WO2001051067A1

WO2001051067A1 - Multiple inactivated blood factor anticoagulant composition

Info

Publication number: WO2001051067A1
Application number: PCT/US2000/034734
Authority: WO
Inventors: Richard J. Johnson; Roger Lundblad
Original assignee: Baxter International Inc.
Priority date: 2000-01-13
Filing date: 2000-12-20
Publication date: 2001-07-19
Also published as: AU2001257893A1

Abstract

An anticoagulant composition is provided which comprises at least two inactivated blood factors in a pharmaceutically acceptable carrier, such inactivated blood factors being present in a therapeutically effective dosage. Methods for utilizing such anticoagulant compositions comprising providing to a patient a coagulation inhibiting amount thereof are also provided, as are methods for the preparation of such compositions.

Description

MULTIPLE INACTIVATED BLOOD FACTOR ANTICOAGULANT

COMPOSITION

FIELD OF THE INVENTION

This invention relates to novel anticoagulant compositions. More specifically, the invention relates to novel cocktails of inactivated blood clotting factors and methods for rapidly and efficiently forming such compositions.

BACKGROUND OF THE INVENTION

The process of blood coagulation involves a series of enzyme-catalyzed reactions which occur on biological surfaces such as activated platelets. This series of reactions involves an enzymatic cascade in which the product resulting from one reaction serves to initiate the next.

A number of blood factors involved in the clotting process, e.g., Factors II, V, Nil, IX, X, XI, XII, Proteins C and S and fibrinogen, are inactive under normal circumstances, and are present in the blood as proenzymes or zymogens. During blood clot formation, such zymogens are transformed to active proteases, containing active site serine, histidine and aspartic acid residues. The activated form of these factors is indicated by addition of a lower case Aa@ following the name (e.g. Vila or IXa).

Three specific reactions are of particular relevance to the problems and opportunities addressed by applicants. The first is the interaction of factor Vila with tissue factor in the presence of calcium ions to form a complex capable of activating factor X, in the Aextrinsic@ coagulation pathway, and factor IX in the Aintrinsic@ pathway. The second is the interaction of factor Xa with factor Na and phospholipid again in the presence of calcium ions to form the Aprothrombinase@ complex which converts prothrombin to thrombin. The third is the interaction of factor IXa with factor Villa and phospholipid in the presence of calcium ions to form the Atenase@ complex which converts factor X to factor Xa via the Aintrinsic@ pathway. A schematic of this Acascade@ of enzyme-driven reactions leading to blood clot formation is shown in Figure 1. Specific focal inhibition of thrombosis and disseminated intravascular coagulation has clinical value in a number of disease and therapeutic settings. These include, but are not limited to, coronary thrombosis leading to myocardial infarction, stroke, pulmonary embolism, renal infarction/ischemia, deep venous thrombosis, various allogeneic transplantations or xenotransplantations and sepsis. Benedict et al. (J. Clin. Invest. 1991, 88:1760) have demonstrated the effectiveness of inactivated factor IXa (IXai) as an anticoagulant and antithrombotic agent. When infused into dogs that had experimentally induced, partially occluded coronary arteries, the IXai inhibited the further occlusion that occurred in all of the controls. IXai did not interfere with hemostasis at extravascular sites (wounds), in contrast to heparin, and thus offers a specific means of blocking intravascular thrombosis and hemostasis. Similarly, Berkner et al., U.S. Patent No. 5,788,965, disclose that inactivated factor Vila (Vllai) may be utilized to impede the blood coagulation cascade. These inactive factors work by competing with the active factors in the formation of the enzyme complexes that are responsible for amplification of the cascade (see Fig. 1). However, prior to applicants, no one had devised an anticoagulant containing a plurality of inactivated blood factors in one composition and, in fact, the prior art contained admonitions to avoid such Amixed@ compositions. See, e.g., U.S. Patent No. 5,589,572 (1996) Col. 8, 1. 64 et seq.

Accordingly, a need exists, in general, for increasingly potent anticoagulant compositions which are selective and non-hemorrhagic and for improved methods for rapidly and efficiently producing such compositions.

SUMMARY OF THE INVENTION

Among the objects of the invention, therefore, may be noted the provision of potent, selective, non-hemorrhagic anticoagulant compositions. The anticoagulant compositions preferably comprise a cocktail of inactivated blood factors which contain (or contained) a biotinylated linker/inactivating moiety for ease of purification of the anti- coagulating components of the composition. Also provided is an improved method for rapidly and efficiently purifying inactivated blood factors and compositions containing cocktails of such proteins. Briefly, therefore, the present invention is directed to an anticoagulant composition comprising a cocktail of at least two inactivated blood factors. In another aspect, the anticoagulant composition of the invention comprises at least one biotinylated inactivated blood factor.

In another aspect of the invention, a method for inhibiting coagulation is provided which comprises providing to a patient a coagulation inhibiting amount of the above compositions. The invention also relates to a method for preparing an inhibited form of a blood factor comprising the steps of obtaining a mixture comprising at least two activated blood factors, treating the activated blood factors with an inactivating moiety capable of inactivating the at least two inactivated blood factors, separating said at least two inactivated blood factors from unwanted components of the mixture to form an anticoagulant precursor, and combining the anticoagulant precursor with a pharmaceutically acceptable carrier, wherein the inactivated blood factors are present in a therapeutically effective dosage.

Other objects and features will be in part apparent and in part pointed out hereinafter.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram showing key components of the blood clotting cascade.

FIG. 2 is a schematic of a preferred process for the preparation of anticoagulant compositions of the present invention. FIG. 3 is a schematic of the multi-phase mechanisms for the anti-clotting activity of preferred embodiments of the anticoagulant composition of the present invention.

Figure 4 is a graphic depiction of a typical elution profile for the purification of inactivated anticoagulant compositions of the invention with a nitrotyrosyl-avidin matrix as described below in Example 4. Figure 5 is a graphic depiction of the dose-dependent inhibition of coagulation in a whole blood ACT test, as described in Example 5 using the anticoagulant composition prepared as described in Example 3 and 4.

Figure 6 is a graphic depiction of the synergistic enhancement of inhibition of coagulation resulting from use of the anticoagulant composition of the invention as described below in Example 6.

DESCRIPTION OF THE PREFERRED EMBODIMENT

In accordance with the present invention it has been discovered that, by utilizing ligand-containing inactivating agents to selectively bind blood coagulating proteins from a mixture containing such blood coagulating proteins, a potent anti-coagulating composition comprising a cocktail of at least two such inactivated blood factors may be rapidly and efficiently produced. Moreover, the resultant cocktail of inactivated blood factors, the corresponding activated factors of which play a role in the coagulation cascade, synergistically combines the anti-coagulating power of the individual inactivated factors into a highly potent, specific, and non-hemorrhagic anticoagulant composition. The anticoagulant composition of the present invention may be advantageously employed in a variety of contexts, including use as a prophylactic in deep vein thrombosis (DVT), in cardiopulmonary bypass and for other conditions where anti-thrombotic therapy is indicated, but where, for example, there is a substantial risk of stroke. The anticoagulant composition may also be used in the inhibition of coronary thrombosis or intravascular thrombosis leading to myocardial infarction, stroke, pulmonary embolism, renal infarction or ischemia. In another use, the anticoagulant composition may be used as an adjunct to allogeneic transplantation or xeno transplantation.

Definitions and Abbreviations

As used herein, the terms Ablood coagulation factor@ and Ablood factor@ refer to blood factors generally, and in particular to any of a number of peptides, factors and cofactors, which comprise the intrinsic and extrinsic blood coagulation cascade in humans, or are involved in modulation, localization or dissolution of blood clots. Blood factors suitable for use in this invention include, but are not limited to, Factors II, Nil, IX, X, XI and XII, Proteins C and plasma kallikrein etc. in their zymogen, non-activated, activated or inhibited forms, as appropriate. The term includes wild-type and modified forms of such factors, including those synthetically or recombinantly produced. The blood factors described in this invention are defined herein to be any polypeptide sequence which possesses a biological property of the naturally occurring blood factor polypeptide comprising a commonly known polypeptide sequence, variants and homologues thereof, and mammal and other animal analogues. Ordinarily, the blood factors described herein will have an amino acid sequence having at least 75% amino acid sequence identity with a commonly known sequence, more preferably at least 80%, even more preferably at least 90%, and most preferably at least 95%. Identity in homology with respect to a commonly known blood factor sequence is defined herein as the percentage of amino acid residues in the candidate sequence that are identical with the known blood factor amino acid residues, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent homology, and not considering any conservative substitutions as part of the sequence identity. None of N-terminal, C-terminal or internal extensions, deletions or insertions into the blood factor sequence shall be construed as affecting homology. ATherapeutically effective amounts@ means those amounts that, when administered to a particular patient in view of the nature and severity of that patient=s disease or condition, will have the desired therapeutic effect, e.g., an amount which will cure, or at least partially arrest the disease or condition.

ALigand@, as used herein, means a molecule that binds to another molecule with high affinity, e.g., such as formed by the avidin-biotin complex, or antigen-antibody interaction.

DVT = deep vein thrombosis Factor Vila (for example) = activated Factor Nil Factor NIlai (for example) = inhibited activated Factor Nil Factor Nllaib (for example) = biotinylated inhibited activated Factor Nil

BEGR-CMK biotinyl-glutamyl-glycyl-arginine-chlorometyl ketone BFPR-CMK biotinyl-phenylalanyl-prolyl-arginine-chloromethyl ketone EGR-CMK glutamyl-glycyl-arginine-chloromethyl-ketone FPR-CMK phenylalanyl-prolyl-arginine-chloromethyl ketone AAllogeneic transplantation® means transplantation of an allograft, i.e., a graft of tissue between individuals of the same species but of different genotypes.

AXenotransplantation@ or Axenogeneic transplantation® refers to transplantation of a xenograft, i.e., a graft of tissue transplanted between individuals of different species. The terms Ainactivator@ and Ainhibitor@ refer to any of a number of molecules which have the ability to covalently bind to the active site of the activated blood coagulation factor and to render the activated blood factor inactive, or to inhibit the coagulation activity of the activated blood factor.

An Ainactivated blood factor@ is one that substantially lacks the coagulation enzymatic activity expected for the blood factor when activated.

Anticoagulant Compositions In a prefeπed embodiment, the present invention is directed to anticoagulant compositions comprising at least two inactivated blood factors. The inactivated factors are formulated in a pharmaceutically acceptable carrier and prepared in a therapeutically effective dosage. The inactivated blood factors can include, but are not limited, to Factors Ilai, NIlai, IXai, Xai, Xlai and Xllai. Preferably, the composition comprises at least a third inactivated factor, more preferably, it comprises at least a fourth, and most preferably it comprises a potent cocktail of inhibited forms of all or nearly all naturally occurring blood factors found in plasma. In preferred embodiments, the at least two inactivated blood factors are selected from the groups consisting of NIlai, IXai, Xai and Xlai. Preferably, the cocktail includes NIlai and IXai or IXai and Xai. The blood factors used in the present invention can be obtained from plasma of animal species such as bovine, equine, goat, porcine, sheep, and human, and are preferably obtained from human plasma. The blood factors can also be altered or synthetic forms of wild-type factors, such as may be synthesized recombinantly using methodologies well known in the art. The blood factors can be activated and then inhibited, or can be simultaneously activated and inhibited. Methods and inhibitors useful for activating and inhibiting blood factors are described, e.g., at U.S. Patent Νos. 5,589,572; 5,120,537; and 5,788,965, which are incorporated herein by reference.

Preferably, the blood factors are modified with one or more serine protease inhibitors. Such inhibitors are reactive with and interfere with the function of the serine and histidine active site and include, but are not limited to, organophosphors, sulfonyl fluorides, antibodies or antibody fragments, fluorophosphates, acylating compounds, peptide aldehydes and peptide halomethyl ketones. Preferably, the inhibitor is a peptide halomethyl ketone. Such compounds include, but are not limited to tosyl-lysyl- chloromethyl ketone, tosyl-phenyl-chloromethyl ketone, glu-gly-arg-chloromethyl ketone, phe-pro-arg-chloromethyl ketone, D-phe-L-phe-L-arg-chloromethyl ketone, dansyl-phe- pro-arg-chloromethyl ketone, dansyl-glu-gly-arg-chloromethyl ketone and dansyl-phe- phe-arg-chloromethyl ketone. More than one inhibiting compound may be used simultaneously. Prefeπed inhibitors are EGR-CMK and FPR-CMK, or a mixture thereof. In a particularly prefeπed embodiment of the invention, the blood factor inhibitor further compromises a ligand moiety which facilitates purification of the inactivated factors. Such ligand-receptor pairings can include avidin-biotin (e.g., biotin-streptavidin), antigen-antibody (e.g. fluorescein-anti-fluorescein), sugar-lectin (e.g., mannoside- concanavalin A), or other ligand-receptor (e.g., TGF-TGF receptor). Preferably, the ligand moiety is biotin or a biotin derivative, and the inhibitor is a biotinylated peptide halomethyl ketone such as biotin - EGR-CMK or biotin - FPR-CMK, or a mixture thereof In one embodiment, the anticoagulant composition can be comprised of a single liganded, e.g., biotinylated, inhibited activated blood factor, although preferably a cocktail of such liganded inactivated factors are used as previously described.

The anticoagulant compositions of the present invention are preferably formulated in a pharmaceutically acceptable carrier. Such carriers include, but are not limited to, water, saline, synthetic plasma, glycine and physiological buffers suitable for intravenous administration. In particular, carriers include water, Ringer=s solution, dextrose solution, 5% human serum albumin, or phosphate buffered saline.

The anticoagulant compositions may contain auxiliary substances used to enhance performance, such as pH modifying substances, buffers and toxicity adjusters. These include various metal salts such as potassium or calcium chloride and sodium acetate and lactate. Methods for devising particular pharmaceutically acceptable formulations are well known in the art as, e.g., detailed in Remington=s Pharmaceutical Science. 16th ed., Mack Publishing Co., Easter, PA (1982), which is hereby incorporated herein by reference.

Preparation of Anticoagulant Compositions

In accordance with the present invention a cocktail of inactivated blood coagulating factors is prepared which may be utilized as a specific focal inhibitor of intravascular thrombosis and coagulation. The present invention provides an efficient and facile isolation procedure for the rapid preparation of these compositions. Specifically, in a prefeπed embodiment, biotinylated-peptide-chloromethyl ketone derivatives or similar liganded inactivating compounds are covalently reacted with specific coagulation proteins to produce inactivated biotin-labeled coagulation factors. A schematic depiction of the method of preparation is set forth in Figure 2. Previous inactivated factors have been made in isolated form, and although workable, do not provide the synergistic advantages provided by the cocktail compositions of the present invention. Using a more complex mixture of coagulation proteins as a starting material, such as in the Autoplex T reagent described below, applicants have produced a more efficient anticoagulant. Moreover, this invention provides the unique advantage of a readily reversible method of purifying the anticoagulant compositions of the present invention, by utilizing a nitrated derivative of avidin. In this aspect of the invention, avidin is nitrated to produce a nitrotyrosyl- derivative that binds biotinylated compounds in a reversible manner. As detailed below, applicants have demonstrated that by utilizing such a reversible binding mechanism, biotin-tripeptide-CMK-coagulation proteins or similar inactivated proteins may be obtained and used as a potent composition for inhibiting clotting as demonstrated in an activated clotting time in vitro assay system. In general, as depicted in Figure 2, the anticoagulant compositions of the present invention are prepared by obtaining a mixture comprising at least two activated blood factors. The activated blood factor mixture is treated with one or more inactivating moieties capable of inactivating the at least two activated blood factors contained in the mixture. In a prefeπed embodiment, the inactivating moieties, or inhibitors, comprise a ligand moiety, preferably biotin or a biotin derivative. The inactivated blood factors are separated from unwanted components of the mixture to form an anticoagulant precursor. The precursor is then combined with an appropriate pharmaceutically acceptable carrier in a therapeutically effective dosage for use in the therapeutic applications described below. Any method which provides a plurality of activated blood clotting factors in accessible form may be utilized as a starting material for the anti-coagulating compositions of the present invention. For example, separately purified activated factors may be obtained commercially (e.g., from Haematologic Technologies, Essex Junction, Nt.) or prepared (see [Sorenson,B.B, et al. J. Biol. Chem. 1997, 272(18):11863; Benedict, C.R. et al. J. Clin. Invest. 1991, 88:1760; Benedict, C.R. et al. Blood 1993, 81(8):2259; Lundblad, R.L. et al. Thromb. Haemostas. 1998, 80:811 ]), and mixed together to form a beginning composition comprising a plurality of activated blood factors. In a prefeπed embodiment, Autoplex T, an activated prothrombin complex concentrate commercially available from Baxter Healthcare is used as the starting material. Autoplex comprises a concentrated mixture of activated blood factors including Factors Ila, Vila, IXa, Xa and XIa. Autoplex T is a coagulant composition used for the treatment of inhibitors in Factor NIII deficiency (hemophilia). A detailed method for the preparation of Autoplex T is set forth in Example 1 below. Ligand-Bound Affinity Purification System

Methods of purifying individual inactivated blood factors, such as those described in U.S. Patent No. 5, 589,572, incorporated herein by reference, are known in the art, and can be utilized in the preparation of the anticoagulant cocktails of the invention. However, applicants have discovered that by utilizing a unique ligand-containing affinity purification system, a rapid and efficient separation of inactivated blood factors from unwanted components may be achieved.

Biotin, a naturally-produced vitamin, and various derivatives thereof known in the art, have an extremely high selectivity and affinity for avidin, a tetrameric, 60,000 dalton protein isolated from egg whites. The Kd for the avidin - biotin complex is on the order of 10^"15M, an essentially iπeversible reaction. Hence, given the ability of the ligand, biotin, to be covalently coupled to a variety of molecules with an aπay of chemistries, the avidin- biotin complex has been widely used for diagnostic and bioanalytical processes. However, the essentially iπeversible nature of the avidin-biotin reaction has also limited its utility for separation and purification purposes.

Applicants have discovered that a modified form of avidin, nitrotyrosyl-avidin, nitrotyrosyl-streptavidin, or related derivatives (see U.S. Pat. No. 5,973,124 incorporated herein by reference), may be advantageously used to bind and separate biotinylated inactivated blood factors, yet the reaction can be readily reversed in order to release the now isolated, inactivated blood factors of the anticoagulant cocktail.

In this embodiment, tetranitromethane (TNM) selectively nitrates the phenolic tyrosine side chain of the avidin protein. The nitrotyrosyl-avidin thus produced has a binding constant which can be altered in a pH-dependent manner. Nitrotyrosine also has a characteristic yellow color (absorbance maximum at 428 with an extinction coefficient of 4200), which allows it to be monitored and analyzed spectrophotometrically.

In this aspect of the invention, e.g., biotinylated-peptide-chloromethyl ketones (b- EGR-CMK and b-FPR-CMK, obtained from Hematological Technologies, Essex Junction, Nt.) can be reacted with Autoplex T, or some other source of a mixture of blood factors, to produce a cocktail of inactivated biotin-labeled coagulation factors. See Figure 2. The mixture is then applied to a nitrotyrosyl -avidin affinity column, which binds the biotinylated compounds in a reversible manner to isolate the mixture of inactivated blood factors.

Optionally, the biotin moiety selected for use in the invention can be a cleavable derivative in order to permit the removal of the biotin moiety following purification. Cleavable biotin derivatives are commercially available, e.g., derivatives containing a disulfide linkage may be used which are then cleaved using a reducing agent such as glutathione or dithiotheitol. Uses of the Anticoagulant Composition

The anticoagulant compositions of the present invention may be utilized to inhibit coagulation by providing a coagulation inhibiting amount of such compositions to a patient. As set out schematically in Fig. 3, the anticoagulant cocktails developed by applicants have the unique advantage not heretofore available of competitively inhibiting activated blood factors at multiple points in the cascade, thus synergistically combining to provide a potent, yet selective, and non-hemoπhagic anticoagulating effect.

The anticoagulant cocktails of the present invention can be used in a wide variety of therapeutic and prophylactic applications for which single-protein inactivated blood factors have previously been employed. See, e.g., U.S. Patent Nos. 5,589,572; 5,120,537; and 5,788,965, which are incorporated by reference.

In particular, the anticoagulant cocktails of the present invention are useful for treatment of a variety of ailments or conditions associated with intravascular coagulation. For example, deep vein thrombosis (DVT) is a common clinical diagnosis with between 170,000 and 300,000 incidents reported in the United States each year. Moreover, deep vein thrombosis can lead to pulmonary embolism, which is estimated to result in 5 to 10% of all hospital deaths in the United States. DVT may result from a variety of causes, including venous stasis or slow blood flow that can occur with obesity or congestive heart failure, endothelial cell (vascular wall) injury resulting from trauma, activated clotting factors formed due to surgery or cancer, activated platelets occurring in myeloproliferative disease, or as a result of a deficiency of natural anticoagulant activity.

Another therapeutic application for applicants= anticoagulant compositions is to ameliorate disseminated intravascular coagulation (DIC) which is a generalized activation of the hemostatic system resulting in widespread fibrin formation, microthrombus formation and consumption of platelets and coagulation, some of which include endothelial cell (vascular wall) damage, sepsis, exposure to immune complexes, protease activity, or toxins, and hypoxia or acidosis.

These anticoagulant compounds are also useful as an adjunct to therapies involving the prophylaxis or treatment of conditions associated with myocardial infarction and stroke due to atherosclerosis. These are well recognized as major causes of disability and death in the United States and other Western societies, estimated to effect 1.1 million each year in the U.S. alone. The evolution of an atherosclerotic plaque is a complex biochemical and cellular process involving damaged endothelium, lipid deposition, accumulation of inflammatory cells (macrophages and T cells), and activation and proliferation of smooth muscle cells. Spontaneous or mechanical disruption of the plaque triggers activation of platelets and coagulation proteins resulting in formation of platelet- rich thrombi which can occlude the artery locally or embolize and cause occlusion downstream in the vasculature.

Use of high doses of heparin is presently the treatment of choice to mitigate severe tissue damage which can occur because of activation of the inflammatory and hemostatic systems upon reinfusion of a patient=s blood during cardio-pulmonary bypass surgery. However, heparin is known to cause bleeding diathesis that requires transfusion of whole blood units to patients post surgery. Thus, since the inactivated blood factors of the anticoagulants of the present invention selectively bind at multiple points in the coagulation cascade, they offer an effective alternative which minimizes the bleeding complications caused by heparin.

In general, the anticoagulant compositions of the present invention are formulated or administered in vivo in therapeutically effective amounts. The particular dosages used depend on the matter and circumstances of the disorder being treated, the concentrations and identities of particular inactivating blood factors which make up the anticoagulation cocktail, the weight and background of the patient being treated, and the therapeutic context of the treatment (e.g., traumatic rescue or prophylactic post-transplantation maintenance).

Typically, the anticoagulant compositions are administered parenterally, although other modes of administration, including topical or local administration are also contemplated. The composition can contain various additives such as buffers and preservatives. The blood factors will typically be formulated in such vehicles at concentrations of approximately 1 ug/ml to 100 mg/ml (100,000 ug/ml). The number of and interval between administrations can vary, depending on the nature of the treatment, from the purely episodic to a planned schedule of treatments over a period of weeks, months or even indefinitely. For example, for use in conjunction with cardio-pulmonary bypass, the compositions will typically be administered within about 24 to 48 hours prior to beginning the operation, and may continue on an as needed basis thereafter. Such administration also can be carried out as part of (and in aid of) a therapeutic regimen such as angioplasty, thrombolisys, stent placement, heart/valve/artery/venous surgery or transplant and may be administered in conjunction with other treatment agents. The following examples illustrate the invention.

EXAMPLE 1

Preparation of Activated Coagulation Protein Mixture Autoplex ®-T is prepared from Cohn Blood Plasma Fraction IN. Cohn blood Plasma Fraction IN is one of the several Cohn Plasma Fractions which can be prepared from human blood plasma by the combination of increasing ethyl alcohol concentration combined with the adjustment of pH and ionic strength. A series of Cohn Blood Plasma Fractions (e.g. Cohn Fraction I, Cohn Fraction II, Cohn Fraction IN and Cohn Fraction N) can be prepared. These Cohn Fractions can then be used as starting materials for the preparation of more highly purified plasma protein fractions such as Autoplex -T® or Baxter Healthcare, Deerfield, Illinois. Fraction IN may be stored at or below -20 °C or immediately processed further to

Anti-Inhibitor Coagulant Complex. The Fraction IV precipitate is dissolved in 0.9% saline while maintaining the pH at 6.0. After mixing thoroughly, the pH of the mixture is adjusted to 7.2 with IN sodium hydroxide and, after mixing again, the material is allowed to settle. The mixture is then centrifuged and the precipitate is discarded. If necessary, the pH of the supernate is readjusted to 7.2 and tri -basic calcium phosphate is added to a concentration of 0.5%. The mixture is mixed thoroughly, centrifuged, and the supernate is discarded. The precipitate is then mixed with 0.1M sodium citrate solution to elute the precipitated prothrombin complex from the tribasic calcium phosphate. After mixing thoroughly, the solution is centrifuged and the precipitate is discarded. The coagulation proteins in the supernate are activated by adding 0.5 to 1.0 g of silica per liter of solution. The activation is monitored by removing samples at intervals, filtering through a .45 millipore filter membrane to remove the silica, and assaying for activation characteristics. When sufficient activation has taken place, the silica is removed by filtration (e.g. Millipore CWSC cartridge filters provided by Milipore Corporation of Bedford, MA). Polyethylene glycol is added to the filtrate to a concentration of 5%. After mixing thoroughly, the mixture is centrifuged and the precipitate is discarded. The pH of the supernate is adjusted to 5.2 and polyethylene is added to a final concentration of 20%. After mixing thoroughly, the mixture is centrifuged and the supernate is discarded. The precipitate is dissolved in heparinized citrated saline (5.9 gm sodium citrate, 7.2 gm sodium chloride, and 2000 units heparin per liter), and the pH is adjusted to 7.0 while mixing. The solution of activated coagulation proteins is clarified and sterilized by filtration.

EXAMPLE 2: Preparation of Νitrotyrosyl-Avidin Affinity Matrix

Avidin was dissolved in 50 mM Tris pH 8.0 buffer to a concentration of 2.5 mg/ml and 2 ml was transfeπed to a polypropylene test tube. TΝM reagent (20 ml) was added to 180 ml of 95% EtOH (ethonolic TΝM). This ethonolic TΝM reagent mixture was further diluted 1:2 with 50 mM Tris buffer. The diluted TΝM/Tris reagent (10 ml) was added to a 2 ml aliquot of avidin. The solution was mixed and the reaction was allowed to proceed for 30 minutes. Another aliquot of the (ethanolic) TΝM reagent was diluted 1:2 with Tris buffer and 10 ml of the diluted TΝM/Tris was added to the avidin solution. After 30 minutes this last step was repeated a third time for a total reaction time of 90 minutes. The reaction solution was concentrated in a Centricon 30 (15 minutes at 5000xg) and purified by Gel Filtration over BioGel P6DG column in PBS. This typically gives a degree of modification in the range of 2 to 2.6 mole NO₂ Tyr/mole of avidin,

Nitrotyrosyl-avidin was coupled to Affigel-10 (BioRad) activated affinity resin. The nitrated avidin was exchanged into a 10 mM NaHCO₃ buffer (pH 8.0) and adjusted to a final concentration of 8.8 mg/ml. Washed Affi-Gel resin was added to the protein at a ratio of approximately 1 ml of resin per 10 mg of nitrated avidin. The tube was mixed at 4EC for 2-4 hours and the resin was washed with PBS to remove uncoupled protein, the nitrotyrosyl-avidin affinity matrix was poured into a column and washed with PBS. The column was blocked with a solution of 1 mM biotin (in 50 mM citrate/phosphate buffer, pH 4) and regenerated with 10 column volumes of 50 mM (Na)₂CO₃ pH 10 buffer. The column was re-equilibrated with 100 mM sodium phosphate buffer, pH 7. Biotinylated BSA was added to the column and the column was washed with 100 mM sodium phosphate buffer, pH 7. The biotin-BSA was eluted with 1 mM biotin.

EXAMPLE 3: Preparation of Inactive Biotinylated Coagulation Factors

A vial of Autoplex (made as described in Example 1 above) was reconstituted with 8 ml of deionized water and the protein solution was incubated with 5-10 fold molar excess of biotin-EGR-CMK plus biotin-FPR-CMK. The reaction was allowed to proceed for two hours at room temperature and the solution was dialized against PBS with 0.5M NaCl added, to remove the biotin-peptide-CMK derivatives. Under these conditions 95- 98% of the enzyme activity, as determined with the Spetrazyme Pro substrate (American Diagnostica), was destroyed.

EXAMPLE 4: Purification of Anticoagulant Composition with Nitrotyrosyl- Avidin Matrix The inactivated Anticoagulant Composition solution prepared in Example 4 was adjusted to pH 4.5 with 0.1 N HCI. The Nitrotyrosyl- Avidin column was equilibrated with 50 mM citrate/5 OmM NaH₂PO₄ pH 4 buffer. The inactivated Anticoagulant Composition solution was loaded onto the column slowly to permit maximum binding of the biotinylated proteins to the matrix. The column was washed with the 50 mM citrate/50mM NaH₂PO₄ pH 4 buffer until the absorbance at 280nm was below 0.05 and not decreasing further. The bound proteins were eluted off with a 100 mM NaHCO₃ pH 9 buffer containing 1 mM biotin. The column was washed with 100 mM Na₂CO₃ pH 10 buffer to elute any strongly bound protein and the column was re-equilibrated with PBS for storage. A typical elution profile is show in Figure 4. EXAMPLE 5: ACT Assay with Anticoagulant Composition

Whole blood was collected from normal human volunteers by venipuncture using heparin as anticoagulant (0.7 U/ml). Anticoagulant Composition stock solution was diluted to the desired concentration using PBS with 1% BSA added. An aliquot of Anticoagulant Composition (40 ul) as prepared and purified in Example 3 and 4 above was added to a Hemacron (glass bead) tube at concentrations ranging from 100 ug/ml to 500 ug/ml (i.e. 10X higher than final blood concentration). Four hundred ul of blood was added to the tube and mixed. The Hemacron tube was placed in the Hemacron device and the timer was started. Each sample was run in duplicate. Baseline ACT were determined by adding 40 ul of PBS with 1% BSA to a Hemacron tube before adding the whole blood. A typical result for this assay is shown in Figure 5, using four normal donors.

EXAMPLE 6: Synergistic Inhibition with Multiple Components

Factors Vila, IXa, and Xa were obtained from Enzyme Research Labs (South Bend, IN) in pure form. The factors were inactivated with a five fold excess of EGR- CMK and FPR-CMK, dialyzed and purified with the nitrotyrosyl-avidin matrix as described above in Example 4. Whole blood was obtained from normal donors (heparinized at 0.7 U/ml) and used to perform ACT assays as described above in example 5. When the individual inactivated factors were added to the blood separately, at relatively low concentrations, each factor produced a modest degree of inhibition (i.e. a small increase in the ACT values) on its own, as shown in Figure 6. When all three inactivated factors were combined and added to the blood at the same concentrations used for the individual factor experiment, a dramatic synergistic inhibition of coagulation was observed (see Figure 6). These results demonstrate that a combination of inactivated factors is more effective anticoagulant than the individual factors are at the same concentrations.

In view of the above, it will be seen that the several objects of the invention are achieved.

As various changes could be made in the above compositions and processes without departing from the scope of the invention, it is intended that all matter contained in the above description be interpreted as illustrative and not in a limiting sense.

Claims

What is claimed is:

1. An anticoagulant composition comprising at least two inactivated blood factors in a pharmaceutically acceptable carrier, wherein the inactivated blood factors are present in a therapeutically effective dosage.

2. The anticoagulant composition of claim 1 wherein the at least two inactivated blood factors are selected from the group consisting of Factors Ilai, NIlai, IXai, Xai and Xlai.

3. The anticoagulant composition of claim 1 wherein the composition further comprises at least a third inactivated blood factor.

4. The anticoagulant composition of claim 1 wherein the composition further comprises at least a third and a fourth inactivated blood factor.

5. The anticoagulant composition of claim 2 wherein the at least two inactivated blood factors are selected from the group consisting of NIlai, IXai, and Xai.

6. The anticoagulant composition of claim 2 wherein the at least two inactivated blood factors are NIlai and IXai.

7. The anticoagulant composition of claim 1 wherein the blood factors are obtained from plasma from an animal species selected from the group consisting of human, bovine, equine, goat, porcine and sheep.

8. The anticoagulant composition of claim 1 wherein the blood factors are obtained from plasma from a human.

9. The anticoagulant composition of claim 1 wherein one or more of the blood factors is produced by recombinant engineering.

10. The anticoagulant composition of claim 1 wherein the at least two inactivated blood factors are modified with one or more serine protease inhibitors.

11. The anticoagulant composition of claim 10 wherein the one or more serine protease inhibitors are selected from the group consisting of an organophosphor, a sulfonyl fluoride, an antibody or fragment thereof, an azapeptide, a fluorophosphate, an acylating compound, and a peptide halomethyl ketone.

12. The anticoagulant composition of claim 11 wherein the one or more serine protease inhibitors is a peptide halomethyl ketone selected from the group consisting of lysyl-chloromethyl ketone, tosyl-phenyl-chloromethyl ketone, glu-gly-arg-chloromethyl ketone, phe-pro-arg-chloromethyl ketone, D-phe-L-phe-L-arg-chloromethyl ketone, dansyl-phe-pro-arg-chloromethyl ketone, dansyl-glu-gly-arg-chloromethyl ketone, and dansyl-phe-phe-arg-chloromethyl ketone.

13. The anticoagulant composition of claim 12 wherein the peptide halomethyl ketone is EGR-CMK or FPR-CMK.

14. The anticoagulant composition of claim 1 wherein the carrier is selected from the group consisting of water, saline, synthetic plasma, and physiological buffers suitable for intravenous administration.

15. The anticoagulant composition of claim 10 wherein the one or more serine protease inhibitors further comprises a ligand moiety which facilitates purification of the inactivated factor.

16. The anticoagulant composition of claim 15 wherein the ligand is selected from the group consisting of an antibody, a lectin, TGF and biotin or a biotin derivative.

17. The anticoagulant composition of claim 16 wherein the ligand is biotin or a biotin derivative.

18. The anticoagulant composition of claim 1 wherein the inactivated factors are administered in a dosage of from about 1 ug/ml to 100 mg/ml.

19. A method for inhibiting coagulation comprising providing to a patient a coagulation inhibiting amount of the anticoagulant composition of claim 1.

20. The method of claim 19 wherein the anticoagulant composition is the composition of claim 10.

21. The method of claim 19 wherein the anticoagulant composition is the composition of claim 15.

22. The method of claim 19 wherein the anticoagulant composition is the composition of claim 17.

23. The method of claim 19 further comprising administering an amount of the anticoagulant composition to a patient effective to inhibit intravascular thrombosis.

24. The method of claim 19 further comprising administering an amount of the anticoagulant composition to a patient effective to inhibit coronary thrombosis.

25. The method of claim 19 further comprising administering an amount of the anticoagulant composition to a patient to inhibit thrombosis following allogeneic or xenotransplantation.

26. The method of claim 19 wherein the anticoagulant composition is administered locally.

27. The method of claim 19 wherein the anticoagulant composition is administered systemically.

28. A method for preparing an anticoagulant composition comprising inhibited forms of at least two blood factors comprising: obtaining a mixture comprising at least two activated blood factors, treating the activated blood factors with an inactivating moiety capable of inactivating the at least two activated blood factors contained in the mixture, separating the at least two inactivated blood factors from unwanted components of the mixture to form an anticoagulant precursor, and combining the anticoagulant precursor with a pharmaceutically acceptable carrier, wherein the inactivated blood factors are present in a therapeutically effective dosage.

29. The method of claim 28 wherein the at least two activated blood factors are selected from the group consisting of Factors Ila, Vila, IXa, Xa and XIa.

30. The method of claim 28 wherein the mixture further comprises at least a third activated blood factor.

31. The method of claim 28 wherein the mixture further comprises at least a third and a fourth activated blood factor.

32. The method of claim 29 wherein the at least two activated blood factors are selected from the group consisting of Vila, IXa, and Xa.

33. The method of claim 29 wherein the at least two activated blood factors are Vila and Xa.

34. The method of claim 28 wherein the blood factors are obtained from plasma from an animal species selected from the group consisting of human, bovine, equine, goat, porcine and sheep.

35. The method of claim 28 wherein the blood factors are obtained from plasma from a human.

36. The method of claim 28 wherein the inactivating moiety or moieties are one or more serine protease inhibitors.

37. The method of claim 36 wherein the one or more serine protease inhibitors are selected from the group consisting of an organophosphor, a sulfonyl fluoride, an antibody or fragment thereof, an azapeptide, a fluorophosphate and a peptide halomethyl ketone.

38. The method of claim 37 wherein the one or more serine protease inhibitors is a peptide halomethyl ketone selected from the group consisting of lysyl-chloromethyl ketone, tosyl-phenyl-chloromethyl ketone, glu-gly-arg-chloromethyl ketone, phe-pro-arg- chloromethyl ketone, D-phe-1-phe-L-arg-chloromethyl ketone, dansyl-phe-pro-arg- chloromethyl ketone, dansyl-glu-gly-arg-chloromethyl ketone, and dansyl-phe-phe-arg- chloromethyl ketone.

39. The method of claim 38 wherein the peptide halomethyl ketone is b-EGR- CMK, b-FPR CMK or a combination thereof.

40. The method of claim 28 wherein the carrier is selected from the group consisting of water, saline, synthetic plasma, and physiological buffers suitable for intravenous administration.

41. The method of claim 36 wherein the one or more serine protease inhibitors further comprises a ligand moiety which facilitates purification of the inactivated factor.

42. The method of claim 41 wherein the ligand is selected from the group consisting of an antibody, a lectin, TGF, biotin or a biotin derivative.

43. The method of claim 42 wherein the ligand is biotin or a biotin derivative.