through interaction with endothelium to produce prostacyclin, thereby reducing neutrophil activation and cytokine production [12—14]. AT precludes generalized microvascular thrombosis, preventing bacteria in the tissues from reaching the systemic circulation via the capillaries. In time, leukocytes will remove bacteria and repair the damaged tissues [15]. Generalized microvascular thrombosis leads to extensive tissue ischemia, which precipitates organ failure and death [16]. In sepsis, the degree of organ injury is related to the quantity of thrombi [17]. AT could interfere with intra-abdominal fibrin formation, which is an adaptive response precluding bacterial spread by encapsulation [17,18]. However, fibrin shields bacteria off, impairs their clearance and enhances abscess formation [17]. Therefore, the role of intraabdominal fibrin is ambiguous.

Severe intra-abdominal infections, such as peritonitis and sepsis, result in activation of coagulation and inflammatory cascades systemically and even in extraperitoneal organs, with the lungs being the first and most frequently affected organ [8,19,20]. Multiple studies have demonstrated distinction between infection and inflammation at the different sites of the body, suggesting that these responses are compartmentalized [19,21].

SUMMARY OF THE INVENTION

Provided herein are methods and compositions related to intraperitoneal administration of antithrombin (i.e., ATIII). In one aspect, a method for producing a systemic anticoagulant or anti-inflammatory effect in a subject is provided. The method comprises administering antithrombin III intraperitoneally in an amount effective to produce the systemic anticoagulant or anti-inflammatory effect in the subject. In one embodiment, the effect is effective in treating the subject. In another embodiment, the effect is effective for prophylaxis in the subject. In another aspect, a method for producing a pulmonary anticoagulant or anti-inflammatory effect in a subject, comprising administering antithrombin III intraperitoneally in an amount effective to produce the pulmonary anticoagulant or antiinflammatory effect in the subject is provided. In one embodiment, the effect is effective in treating the subject. In another embodiment, the effect is effective for prophylaxis in the subject. The treatment or prophylaxis can be for the treatment or prophylaxis of any of the disease, conditions or indications provided herein.

In still another aspect, a method of treating sepsis in a subject, comprising administering antithrombin III intraperitoneally in an amount effective to treat sepsis in the subject is provided. In yet another aspect, a method of preventing sepsis in a subject, comprising administering antithrombin III intraperitoneally in an amount effective to prevent sepsis in the subject is provided. In one embodiment,, the amount effective is also effective in treating an injury local to the peritoneum in the subject., In yet another embodiment, the amount is also effective to produce a systemic or pulmonary anticoagulant or antiinflammatory effect in the subject. ,

In a further aspect, a method of treating a subject with an injury local to the peritoneum is provided. In one embodiment, the method comprises administering antithrombin III intraperitoneally in an amount effective to treat the injury is provided. In another embodiment, the antithrombin III is administered by intraperitoneal injection or intraperitoneal lavage. In still another embodiment, the amount is also effective to treat or prevent sepsis. In yet another embodiment, the amount is also effective to produce a systemic or pulmonary anticoagulant or anti-inflammatory effect in the subject.

In still a further aspect, a method of treating a subject with disseminated intravascular coagulation (DIC) is provided. In one embodiment, the method comprises administering antithrombin III intraperitoneally in an amount effective to treat the subject- In yet a further aspect, a method of preventing disseminated intravascular coagulation (DIC) is provided. In one embodiment, the method comprises administering antithrombin III intraperitoneally in an amount effective to prevent DIC in the subject. In an embodiment of these methods, the subject has an injury local to the peritoneum. In one embodiment, the amount effective is also effective in treating the injury local to the peritoneum, hi another embodiment, the subject has or is at risk of sepsis, hi one embodiment, the amount effective is also effective in treating or preventing sepsis. In still another embodiment, the subject does not have pancreatitis.

The methods provided can, in one embodiment, further comprise assessing the effect of the administration in the subject. In another embodiment, the effect is anticoagulation or anti-inflammation. In still another embodiment, anticoagulation or anti-inflammation is assessed in the subject systemically (e.g., using plasma samples). In a further embodiment, anticoagulation or anti-inflammation in the pulmonary compartment of the subject is assessed.

In one embodiment, the antithrombin III is administered by peritoneal lavage or intraperitoneal injection.

In another embodiment, the antithrombin III is administered at a high dose.

In a further embodiment, the subject has sepsis. In one embodiment, the subject has severe sepsis or septic shock, In still a further embodiment, the subject has an injury local to the peritoneum. In yet a further embodiment, the subject has disseminated intravascular coagulation (DIC). In another embodiment, the subject does not have DIC. In yet another embodiment, the subject is not at risk of DIC. In still another embodiment, the subject does not have and is not at risk of DIC. In still another embodiment, the subject does not have pancreatitis. In a further embodiment, the subject has an injury local to the peritoneum and has or is at risk of sepsis, hi one embodiment, the subjeέt that has an injury that is local to the peritoneum and/or has or is at risk of sepsis, does not have pancreatitis and/or has or is not at risk of DIC. In another embodiment, the subject has not been and/or is not being treated with heparin. In still another embodiment, the subject has been treated with heparin or is being treated with heparin.

In one embodiment, the antithrombin III is plasma-derived antithrombin III. In another embodiment, the antithrombin III is recombinant antithrombin III. In still another embodiment, the antithrombin III is antithrombin III that is milk-derived (e.g., transgenically produced in the milk of a non-human mammal). The non-human mammal, in one embodiment, is a goat.

In another embodiment, the subject is human.

In still another embodiment, the methods provided can further comprise administering another therapeutic agent to the subject. In one embodiment, the other therapeutic agent is an agent for treating sepsis, DIC, peritonitis or ovarian cancer. In another embodiment, the other therapeutic agent is for treating a gastrointestinal injury or infection. In still another embodiment, the other therapeutic agent is for treating an injury local to the peritoneum. In another embodiment, the other therapeutic agent is an anticoagulant. In still another embodiment, the other therapeutic agent is an anti-inflammatory agent. In a further embodiment, the other therapeutic agent can be administered concomitantly with, subsequent to or prior to the administration of antithrombin III. In one embodiment, the other therapeutic agent is administered locally. In a further embodiment,' the other therapeutic agent is administered intraperitoneally. In another embodiment, the other therapeutic agent is administered by a mode other than intraperitoneally. In one embodiment, the other therapeutic agent is administered intravenously, parenterally, intramuscularly, intracavity or subcutaneously. In another embodiment, the administration is by inhalation. In still another embodiment, the administration is intranasal, oral or sublingual.

In another aspect, compositions comprising antithrombin III are provided. In one embodiment, the antithrombin III is formulated for intraperitoneal administration. In another embodiment, the composition is formulated for administration by intraperitoneal lavage. In a further embodiment, the composition is formulated for administration by intraperitoneal injection.

In another embodiment, the compositions of antithrombin III further comprise another therapeutic agent. In one embodiment, the other therapeutic agent is an agent for treating sepsis, DIC, peritonitis or ovarian cancer. In another embodiment, the other therapeutic agent is for treating a gastrointestinal injury or infection. In still another embodiment, the other therapeutic agent is for treating an injury local to the peritoneum. In another embodiment, the other therapeutic agent is an anticoagulant. In still another embodiment, the other therapeutic agent is an anti-inflammatory agent.

In yet a further embodiment, compositions comprising another therapeutic agent can also be formulated for administration by intraperitoneal administration. In another embodiment, compositions comprising other therapeutic agents (e.g., without antithrombin) can be formulated for administration by other than intraperitoneal administration.

Each of the limitations of the invention can encompass various embodiments of the invention. It is, therefore, anticipated that each of the limitations of the invention involving any one element or combinations of elements can be included in each aspect of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 depicts the time line of the experiment with surgical treatment and lavage intervention.

Fig. 2 shows the increased abdominal and reduced pulmonary clotting times after antithrombin (AT) lavage. CLP=cecal ligation and puncture; solid squares=CLP-saline; empty circles^CLP-AT. Values are expressed as means ± SEM, sham level is indicated by dotted line. P-values represent differences between CLP-saline and CLP-AT (ANOVA).

Fig.3 shows that abdominal thxombin-anti-thrombin complexes (TAT) were reduced after AT lavage. Solid squares=CLP-saline; open circles=CLP-AT. Values are expressed as means ± SEM, sham level is indicated by dotted line. P,-values represent differences between CLP-saline and CLP-AT (ANOVA). ,

Fig. 4 provides fibrin degradation product D-dimer levels, measured in peritoneal lavage fluid (PLF), plasma and lungs, are reduced after AT lavage. Solid squares=CLP- saline; open circles=CLP-AT. Dotted line represents sham group. Values are expressed as means ± SEM. P-values represent differences between CLP-saline and CLP-AT (ANOVA).

Fig. 5 shows that AT lavage decreased abdominal and pulmonary cytokine and cellular inflammatory responses. Abdominal and pulmonary concentrations of interleukin-6 (IL-6; Fig. 5A and Fig.5D, respectively) and keratinocyte-derived chemokine (KC; Fig.5B and Fig. 5E, respectively) were measured to assess pro-inflammatory cytokine responses. Leukocyte count in peritoneal lavage fluid (Fig. 5C) and myeloperoxidase activity (MPO; Fig. 5F) assessed cellular response. Solid squares=CLP-saline; open circles=CLP-AT. Values are expressed as means ± SEM. P-values represent differences between CLP-saline and CLP-AT (ANOVA). Fig. 6 shows the results of the histological and immunostaining of peritoneum. Slices of peritoneum of CLP-AT (Fig. 6 A and Fig. 6B), CLP-saline (Fig. 6C and Fig. 6D) and sham mice (Fig. 6E and Fig. 6F) were stained with hematoxillin-eosin (upper panel), and fibrin depositions were stained immunohistochemically (lower panel). Note that CLP-AT mice had less inflammatory cells and thickened peritoneal layer (Fig. 6A) compared to CLP- saline (Fig. 6C). Moreover, the lower panel showed a thinner dark layer, representing fibrin, in CLP-AT mice (Fig. 6B) compared to CLP-saline mice (Fig. 6D); sham controls had the fewest number of inflammatory cells (Fig. 6E) and no fibrin deposition (Fig. 6F); t=48 hours after CLP; magnification 2Ox.

Fig. 7 provides Kaplan-Meier curves of sham, CLP-saline and CLP-AT groups. 100% survival rate in sham animals (-— ), 62% survival in CLP-saline animals ( — ), 83% survival in CLP-AT ( — ). P<0.03 CLP-saline vs. CLP-÷AT (cumulative survival, LogRank).

DETAILED DESCRIPTION OF THE INVENTION

Systemic AT use is complicated by increased risk of bleeding (odds ratio 1.7) and clinically important survival increase is seen only in the non-heparinized subgroup. The effects of intra-abdominal high-dose recombinant human AT (rhAT) lavage on coagulation and inflammation in experimental polymicrobial sepsis was investigated with a murine cecal ligation and puncture model used with peritoneal lavage after 24 h, containing rhAT (3 IU/mL) or saline. Clotting time, thrombin— antithrombin complexes (TAT), D-dϊmers, tissue- type plasminogen activator and plasminogen activator inhibitor- 1 were used to assess coagulation and fibrinolysis responses. Inflammation was assessed by keratinocyte-derived chemokine (KC), interleukin-lβ (IL-I β), IL-6, tumor necrosis factor-alpha (TNF-α). leukocyte count, myeloperoxidase and bacterial load. rhAT lavage was found to prolong abdominal clotting times and reduced D-dimers and TAT levels, indicating inhibited abdominal coagulation. Further, pulmonary clotting time and D-dimers decreased towards normal. Abdominal fibrinolysis was also reduced after rhAT lavage, as were abdominal IL- 1 β, KC, leukocytes and bacterial load. Pulmonary TNF-Ci, KC, myeloperoxidase and histopathological injury were decreased. Survival improved from 62% (saline lavage) to 83% (rhAT lavage, P < 0.05). The high-dose rhAT lavage inhibits coagulation activation and reduced inflammatory responses in both abdominal and pulmonary compartments, ultimately improving survival. In addition, higher doses can be used without the risk of complications. The interaction with systemic (i.v.) heparin, thought to be the cause of increased hemorrhages, is less likely [5].

Therefore, provided herein are methods of administering antithrombin (also referred to herein as antithrombin III) intraperitoneally as well as compositions and other methods related thereto. "Intraperitoneally" is intended herein to refer to administration via the peritoneal compartment (i.e., administration that results in contact with the peritoneum or the compartment that is enclosed by the peritoneum). Such administration includes intraperitoneal injection, peritoneal lavage, etc. Such forms of administration are well known in the art. For example, peritoneal lavage with saline is routinely performed during laparotomy for peritonitis [8].

The administration of antithrombin can be used to produce a number of effects in a subject as well as for a number of therapeutic purposes.' The methods and compositions provided can be used, for example, to produce a systemic or pulmonary anticoagulant or antiinflammatory effect in a subject. Preferably, such effects are in addition to local effects and are effective in treating and/or preventing sepsis in the subject, but are not necessarily so. As used herein, "sepsis" refers to the serious medical condition resulting from the immune ^• response to an infection. "Severe sepsis" refers to sepsis with acute organ dysfunction, and "septic shock" refers to sepsis with refractory arterial hypotension.

The resulting effects on coagulation can be assessed by clotting time by standard procoagulant assay or the measurement of TAT complexes, D-dimer concentrations, etc. Anti-inflammatory effects can be assessed by determining leukocyte count, neutrophil count (e.g., by measuring myeloperoxidsae activity) or by measuring IL-6, IL-I β, tumor necrosis factor or keratinocyte-derived chemokine (KC) concentrations, for example. The methods provided, therefore, can also include a step of assessing the effect in a subject. As used herein, "assessing an effect in a subject" refers to determining whether or not subsequent to the administration of antithrombin, anticoagulation or anti-inflammation occurs locally, systemically or in the pulmonary compartment. In one embodiment, systemic or pulmonary anticoagulation or anti-inflammation effects are assessed. Such an assessment can be performed using one of the methods provided above or those provided in the Examples. Further methods will be known to those of ordinary skill. in the art.

Producing anticoagulant and anti-inflammatory effects with antithrombin administration can be beneficial for treatment or prophylaxis in a subject. As used herein, "treat", "treating" or "treatment" refers to reducing the symptoms related to an existing disease, condition or indication provided herein; slowing or halting of the progression of the existing disease, condition or indication; curing the existing disease, condition or indication; or otherwise providing some benefit to a subject that has the existing disease, condition or indication. "Prophylaxis", as used herein, refers to preventing or delaying the onset of a disease, condition or indication or reducing the risk of developing the disease, condition or indication. The methods and compositions provided can, therefore, be used in the treatment or prophylaxis of a number of diseases, conditions or indications. The methods and compositions provided can be used, for example, for the treatment or prophylaxis of sepsis, disseminated intravascular coagulation (DIC), peritonitis, ovarian cancer, or for any gastrointestinal injury or infection. The methods and compositions can be used for any injury that is local to the peritoneum. "Injuries local to the peritoneum" refer to any injury, such as trauma or infection, that occurs in the vicinity of the peritoneum such that intraperitoneal administration of a therapeutic agent would be expected to have some local effect. In some embodiments, the treatment of any of the diseases, conditions or indications provided with antithrombin can result in the treatment of the disease or condition itself as well as the treatment or prophylaxis of sepsis. Therefore, the methods and compositions provided can, in some embodiments, be used to treat any disease or condition local to the peritoneum that can result in sepsis. The diseases, conditions or indications are known to those of ordinary skill in the art and/or are described, for instance, in Harrison 's Principles of Internal Medicine (McGraw Hill, Inc., New York).

The antithrombin can be plasma-derived, recombinantly produced, transgenically produced or produced by any technique known to those of ordinary skill in the art. Plasma- derived and recombinantly produced antithrombin can be obtained commercially and/or obtained by methods known in the art. Antithrombin that is transgenically produced can be produced in the milk of a non-human mammal. An example of such an antithrombin is ATryn®, which can be obtained commercially from GTC Biotherapeutics, Inc. (Framingharn, MA). Such antithrombin can also be produced by methods known to those of skill in the art. Examples of transgenically produced antithrombin and/or methods of transgenic production can be found in U.S. Patent Nos. 5,843,705; 6,441,145; 6,727405; 7,019193; and 7,045,676. Such antithrombin molecules as well as the methods of their production are expressly incorporated herein by reference. The antithrombin can also be in mutated form. Examples of such mutated versions of antithrombin are provided in EP Publication No. EPl 194033 and are expressly incorporated herein by reference. Further, the antithrombin can be functional variants of any of the above examples. Such functional variants include conservatively substituted versions, analogues, mimetics as well as antithrombin molecules with altered glycosylation.

As used herein, a "conservative amino acid substitution" or "conservative substitution" refers to an amino acid substitution in which the substituted amino acid residue is of similar charge as the replaced residue and is of similar or smaller size than the replaced residue. Conservative substitutions of amino acids include substitutions made amongst amino acids within the following groups: (a) the small non-polar amino acids, A, M, I, L₃ and V; (b) the small polar amino acids, G, S, T and C; (c) the amido amino acids, Q and N; (d) the aromatic amino acids, F, Y and W; (e) the basic amino acids, K, R and H; and (f) the acidic amino acids, E and D. Substitutions which are charge neutral and which replace a residue with a smaller residue may also be considered "conservative substitutions" even if the residues are in different groups (e.g., replacement of phenylalanine with the smaller isoleucine). The term "conservative amino acid substitution" also refers to the use of amino acid analogs.

The antithrombin can be administered at a high dose. "High dose" is intended to refer to any dose that when given would be expected to increase the plasma antithrombin level to a level above that of normal plasma. In one embodiment, the dose is a dose that when given intravenously would be expected to increase the plasma antithrombin level to a level above that of normal plasma. In some embodiments, the dose results in or would be expected to result in a plasma level that is at least 2-, 3-, 4-, or 5-fold higher than normal plasma levels. In other embodiments, the dose is at least 100 U/kg, 125 U/kg, 150 U/kg, 200 U/kg, 250 U/kg, 300 U/kg, 350 U/kg, 400 U/kg, 500 U/kg or more. In still other embodiments, the dose is at least 1, 2, 3, 4, 5 U/ml or more. The absolute amount will depend upon a variety of factors (including whether the administration is in conjunction with other methods of treatment, the number of doses and individual patient parameters including age, physical condition, size and weight) and can be determined with routine experimentation. It is preferred, generally, that a maximum dose be used, that is, the highest safe dose according to sound medical judgment.

The methods provided can further include the administration of another therapeutic agent. The other therapeutic agents include any agent that would provide some benefit to a subject that has or is at risk of having any of the diseases, conditions or indications provided herein. The other therapeutic agent can also be used for treatment or prophylaxis. As used herein, "at risk of having" refers to any subject that would be considered to have some increased probability of developing the disease, condition or indication. Conversely, as used herein, "not at risk of having" is intended to refer to any subject that would be considered to not have an increased probability of developing the disease, condition or indication. It is within the ordinary skill of those in the art to be able to determine whether or not a subject is at risk of having a disease, condition or indication.

The other therapeutic agent can be, for example, activated protein C (e.g., recombinant activated protein C (rhAPC), Xigris); thrombomodulin; tissue factor pathway inhibitor (TFPI); anti-TNF receptor antibodies; antibodies that modulate the TNF signalling cascade (e.g., Remicade, Humira, MAKl 95); Enbrel; or antibodies that inhibit immune cells from crossing blood vessel walls to reach various tissues (e.g., Tysabri).

The other therapeutic agent can be an anticoagulant. Anticoagulants include heparin, warfarin, Coumadin, dicumarol. phenprocournon, acenocoumarol, ethyl biscoumacetate and indandione derivatives.

The other therapeutic agent can be an anti-inflammatory agent. Anti-inflammatory agents include Alclofenac; Alclometasone Dipropionate; Algestone Acetonide; Alpha Amylase; Amcinafal; Amcinafide; Amfenac Sodium; Amiprilose Hydrochloride; Anakinra; Anirolac ; Anitrazafen; Apazone; Balsalazide Disodium; Bendazac; Benoxaprofen; Benzydamine Hydrochloride; Bromelains; Broperamole; Budesonide; Carprofen; Cicloprofen; Cintazone; Cliprofen; Clobetasol Propionate; Clobetasone Butyrate; Clopirac; Cloticasone Propionate; Cormethasone Acetate; Cortodoxone; Deflazacort; Desonide; Desoximetasone; Dexamethasone Dipropionate; Diclofenac Potassium; Diclofenac Sodium; Diflorasone Diacetate; Diflumidone Sodium; Diflunisal ; Difluprednate; Diftalone; Dimethyl Sulfoxide; Drocinonide; Endrysone; Enlimomab ; Enolicam Sodium ; Epirizole ; Etodolac; Etofenamate ; Felbinac; Fenamole; Fenbufen; Fenclofenac; Fenclorac; Fendosal; Fenpipalone; Fentiazac; Flazalone; Fluazacort; Flufenarnic Acid; Flumizole; Flunisolide Acetate; Flunixin ; Flunixin Meglumine ; Fluocortin Butyl; Fhiorometholone Acetate; Fluquazone; Flurbiprofen ; Fluretofen; Fluticasone Propionate; Furaprofen; Furobufen; Halcinonide; Halobetasol Propionate; Halopredone Acetate; Ibufenac ; Ibuprofen; Ibuprofen Aluminum; Ibuprofen Piconol; Ilonidap; Indomethacin;; Indomethacin Sodium; Indoprofen ; Indoxole ; Intrazole; Isoflupredone Acetate; Isoxepac; Isoxicam; Ketoprofen; Lofemizole Hydrochloride ; Lornoxicam ; Loteprednol Etabonate; Meclofenamate Sodium; Meclofenamic Acid; Meclorisone Dibutyrate; Mefenamic Acid ; Mesalamine; Meseclazone; Methylprednisolone Suleptanate; Morniflumate; Nabumetone; Naproxen ; Naproxen Sodium; Naproxol ; Nimazone; Olsalazine Sodium; Orgotein ; Orpanoxin; Oxaprozin; Oxyphenbutazone; Paranyline Hydrochloride; Pentosan Polysulfate Sodium; Phenbutazone Sodium Glycerate; Pirfenidone ; Piroxicam; PiroxicamlCinπamate; Piroxicam Olamine; Pirprofen; Prednazate; Prifelone; Prodolic Acid; Proquazone; Proxazole; Proxazole Citrate ; Rimexolone; Romazarit ; Salcolex ; Salnacedin; Salsalate ; Salycilates; Sanguinarium Chloride ; Seclazone ; Sermetacin; Sudoxicam; Sulindac; Suprofen; Talmetacin; Talniflumate; Talosalate ; Tebufelone ; Tenidap; Tenidap Sodium; Tenoxicam; Tesicam; Tesimide; Tetrydamine; Tiopinac; Tixocortol Pivalate; Tolmetin; Tolmetin Sodium; Triclonide; Triflumidate; Zidometacin; Glucocorticoids and Zomepirac Sodium.

Other anti-inflammatory agents include the following aforementioned agents: activated protein C (e.g., recombinant activated protein C (rhAPC), Xigris); thrombomodulin; tissue factor pathway inhibitor (TFPI); anti-TNF receptor antibodies; antibodies that modulate the TNF signalling cascade (e.g., Remicade, Humira, MAK 195); Enbrel; and antibodies that inhibit immune cells from crossing blooά vessel walls to reach various tissues (e.g., Tysabri).

The other therapeutic agent can be an antibacterial agent. Antibacterial agents include Acedapsone; Acetosulfone Sodium; Alamecin; Alexidine; Amdinocillin; Amdinocillin Pivoxil; Amicycline; Amifloxacin; Amifloxacin Mesylate; Amikacin; Amikacin Sulfate; Aminosalicylic acid; Aminosalicylate sodium; Amoxicillin; Amphomycin; Ampicillin; Ampicillin Sodium; Apalcillin Sodium; Apramycin; Aspartocin; Astromicin Sulfate; Avilamycin; Avoparcin; Azithromycin; Azlocillin; Azlocillin Sodium; Bacampicillin Hydrochloride; Bacitracin; Bacitracin Methylene Disalicylate; Bacitracin Zinc; Bambermycins; Benzoylpas Calcium; Berythromycin ; Betamicin Sulfate; Biapenem; Biniramycin; Biphenamine Hydrochloride ; Bispyrithione Magsulfex; Butikacin; Butirosin Sulfate; Capreomycin Sulfate; Carbadox; Carbenicillin Disodium; Carbenicillin Indanyl Sodium; Carbenicillin Phenyl Sodium; Carbenicillin Potassium; Carumonam Sodium; Cefaclor; Cefadroxil; Cefamandole; Cefamandole-Nafate; Cefamandole Sodium; Cefaparole; Cefatrizine; Cefazaflur Sodium; Cefazolin; Cefazolin Sodium; Cefbuperazone; Cefdinir; Cefepime; Cefepime Hydrochloride; Cefetecol; Cefϊxime; Cefmenoxime Hydrochloride; Cefmetazole; Cefrnetazole Sodium; Cefonicid Monosodium; Cefonicid Sodium; Cefoperazone Sodium; Ceforanide; Cefotaxime Sodium; Cefotetan; Cefotetan Disodium; Cefotiam Hydrochloride; Cefoxitin; Cefoxitin Sodium; Gefpimizole; Cefpimizole Sodium; Cefpiramide; Ceφiramide Sodium; Cefpirome Sulfate; Gefpodoxime Proxetil; Cefprozil; Cefroxadine; Cefsulodin Sodium; Ceftazidime; Ceftibuten; Ceftizoxime Sodium; Ceftriaxone Sodium; Cefuroxime;. Cefuroxhne Axetil; Cefuroxime Pivoxetil; Cefuroxime Sodium; Cephacetrile Sodium; Cephalexin; Cephalexin Hydrochloride; Cephalόglycin; Cephaloridine; Cephalothin Sodium; Cephapirin Sodium; Cephradine; Cetocycline Hydrochloride; Cetophenicol; Chloramphenicol; Chloramphenicol Palmitate; Chloramphenicol Pantothenate Complex ; Chloramphenicol Sodium Succinate; Chlorhexidine Phosphanilate; Chloroxylenol; Chlortetracycline Bisulfate; Chlortetracycline Hydrochloride; Cinoxacin; Ciprofloxacin;

Ciprofloxacin Hydrochloride; Cirolemycin ; Clarithromycin; Clinafloxacin Hydrochloride; Clindamycin; Clindamycin Hydrochloride; Clindamycin Palmitate Hydrochloride; Clindamycin Phosphate; Clofazimine ; Cloxacillin Benzathine; Cloxacillin Sodium; Cloxyquin; Colistimethate Sodium; Colistin Sulfate; Coumermycin; Coumermycin Sodium; Cyclacillin; Cycloserine; Dalfopristin; Dapsone ; Daptomycin; Demeclocycline; Demeclocycline Hydrochloride; Demecyciine; Denofungin ; Diaveridine; Dicloxacillin; Dicloxacillin Sodium; Dihydrostreptomycin Sulfate; Dipyrithione; Dirithromycin; Doxycycline; Doxycycline Calcium ; Doxycycline Fosfatex; Doxycycline Hyclate; Droxacin Sodium; Enoxacin; Epicillin; Epitetracycline Hydrochloride; Erythromycin; Erythromycin Acistrate; Erythromycin Estolate; Erythromycin Ethylsuccinate; Erythromycin Gluceptate; Erythromycin Lactobionate; Erythromycin Propionate; Erythromycin Stearate; Ethambutol Hydrochloride; Ethionamide; Fleroxacin; Floxacillin; Fludalanine; Flumequine; Fosfomycin; Fosfomycin Tromethamine; Fumoxicillin; Furazolium Chloride; Furazolium Tartrate; Fusidate Sodium; Fusidic Acid; Gentamicin Sulfate; Glόximonam; Gramicidin; Haloprogin; Hetacillin; Hetacillin Potassium; Hexedine; Ibafloxacin; Imipenem; Isoconazole; Isepamicin; Isoniazid; Josamycin; Kanamycin Sulfate; Kitasamycin; Levofuraltadone; Levopropylcillin Potassium; Lexithromycin; Lincomycin; Lincomycin Hydrochloride; Lomefloxacin; Lomefloxacin Hydrochloride; Lomefloxacin Mesylate; Loracarbef; Mafenide; Meclocycline; Meclocycline Sulfosalicylate; Megalomicin Potassium Phosphate; Mequidox; Meropenem; Methacycline; Methacycline Hydrochloride; Methenamine; Methenamine Hippurate; Methenamine Mandelate; Methicillin Sodium; Metioprim; Metronidazole Hydrochloride; Metronidazole Phosphate; Mezlocillin; Mezlocillin Sodium; Minocycline; Minocycline Hydrochloride; Mirincamycin Hydrochloride ; Monensin ; Monensin Sodium ; Nafcillin Sodium; Nalidixate Sodium; Nalidixic Acid; Natamycin; Nebramycin; Neomycin Palmitate; Neomycin Sulfate; Neomycin Undecylenate ; Netilmicin Sulfate;- Neutramycin; Nifuradene; Nifuraldezone; Nifuratel ; Nifuratrone; Nifurdazil; Nifufimide; Nifurpirinol; Nifurquinazol; Nifurthiazole; Nitrocycline; Nitrofurantoin; Nitromide; Norfloxacin; Novobiocin Sodium; Ofloxacin; Ormetoprim; Oxacillin Sodium; Oximonam; Oximonam Sodium; Oxolinic Acid; Oxytetracycline; Oxytetracycline Calcium; Oxytetracycline Hydrochloride; Paldimycin; Parachlorophenol; Paulomycin; Pefloxacin; Pefloxacin Mesylate; Penamecillin; Penicillin G Benzathine; Penicillin G Potassium; Penicillin G Procaine; Penicillin G Sodium; Penicillin V; Penicillin V Benzathine; Penicillin V Hydrabamine; Penicillin V Potassium; Pentizidone Sodium; Phenyl Aminosalicylate; Piperacillin Sodium;! Pirbenicillin Sodium; Piridicillin Sodium; Pirlimycin Hydrochloride; Pivampicillin Hydrochloride; Pivampicillin Pamoate; Pivampicillin Probenate; Polymyxin B Sulfate; Porfϊromycin ; Propikacin; Pyrazinamide; Pyrithione Zinc; Quindecamine Acetate; Quinupristin; Racephenicol; Ramoplanin; Ranimycin; Relomycin; Repromicin; Rifabutin; Rifametane; Rifamexil; Rifamide; Rifampin; Rifapentine; Rifaximin; Rolitetracycline; Rolitetracyclihe Nitrate; Rosaramicin; Rosaramicin Butyrate; Rosaramicin Propionate; Rosaramicin Sodium Phosphate; Rosaramicin Stearate; Rosoxacin; Roxarsone; Roxithromycin; Sancycline; Sanfetrinem Sodium; Sarmoxicillin; Sarpicillin; Scopafungin ; Sisomicin; Sisomicin Sulfate; Sparfloxacin; Spectinomycin Hydrochloride; Spiramycin; Stallimycin Hydrochloride; Steffimycin; Streptomycin Sulfate; Streptonicozid; Sulfabenz ; Sulfabenzamide; Sulfacetamide; Sulfacetamide Sodium; Sulfacytine; Sulfadiazine; Sulfadiazine Sodium; Sulfadoxine; Sulfalene; Sulfamerazine; Sulfameter; Sulfamethazine; Sulfamethizole; Sulfamethoxazole; Sulfamonomethoxine; Sulfamoxole; Sulfanilate Zinc; Sulfanitran ; Sulfasalazine; Sulfasomizole; Sulfathiazole; Sulfazamet; Sulfisoxazole; Sulfisoxazole Acetyl; Sulfisbxazole Diolamine; Sulfomyxin; Sulopenem; Sultamicillin; Suncillin Sodium; Talampicillin Hydrochloride; Teicoplanin; Temafloxacin Hydrochloride; Temocillin; Tetracycline;^' Tetracycline Hydrochloride ; Tetracycline Phosphate Complex; Tetroxoprim; Thiamphenicol; Thiphencillin Potassium; Ticarcillin Cresyl Sodium; Ticarcillin Disodium; Ticarcillin Monosodium; Ticlatone; Tiodonium Chloride; Tobramycin; Tobramycin Sulfate; Tosufloxacin; Trimethoprim; Trimethoprim Sulfate; Trisulfapyrimidines; Troleandorήycin; Trospectomycin Sulfate; Tyrothricin; Vancomycin; Vancomycin Hydrochloride; Virginiamycin and Zorbamycin.

The other therapeutic agent can be an antimicrobial agent. Antimicrobial agents include Aztreonam; Chlorhexidine Gluconate; Imidurea; Lycetamine; Nibroxane; Pirazmonam Sodium; Propionic Acid ; Pyrithione Sodium; Sanguinarium Chloride ; and Tigemonam Dicholine.

The other therapeutic agent can be an antiviral agent. Antiviral agents include nucleotide analogues which include acyclovir, gancyclovir, idoxuridine, ribavirin, dideoxyinosine, dideoxycytidine and zidovudine (azidothymidine).

The other therapeutic agent can be an anti-infective, which include Difloxacin Hydrochloride ; Lauryl Isoquinolinium Bromide; Moxalactam Disodium; Ornidazole; Pentisomicin; Sarafloxacin Hydrochloride; Protease inhibitors of HIV and other retroviruses; Integrase Inhibitors of HIV and other retroviruses; Cefaclor (Ceclor); Acyclovir (Zovirax); Norfloxacin (Noroxin); Cefoxitin (Mefoxin); Cefuroxirne axetil (Ceftin); and Ciprofloxacin (Cipro).

The other therapeutic agent can be an anti-cancer agent. Anti-cancer agents include Acivicin; Aclarubicin; Acodazole Hydrochloride; Acronine; Adriamycin; Adozelesin; Aldesleukin; Altretamine; Ambomycin; Ametantrone Acetate; Aminoglutethimide; Amsacrine; Anastrozole; Anthramycin; Asparaginase; Asperlin; Azacitidine; Azetepa; Azotomycin; Batimastat; Benzodepa; Bicalutamide; Bisantrene Hydrochloride; Bisnaflde Dimesylate; Bizelesin; Bleomycin Sulfate; Brequinar Sodium; Bropirimine; Busulfan; Cactinomycin; Calusterone; Caracemide; Carbetimer; Carboplatin; Carmustine; Carubicin Hydrochloride; Carzelesin; Cedefingol; Chlorambucil; Cirolemycin; Cisplatin; Cladribine; Crisnatol Mesylate; Cyclophosphamide; Cytarabine; Dacarbazine; Dactinomycin; Daύnorubicin Hydrochloride; Decitabine; Dexormaplatin; Dezaguanine; Dezaguanine Mesylate; Diaziquone; Docetaxel; Doxorubicin; Doxorubicin Hydrochloride; Droloxifene; Droloxifene Citrate; Dromostanolone Propionate; Duazomycin; Edatrexate; Efiornithine Hydrochloride; Elsamitrucin; Enloplatin; Enpromate; Epipropidine; Epirubicin Hydrochloride; Erbulozole; Esorubicin Hydrochloride; Estramustine; Estramustine Phosphate Sodium;. Etanidazole; Etoposide; Etoposide Phosphate; Etoprine; Fadrozole Hydrochloride; Fazarabine; Fenretinide; Floxuridine; Fludarabine Phosphate; Fluorouracil; Flurocitabine; Fosquidone; Fostriecin Sodium; Gemcitabine; Gemcitabine Hydrochloride; Hydroxyurea; Idarubicin Hydrochloride; Ifosfamide; Ilmofosine; Interferon Alfa-2a; Interferon Alfa-2b; Interferon Alfa-nl ; Interferon Alfa-n3; Interferon Beta- I a; Interferon Gamma- 1 b; Iproplatin; Irinotecan Hydrochloride; Laπreotide Acetate; Letrozole; Leuprolide Acetate; Liarozole Hydrochloride; Lometrexol Sodium; Lomustiήe; Losoxantrone Hydrochloride; Masoprocol; Maytansine; Mechlorethamine Hydrochloride; Megestrol Acetate; Melengestrol Acetate; Melphalan; Menogaril; Mercaptopurine; Methotrexate; Methotrexate Sodium; Metoprine; Meturedepa;. Mitindomide; Mitocarcin; Mitocromin; Mitogillin; Mitomalcin; Mitomycin; Mitosper; Mitotane; Mitoxantrone Hydrochloride; Mycophenolic Acid; Nocodazole; Nogalamycin; Ormaplatin; Oxisuran; Paclitaxel; Pegaspargase; Peliomycin; Pentamustine; Peplomycin Sulfate; Perfosfamide; Pipobroman; Piposulfan; Piroxantrone Hydrochloride; Plicamycin; Plomestane; Porfϊmer Sodiuim; Porfϊromycin; Prednimustine; Procarbazine Hydrochloride; Puromyciή; Puromycin Hydrochloride; Pyrazofurin; Riboprine; Rogletimide; Safingol; Safingol Hydrochloride; Semustiήe; Simtrazene; Sparfosate Sodium; Sparsomycin; Spirogermanium Hydrochloride; Spiromustine; Spiroplatin; Streptonigrin; Streptozocin; Sulofenur; Talisomycin; Tecogalan Sodium; Tegafiir; Teloxantrone Hydrochloride; Teraoporfin; Tenϊposide; Teroxirone; Testolactone; Thiamiprine; Thioguanine; Thiotepa; Tiazofurin; Tirapazamine; Topotecan Hydrochloride; Toremifene Citrate; Trestolone Acetate; Triciribine Phosphate; Trimetrexate; Trimetrexate Glucuronate; Triptorelin; Tubulozole Hydrochloride; Uracil Mustard; Uredepa; Vapreotide; Verteporfin; Vinblastine Sulfate; Vincristine Sulfate; Vindesine; Vindesine Sulfate; Vinepidine Sulfate; Vinglycinate Sulfate; Vinleurosine Sulfate; Vinorelbine Tartrate; Vinrosidine Sulfate; Vinzolidine Sulfate; Vorozole; Zeniplatin; Zinostatin; and Zorubicin Hydrochloride.

The other therapeutic agent can be an analgesic. Analgesics include Acetaminophen; AIfentanil Hydrochloride; Aminobenzoate Potassium; Aminobenzoate Sodium; Anidoxime; Anileridine; Anileridine Hydrochloride; Anilopam Hydrochloride; Anirolac; Antipyrine; Aspirin; Benoxaprofen; Benzydamine Hydrochloride; Bicifadine Hydrochloride; Brifentanil Hydrochloride; Bromadoline Maleate; Bromfenac Sodium; Buprenorphine Hydrochloride; Butacetin; Butixirate; Butorphanol; Butoφhanol Tartrate; Carbamazepine; Carbaspirin Calcium; Carbiphene Hydrochloride; Carfentanil Citrate; Ciprefadol Succinate; Ciramadol; Ciramadol Hydrochloride; Clonixeril; Clonixin; Codeine ; Codeine Phosphate; Codeine Sulfate; Conorphone Hydrochloride; Cyclazocine; Dexόxadrol Hydrochloride; Dexpemedolac; Dezocine; Diflunisal; Dihydrocodeine Bitartrate; Dimefadane; Dipyrone; Doxpicomine Hydrochloride; Drinidene; Enadoline Hydrochloride; Epirizole; Ergotamine Tartrate; Ethoxazene Hydrochloride; Etofenamate; Eugenol; Fenoprofen; Fenoprofen Calcium; Fentanyl Citrate; Floctafenine; Flufenisal; Flunixin; Flunixin Meglumine; Flupirtine Maleate; Fluproquazone; Fluradoline Hydrochloride; Flurbiprofen ; Hydromorphone Hydrochloride; Ibufenac; Indoprofen; Ketazocine; Ketorfanol; Ketorolac Tromethamine; Letimide Hydrochloride; Levomethadyl Acetate; Levomethadyl Acetate Hydrochloride; Levonantradol Hydrochloride; Levorphanol Tartrate; Lofemizole Hydrochloride; Lofentanil Oxalate; Lorcinadol; Lornoxicam; Magnesium Salicylate; Mefenamic Acid; Menabitan Hydrochloride; Meperidine Hydrochloride; Meptazinol Hydrochloride; Methadone Hydrochloride; Methadyl Acetate; Methopholine; Methptrimeprazine; Metkephamid Acetate; Mimbane Hydrochloride; Mirfentanil Hydrochloride; Molinazone; Morphine Sulfate; Moxazocine; Nabitan Hydrochloride; Nalbuphine Hydrochloride; Nalmexone Hydrochloride ; Namoxyrate; Nantradol Hydrochloride; Naproxen ; Naproxen Sodium ; Naproxol; Nefopam Hydrochloride; Nexeridine Hydrochloride; Noracymethadol Hydrochloride; Ocfentanil Hydrochloride; Octazamide; Olvanil; Oxetorone Fumarate; Oxycodone; Oxycodone Hydrochloride; Oxycodone Terephthalate; Oxymorphone Hydrochloride; Pemedolac; Pentamorphone; Pentazocine; Pentazocine Hydrochloride; Pentazocine Lactate; Phenazopyridine Hydrochloride; Phenyramidol Hydrochloride; Picenadol Hydrochloride; Pinadoline; Pirfenidone; Piroxicam Olamine; Pravadoline Maleate; Prodilidine Hydrochloride; Profadol Hydrochloride; Propiram Fumarate; Propoxyphene Hydrochloride; Propoxyphene Napsylate; Proxazole ; Proxazole Citrate ; Proxorphan Tartrate; Pyrroliphene Hydrochloride; Remifentanil Hydrochloride; Salcolex ; Salethamide Maleate; Salicylamide; Salicylate Meglumine; Salsalate ; Sodium Salicylate; Spiradoline Mesylate; Sufentanil; Sufentanil Citrate; Talmetacin ; Talnifiumate ; Talosalate ; Tazadolene Succinate; Tebufelone ; Tetrydamine ; Tifurac Sodium; Tilidine Hydrochloride; Tiopinac; Tonazocine Mesylate; Tramadol Hydrochloride; Trefentanil Hydrochloride; Trolamine; Veradoline Hydrochloride; Verilopam Hydrochloride; Volazocine; Xorphanol Mesylate; Xylazine Hydrochloride; Zenazocine Mesylate; Zomepirac Sodium; and Zucapsaicin.

Other therapeutic agents also include agents for treating sepsis, DIC, peritonitis or ovarian cancer.

Agents for treating sepsis include antibiotics, vasopressors (e.g., dopamine, dobutamine, norepinephrine (noradrenaline), epinephrine (adrenaline), vasopressin, dopexamine, phenylephrine, phosphodiesteradse inhibitors, etc.), activated protein C, etc.

Agents for treating DIC include plasma or blood (e.g., by transfusion), a fibrinogen replacements and anti-thrombotics.

Agents for treating peritonitis include antibiotics and morphine.

Agents for treating ovarian cancer include cisplatin, carboplatin, paclitaxel, etc. In some embodiments the subject is treated with a combination of paclitaxel and a platinum- based drug. In still other embodiments, the subject is treated with an agent, surgery, radiation or a combination thereof.

Compositions, therefore, are also provided that include other therapeutic agents. The compositions can also include antithrombin, in some embodiments.

Effective amounts of the compositions provided (or combinations thereof) are administered to subjects in need of such treatment. Effective amounts are those amounts (of one or a combination of therapeutics) which will result in a desired improvement in the condition, disease or disorder or symptoms of the condition, disease or disorder. Effective amounts also include those amounts that lead to the desired endpoint. Such amounts can be determined with no more than routine experimentation. The effective amounts can be the high doses provided above. In general, when administered for therapeutic purposes, the formulations of the invention are applied in pharmaceutically acceptable solutions. Such preparations may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, adjuvants and optionally other therapeutic ingredients.

The compositions of the invention may be administered per sβ (neat) or in the form of a pharmaceutically acceptable salt. When used in medicine the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically acceptable salts thereof and are not excluded from the scope of the invention. Such pharmacologically and pharmacjeutically acceptable salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulphuric, nitric, phosphoric, maleic, acetic, salicylic, p-toluene sulphonic, tartaric, citric, methane sulphonic, formic, malonic, succinic, naphthalene-2-sulphonic, and benzene sulphonic. Also, pharmaceutically acceptable salts can "be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts of the carboxylic acid group.

Suitable buffering agents include: acetic acid and a salt (1-2% W/V); citric acid and a salt (1-3% W/V); boric acid and a salt (0.5-2.5% W/V); and phosphoric acid and a salt (0.8-2% W/V). Suitable preservatives include benzalkonium chloride (0.003-0.03% W/V); chlorobutanol (0.3-0.9% W/V); parabens (0.01-0.25% W/V) and thimerosal (0.004-0.02% W/V).

The present invention provides compositions, for medical use, which comprise one or more agents together with one or more pharmaceutically acceptable carriers and optionally other therapeutic ingredients. The pharmaceutical compositions can also, in some embodiments, include one or more additional therapeutic agents. The term "pharmaceutically-acceptable carrier" as used herein, means one or more compatible solid or liquid filler, diluents or encapsulating substances which are suitable for administration to a human or other animal. In the present invention, the term "carrier" denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application. The components of the pharmaceutical compositions also are capable of being commingled with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficiency. The pharmaceutically acceptable carrier can, in some embodiments, be sterile.

A variety of other administration routes are also available, such as, for example, for the administration of one or more therapeutic agents in addition to the antithrombin. The particular mode selected will depend, of course, upon the particular active agent(s) selected, the desired results, the particular condition being treated and the dosage required for therapeutic efficacy. The methods of this invention, generally speaking, may be practiced using any mode of administration that is medically acceptable, meaning any mode that produces effective levels of a desired response without causing clinically unacceptable adverse effects. One mode of administration is the parenteral route. The term "parenteral" includes subcutaneous injections, intravenous, intramuscular, intraperitoneal, intrasternal injection or infusion techniques. Other modes of administration include oral, mucosal, rectal, vaginal, sublingual, intranasal, intratracheal, inhalation, ocular, transdermal, etc.

The compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g. , in ampoules or in multi-dose containers, with an added preservative. In some embodiments the compounds provided are administered by infusion pump. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the, compounds to allow for the preparation of highly concentrated solutions.

Alternatively, the active compounds may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

In addition to the formulations described previously, the compounds may also be formulated as a depot preparation. Such long acting formulations may/ be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

Suitable liquid pharmaceutical preparation forms are, for example, aqueous or saline solutions, microencapsulated, encochleated, coated onto microscopic gold particles, contained in liposomes, etc. The pharmaceutical compositions also include granules, (micro)capsules, syrups, emulsions, suspensions or preparations with protracted release of active compounds, in whose preparation excipients and additives and/or auxiliaries such as disintegrants, binders, coating agents, swelling agents, lubricants or solubilizers are customarily used as described above. The pharmaceutical compositions are suitable for use in a variety of drug delivery systems. For a brief review of methods for drug delivery, see Langer, Science 249:1527-1533, 1990 and Langer and Tirrell, Nature, 2004 Apr 1; 428(6982): 487-92, which are incorporated herein by reference.

The compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy.

Other delivery systems can include time-release, delayed release or sustained release delivery systems. Such systems can avoid repeated administrations of the compounds of the invention, increasing convenience to the subject and the physician. Many types of release delivery systems are available and known to those of ordinary skill in the art. They include polymer based systems such as polylactic and polyglycolic acid, polyanhydrides and polycaprolactone; nonpolymer systems that are lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono-, di -and triglycerides; hydrogel release systems; silastic systems; peptide based systems and the like. Specific examples include, but are not limited to: (a) erosional systems in which the agent is contained in a form within a matrix, found in U.S. Patent Nos. 4,452,775 (Kent); 4,667,014 (Nestor et al.); and 4,748,034 and 5,239,660 (Leonard) and (b) diffusional systems in which an agent permeates at a controlled rate through a polymer, found in U.S. Patent Nos. 3,832,253 (Higuchi et al.) and 3,854,480 (Zaffaroni). In addition, a pump-based hardware delivery system can be used, some of which are adapted for implantation.

Controlled release can also be achieved with appropriate excipient materials that are biocompatible and biodegradable. These polymeric materials which effect slow release may be any suitable polymeric material for generating particles, including, but not limited to, nonbioerodable/non-biόdegradable and bioerodable/biodegradable polymers. Such polymers have been described in great detail in the prior art. They, include, but are not limited to: polyamides, polycarbonates, polyalkylenes, polyalkylene glycols, polyalkylene oxides, polyalkylene terepthalates, polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, polyvinyl halides, polyvinylpyrrolidone, polyglycolides, polysiloxanes, polyurethanes and copolymers thereof, alkyl cellulose, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitro celluloses, polymers of acrylic and methacrylic esters, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxy-propyl methyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxylethyl cellulose, cellulose triacetate, cellulose sulfate sodium salt, poly (methyl methacrylate), poly(ethylmethacrylate), poly(butylmethacrylate), poly(isobutylmethacrylate), poly(hexlmethacrylate), poly(isodecylmethacrylate), poly(lauryl methacrylate), poly (phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acirylate), poly(isobutyl acrylate), poly(octadecyl acrylate), polyethylene, polypropylene poly(ethylene glycol), poly(ethylene oxide), poly(ethylene terephthalate), poly(vinyl alcohols), poly( vinyl acetate, poly vinyl chloride polystyrene, polyvinylpryrrolidone, hyaluronic¹ acid, and chondroitin sulfate. In one embodiment the slow release polymer is a block copolymer, such as poly(ethylene glycol) (PEG)/poly(lactic-co-glycolic acid) (PLGA) block copolymer.

Examples of preferred non-biodegradable polymers include ethylene vinyl acetate, poly(meth) acrylic acid, polyamides, copolymers and mixtures thereof.

Examples of preferred biodegradable polymers include synthetic polymers such as polymers of lactic acid and glycolic acid, polyanhydrides, poly(ortho)esters, polyurethanes, poly(butic acid), poly(valeric acid), poly(caprolactone), poly(hydroxybutyrate), poly(lactide- co-glycolide) and poly(lactide-co-caρrolactone), and natural polymers such as alginate and other polysaccharides including dextran and cellulose, collagen, chemical derivatives thereof (substitutions, additions of chemical groups, for example, alkyl, alkylene, hydroxylations, oxidations, and other modifications routinely made by those skilled in the art), albumin and other hydrophilic proteins, zein and other prolamines and hydrophobic proteins, copolymers and mixtures thereof. In general, these materials, degrade either by enzymatic hydrolysis or exposure to water in vivo, by surface or bulk erosion. Th'e foregoing materials may be used alone, as physical mixtures (blends), or as co-polymers. The most preferred polymers are polyesters, polyanhydrides, polystyrenes and blends thereof.

A subject is any human or non-human vertebrate, e.g., dog, cat, horse, cow, monkey, pig, mouse, rat.

The present invention is further illustrated by the following Examples, which in no way should be construed as further limiting. The entire contents of all of the references (including literature references, issued patents, published patent applications, and co-pending patent applications) cited throughout this application are hereby expressly incorporated by reference. EXAMPLES

Materials and Methods

Experimental Design

Male specific pathogen-free C57BL/6 mice (25-28 g, n = 114; Harlan Horst, the Netherlands) were acclimated for 1 week and housed in filter-top cages in a temperature- controlled room (22-24 °C), with an alternating 12 h-light/dark cycle. The mice had standard mouse chow and water ad libitum throughout the experiment. The time line of the experiment with surgical and intervention treatment and sampling is depicted in Fig. 1.

Antithrombin

Recombinant human AT (ATryn®; GTC Biotherapeutics, Framingham, MA) was used in this experiment [22]. Lyophilized ATryn® was'reconstituted with sterile water for injections to a concentration of 50 mg/mL (350 U/ml) according to the manufacturer's descriptions. Further dilutions (up to 3.0 U/mL) were made with sterile saline (0.9% NaCl, Fresenius Kabi's-Hertogenbosch, the Netherlands). A dose-finding study with doses between 3 and 12 U/mL rhAT demonstrated that all doses reduced aerobe and anaerobe bacterial load significantly. Intra-abdominal leukocyte counts were reduced in the highest (12 U/mL) and lowest (3 U/mL) dose groups. Abdominal keratinocyte-derived chemokine (KC) concentrations were most reduced in the 3 and 6 U/mL groups, but not in the 12 U/mL group, which also showed slightly higher mortality (two of six) as compared with the 3 and 6 U/mL groups (both none of six). No signs of major bleeding could be detected at necropsy. Therefore, a dose of 3 U/mL was chosen, which is approximately 30 times greater than the recommended human i.v. dose.

Operative Procedures

A cecal ligation and puncture (CLP) model was used as described previously to induce a standardized sublethal peritonitis [23]. Briefly, after induction of anesthesia with 2- 3% isoflurane (Florene, Abott Laboratories, Queensborough, UK)/N2O:O2 (1:1 v/v), the cecum was ligated just distal to the ileocecal junction and punctured through-and-through with a 23 G needle. Twenty-four hours after the index operation, mice were reanesthetized for relaparotomy and therapeutic peritoneal lavage was performed by rinsing the abdominal cavity three times with 2.0 mL rhAT (3 U/mL). Fluid from the first lavage was collected for analysis. Control mice received saline lavage (3 x 2.0 mL). The sham group received a sham operation: cecal manipulations without ligation or puncture. Fluid resuscitation was given (1 mL saline twice daily) until the mice were killed. For survival analysis, the observation period was 96 h (n = 22/group) (Fig. 1). All procedures were performed in series, with at least two different treatment strategies (AT, saline and sham) in each series. Treatment strategy was blinded to animal caretakers.

Sample Collection

After 48, 72 and 96 h, respectively, groups of mice were randomly killed for sample harvesting (n = 6/group/time point). Diagnostic lavage was performed by injecting 2 mL sterile saline i.p., and peritoneal lavage fluid (PLF) was subsequently collected in sterile tubes (Sarstedt, Niimbrecht, Germany). Blood was drawn from the inferior caval vein, 0.109 m sodium citrate (1 :10 v/v) was added to prevent clotting, and centrifuged (1200 x g, 10 min, 4 °C). Organs (liver, lung and peritoneum) were removed immediately after blood was drawn. Lungs were homogenized in sterile saline (200 mg/mL) using an ultrathorax (Heidolph, Schwabach, Germany) [23].

Assessment of Coagulation and Fibrinolysis

AT activity was measured in PLF, plasma and lung homogenates (Berichrom®; Dade Behring, Deerfield, IL, USA). One unit was defined as .activity of 1 mL normal plasma pool (IU/mL). The detection limit was 0.12 IU/mL. Clotting time was determined in PLF and lung homogenates using a standard procoagulant assay as described elsewhere [24]. Briefly, 50 μL PLF or (diluted) lung homogenate was added to 50 μL mouse plasma (Sigma, St. Louis, MO, USA) and incubated for 2 min at 37 °C. Calcium chloride (50 μL, 25 mm) was added and clotting time was measured using a KC-10 coagulometer (Amelung GmbH, Lemego, Germany). Brain homogenate was used as standard positive control. The cut-off time for clotting was 500 s. TAT complexes and D-dimer concentrations were measured in plasma, PLF and lung homogenate by ELISA (Enzygno'st®; Dade Behring and Asserachrom, Diagnostica Stago, Asniere, France, respectively) according to manufacturers' descriptions (detection limits 2 μg Ll and 5 μg Ll, respectively). Fibrinolysis was assessed by active tissue-type plasminogen activator (t-PA) and active plasminogen activator inhibitor- 1 (PAI-I) ELISA according to the manufacturer's instructions. The detection limit was 0.05 ng/mL for both t-PA and PAI-I (Molecular Innovations®; Southfield, MI, USA). Assessment of Inflammatory Response and Histopathological Examination

Just after collection, leukocyte count was determined in PLF using Turk dye and a Btirker counting frame. To assess pulmonary neutrophil count, myeloperoxidase (MPO, enzyme in granulocytes of neutrophils) activity in lung homogenates was measured as described previously [23]. Briefly, lung homogenates were dissolved in assay mix of a sodium-ethylenediaminetetraacetic acid buffer containing hexadecyl-trimethyl-ammonium- bromide and TMB. After incubation, H2O2 was added, and the reaction was stopped with 0.2 m glacial acetic acid. Color intensity was determined by photospectrometer at 655 urn. One unit (U) is defined as one OD change per minute. Results were expressed as units per milligram tissue (wet-weight).

Interleukin (IL)-Iβ, IL-6, tumor necrosis factor-α (TNF-α) and KC concentrations were measured in PLF, plasma and lung homogenates by multiplex system (R&D Systems, Abingdon, UK) according to the manufacturer's descriptions (detection limits 1.2, 9.0, 6.0 and 12 pg/mL, respectively).

Parts of liver, lung and peritoneum were immediately fixed in 4% buffered formaldehyde after collection, and routinely processed for hematoxylin and eosin (H&E) staining in paraffin sections. Sections of all organs were blindly scored by two independent observers. The liver was scored for thrombus formation, steatosis, portal inflammation, parenchymal inflammation and necrosis. The lung was scored for pleuritis, thrombus formation, edema, thickened septa and inflammation. The peritoneum was evaluated for inflammation and fibrin depositions. Each parameter was graded on a scale of 0-4, with 0 meaning 'absent', 1 meaning 'occasional', 2 meaning 'mild', 3 meaning 'moderate' and 4 meaning 'severe'. The total organ injury score was expressed as the sum of scores for all parameters per organ [25,26].

Quantification of Intra-abdominal Bacterial Load

Aerobe and anaerobe colony forming unit (CFU) counts were determined in PLF. Serial log dilutions, using sterile saline, were plated on blood— agar plates and incubated at 37 °C. For anaerobe cultures, a GasPack® (BBL₃ Kansas City, MO) was used. CFUs were counted after 1 day (aerobic conditions) and 2 days (anaerobic conditions). Quantitative cultures are expressed as CFU per milliliter PLF. Data Analysis

Statistical Package for the Social Sciences (SPSS 12.0; SPSS Inc, Chicago, IL) was used for data analysis. Data are expressed as mean ± SEM, unless otherwise indicated. For time series, ANOVA for repeated measurements was performed followed by an LSD post hoc test. Kaplan-Meier survival curves were compared using a LogRank test. Significance was assumed when P < 0.05.

Results

rhAT Recovery

Abdominal AT activity levels were 3.1—3.6 IU/mL immediately after rhAT lavage, whereas AT activity was below detection limit in CLP-saline and sham groups. After 48 h (i.e., 24 h after lavage) and onward, intra-abdominal AT activities were below detection levels in the AT lavage group, similar to CLP-saline and sham groups. Plasma AT activity was reduced in both CLP groups (0.8—1.0 IU/mL) as compared with sham mice (1.2—1.4 IU/mL; P < 0.05). Between saline and AT lavage CLP jgroups, no significant differences in plasma AT activity were observed. In lung homogenates, AT activities were below the . detection limit at all time points in all groups.

Coagulation Activation and D-dimers

At each operative procedure, all animals were checked for intra-abdominal bleedings. No major bleedings (e.g., > 0.5 mL of blood intra-abdominally) were observed. Intraabdominal clotting time was prolonged in both CLP groups (P < 0.05 vs. sham; Fig. 2), with extremely prolonged clotting times immediately after AT lavage throughout the rest of the experiment as compared with saline lavage (P < 0.01). Pulmonary clotting time was also prolonged in CLP groups compared with sham groups (P < 0.05). However, AT lavage reduced pulmonary clotting times (P < 0.05 vs. CLP-saline) to normal values (P = NS vs. sham). Abdominal TAT complexes were highly elevated after CLP (15-70 μg/mL) compared with the sham group that had very low TAT complexes (< 2 μg/mL; P < 0.05 sham vs. CLP). The CLP-AT group had reduced abdominal TAT levels compared with the CLP- saline group (P < 0.05, Fig. 3). In systemic and pulmonary compartments, concentrations of TAT complexes varied between 9-17 μg Ll and 2-4 μg Ll, respectively, similar between groups. Concentrations of both fibrinolysis markers, t-PA and PAI-I, were significantly increased after CLP compared with sham (P < 0.05). In CLP (irrespective of treatment protocol), abdominal t-PA concentrations were between 0.07 and 0.38 ng/mL, plasma t-PA concentrations were between 0.25 and 1.4 ng/mL, and pulmonary t-PA concentrations were between 3.6 and 64.5 ng/mL. In particular, rhAT-treated animals had increasing pulmonary t-PA levels, whereas saline controls had decreasing t-PA levels (P < 0.01 rhAT vs. saline). In sham controls, t-PA concentration was 0.1 ng/mL in the abdominal compartment and 0.4 ng/mL in the systemic (plasma) compartment. In CLP lungs, PAI-I concentrations were between 20 and 120 ng/mL, which is above sham controls (approximately 20 ng/mL). Reduction of the t-PA/PAI-1 ratio indicated inhibition of fibrinolysis. rhAT lavage significantly decreased the t-PA/PAI-1 ratio in the abdominal compartment (0.007 ± 0.006 vs. 0.035 ± 0.007; P < 0.01 vs. CLP-saline). In the systemic compartment, the t-PA/PAI-1 ratio was also reduced (0.002 ± 0.002 vs. 0.041 ± 0.007; P < 0.01). The pulmonary t- PA/PAI-1 ratio was unaffected (6.8 ± 2.0 vs. 4.6 ± 1.9; P = NS).

D-dimer levels, reflecting both fibrin formation and degradation, were elevated after CLP at all time points in all groups in all compartments (P < 0.01 vs. sham). After CLP, animals in the AT group had significantly reduced D-dimer concentrations in plasma and lungs as compared with saline-treated mice (P < 0.05 and P < 0.01, respectively, Fig. 4). In PLF, the difference in D-dimer levels between CLP-AT and CLP-saline failed to reach statistical significance because, after a nadir at 72 h, levels of D-dimer started to rise again in the AT group.

Inflammatory Responses

Both cytokine responses and cellular inflammatory responses were assessed in the abdominal, systemic and pulmonary compartments (Fig. 5A-F). Cytokine production was increased after CLP and peritoneal lavage compared with sham-operated controls, which had very low or undetectable cytokine levels (P < 0.01). Abdominal production of IL-6 and KC was significantly reduced after rhAT lavage as compared with saline lavage (P < 0.01 and P < 0.05, respectively, Fig. 5A,B); IL-β production was also decreased, with peak levels being reduced from 27÷0 ± 6.2 pg/mL to 2.5 ± 2.4 pg/mL (P = 0.01). Intra-abdominal TNF-α production was not affected by rhAT lavage. In plasma; IL-6 production was significantly reduced from 1620 ± 297 pg/mL to 580 ± 192 pg/mL (P < 0.05 vs. saline lavage) and KC production from 1390 ± 80 pg/mL to 11.3 ± 11.2 pg/mL (P < 0.05). Systemic IL-I β and TNF-α concentrations were not affected (P = NS). Although pulmonary IL-6 concentrations were not affected by rhAT lavage, KC concentrations were decreased after rhAT lavage (P < 0.05, Fig. 5D^E). Pulmonary TNF-α was also reduced toy rhAT lavage from 1510 ± 929 pg/mL to 545 ± 312 pg/mL, but not IL-β. Leukocyte counts in PLF and MPO activity in the lungs were measured, to assess influx of intra-abdominal inflammatory cells and pulmonary neutrophils, respectively (Fig. 5C,F). Intra-abdominal leukocyte counts were elevated in both CLP groups as compared with sham (P < 0.01)! rriAT lavage significantly reduced intra-abdominal leukocyte counts, especially after 48 h and onward (P < 0.01 vs. CLP -saline). Similar responses were observed in the lungs. Elevated MPO activity, indicating elevated neutrophil counts, was demonstrated in both CLP groups (P < 0.01 vs. sham). Again, rhAT lavage reduced pulmonary MPO activity as compared with saline lavage (P < 0.05).

Evaluation of H&E slices of peritoneum, liver and lungs demonstrated significantly higher histopathological scores, indicating significant tissue damage in all organs after CLP as compared with sham (P < 0.01; Table 1). AT lavage significantly reduced peritoneal injury (Fig. 6A) and fibrin deposition (Fig. 6C) as welljas liver and pulmonary injury (Table 1), as compared with saline lavage (Fig. 6B and Fig. 6D for peritoneal fibrin deposition; Table 1 for all tissue histopathological scores).

Bacterial Load

High numbers of aerobe and anaerobe bacteria were counted in the abdominal cavity after CLP, whereas no bacteria were cultured in shams (P < 0.01). Aerobe and anaerobe bacterial load were reduced after rhAT lavage (0.4 ± 0.3 *10⁶ CFU/mL and 1.1 ± 0.4 *10⁶ CFU/mL) as compared with saline lavage (19.1 ± 7.9 *,10⁶ CFU/mL and 13.9 ± 7.6 *10⁶ CFU/mL; P < 0.05 and P < 0.01 , respectively).

Survival

This sublethal model of peritonitis showed a survival rate of 62% in the saline-treated group. After rhAT lavage, survival significantly improved to almost 83% (P < 0.03, Fig. 7). Sham mice showed 100% survival.

Discussion

The present study assessed peritoneal rhAT lavage in experimental polymicrobial peritonitis. There is a therapeutic rationale for suppletiόn of AT in sepsis, because AT plasma concentration declines rapidly during severe intra-abdominal infection and is a good predictor of mortality in sepsis patients [27]. Although systemic AT treatment has been very promising in animal sepsis models [2-4], in patients, there is no convincing survival benefit [5]. However, heparin may reduce the rhAT effect, as subgroup analysis of patients without concomitant heparin indeed shows improved 90 days survival after rhAT treatment. Moreover, heparin-treated patients show significantly more bleeding complications [5], Currently, there is no indication for systemic rhAT treatment in sepsis. This is the first study reporting rebalanced coagulation and improved survival; after high-dose intra-abdominal rhAT treatment in peritonitis. Severe bleeding was not observed in this study, nor were serious derangements of the coagulation system (TAT, D-dimer and clotting time).

Peritoneal rhAT lavage efficiently inhibited intra-abdominal coagulation for several days, as was demonstrated by prolonged intra-abdominal clotting times up to 72 h after rhAT lavage (being 96 h after induction of peritonitis by CLP), although AT activity itself was below detection limits. In normal plasma, the half-life time of AT (or elimination time) is 48-56 h, but in severe sepsis plasma half-life time decreases to 8-12 h [28]. Despite this shortened half-life, the consequences of AT administration have been demonstrated over a longer period of time [7,29]. It is most likely that rhAT was consumed by intra-abdominal thrombin, other clotting factors and elastase from neutrophils [I]. Others have demonstrated increased intra-abdominal TAT complexes up to 12 h after AT administration, which reduced later on [29]. Indeed, in this study, intra-abdominal TAT complexes were reduced as of 24 h after rhAT lavage. AT inhibits the TF/FVIIa complex, ;and FDCa, FXa and FXIa, thereby inhibiting thrombin formation [4,9, 1O]₄ Thus, less thrombin' could be complexed into TAT and, in addition, fibrin formation will be reduced, which was demonstrated at histopathology (peritoneum and liver) and by lower D-dimer concentrations [28,30,31]. Furthermore, intraabdominal cytokine production and leukocyte recruitment were both reduced, as were histopathology scores of peritoneum, encompassing the degree of influx of inflammatory cells. This indicates reduced local inflammation [30]. Intra-abdominal fibrinolysis (t- PA/PAI-1 ratio) was even further decreased by rhAT lavage. This is likely to be due to reduced intra-abdominal fibrin deposition, thereby reducing the need for activation of fibrinolysis [32]. Formation of thrombin, a target protease for PAI-I, is reduced due to complexation with AT [28], resulting in augmented PAI-I concentrations and subsequently reduced t-PA concentrations. Thus, peritoneal rhAT lavage reduced intra-abdominal coagulation and inflammation activation.

In this study, local control of coagulation and inflammation consistently reduced systemic and pulmonary coagulation and inflammation [2,33]. In plasma, IL-6 and KC concentration were reduced after rhAT lavage. In the lungs, rhAT lavage resulted in reduced TNF-α and neutrophil chemoattractant KC concentrations, indicating reduced recruitment of neutrophils [34]. This was confirmed by reduced pulmonary neutrophil activity (MPO) and reduced pulmonary injury at histology. Similarly, in previous sepsis studies in animals, systemic rhAT treatment decreases IL-6 and IL-8 plasma concentrations [2], leukocyte adherence and pulmonary neutrophil accumulation and injury [35,36]. AT lavage had no effect on fibrinolysis parameters in circulatory and pulmonary compartments, as has been demonstrated before [37]. This is the first study reporting pulmonary anti-inflammatory effects after intra-abdominal rhAT administration for peritonitis.

Because coagulation and inflammation are cross-linked and AT has both anticoagulant and anti-inflammatory characteristics, it is difficult to distinguish between anticoagulant and anti-inflammatory effects. In this study, anticoagulant properties of AT reduced the generation of thrombin [4,9,10], which plays a role in inflammation by inducing cytokine production and neutrophil attraction [1,16]. AT itself stimulates endothelial cells to produce prostacyclin, which inhibits recruitment and activation of neutrophils as well as synthesis of pro-inflammatory cytokines such as IL-8 arid. IL-6, the latter of which is a well- known activator of coagulation in sepsis [13,38]. Inhibition of fibrin formation by rhAT lavage resulted in enhanced bacterial clearance (CFU count), which correlates with survival [2,7,39]. All and all, this suggests that, in this model, anticoagulant mechanisms are more prominent than anti-inflammatory mechanisms. However, the combination of both results in improved survival.

Local therapy with rhAT did not affect AT activity in plasma or lungs. Others have reported very mildly increased plasma AT activity shortly (hours) after intra-peritoneal administration in experimental pancreatitis [7]. Conversely, i.v. AT increases intraabdominal AT concentrations mildly and shortly (hours) after administration [7,29]. Therefore, massive diffusion of AT from one compartment to another does not occur, i because of the size and structure of the AT molecule [40]. This makes occurrence of severe bleeding complications less likely, and favors local AT treatment.

Clinical, biochemical and histological differences in experimental models of infection may be partially gender-related. Several experimental studies have shown a gender dimorphism of the immune and organ responsiveness in the susceptibility to and morbidity

! from shock, trauma, and sepsis [41-43]. Sex hormone receptors have been identified on various immune cells, suggesting direct effects. Nonetheless, the precise underlying mechanisms for these immunomodulatory effects of sex steroids are still not completely elucidated. In the literature there is still controversy on _[the subject of which patients benefit more or less from anticoagulant therapy in sepsis. There is some evidence that gender does

) not predict 28-day mortality [44].

In conclusion, in this model of experimental polymicrobial peritonitis, intraabdominal administration of a high dose of rhAT by peritoneal lavage reduced intraabdominal coagulation activation and fibrin formation, without severe derangements in coagulation that could lead to serious bleeding complications, and concomitantly improved i bacterial clearance and inflammatory responses. This resulted in reduced inflammation and coagulation activation in the pulmonary compartment, and ultimately in survival.

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The foregoing written specification is considered'to be sufficient to enable one skilled in the art to practice the invention. The present invention is not to be limited in scope by examples provided, since the examples are intended as a single illustration of one aspect of the invention and other functionally equivalent embodiments are within the scope of the invention. Various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. The advantages and objects of the invention are not necessarily encompassed by each embodiment of the invention. The listing of references or discussion of references herein is not intended to be an admission that any of the references is a prior art reference.

What is claimed is:

Claims

1. A method for producing a systemic or pulmonary anticoagulant or antiinflammatory effect in a subject, comprising: administering antithrombin III intraperitoneally in an amount effective to produce the systemic or pulmonary anticoagulant or antiinflammatory effect in the subject, wherein the effect is effective in treating the subject.

2. The method of claim 1 , wherein the antithrombin III is administered by peritoneal lavage or intraperitoneal injection.

3. The method of claim 1, wherein the antithrombin III is administered at a high dose.

4. The method of claim 1, wherein the subject has sepsis.

5. The method of claim 4, wherein the sepsis is severe sepsis.

6. The method of claim 4, wherein the sepsis is septic shock.

0 7. The method of claim I, wherein the subject has an injury local to the peritoneum.

8. The method of claim 1, wherein the subject has disseminated intravascular coagulation (DIC).

5 9. The method of claim 1, wherein the subject does not have pancreatitis.

10. The method of claim 1 , further comprising assessing the effect in the subject.

11. The method of claim 1, wherein the antithrombin III is transgenically produced in the ) milk of a non-human mammal.

12. The method of claim 11, wherein the non-human mammal is a goat.

13. The method of claim 1 , wherein the subject is human.

14. A method of treating sepsis in a subject, comprising: administering antithrombin III intraperitoneally in an amount effective to treat sepsis in the subject.

15. The method of claim 14, wherein the sepsis is severe sepsis.

16. The method of claim 14, wherein the sepsis is septic shock.

17. The method of claim 14, wherein the antithrombin III is administered by peritoneal lavage or intraperitoneal injection.

18. The method of claim 14, wherein the antithrombin III is administered at a high dose.

19. The method of claim 14, wherein the subject has disseminated intravascular coagulation (DIC).

20. The method of claim 14, wherein the subject does not have pancreatitis.

21. The method of claim 14, wherein the antithrombin III is transgenically produced in the milk of a non-human mammal.

22. The method of claim 14, wherein the subject is iuman.

23. A method of treating a subject with an injury local to the peritoneum, comprising: administering antithrombin III by intraperitoneal lavage in an amount effective to treat subject, wherein the amount is effective to treat or .prevent sepsis.

24. The method of claim 23, wherein the antithrombin III is administered at a high dose.

25. The method of claim 23, wherein the subject has DIC.

26. The method of claim 23, wherein the subject does not have pancreatitis.

27. The method of claim 23, wherein the antithrombin III is transgenically produced in the milk of a non-human mammal.

28. The method of claim 23, wherein the subject is human.

29. A method of treating a subject with disseminated intravascular coagulation (DIC), comprising: administering antithrombin III intraperitoneally in an amount effective to treat the subject.

30. The method of claim 29, wherein the antithrombin III is administered by peritoneal lavage or intraperitoneal injection.

31. The method of claim 29, wherein the subject do'es not have pancreatitis. 5

32. The method of claim 29, wherein the antithrombin III is administered at a high dose.

33. The method of claim 29, wherein the antithrombin III is transgenically produced in the milk of a non-human mammal.

)

34. The method of claim 29, wherein the subject is human.