The present invention relates to anticoagulant compositions based on coagulant factor Xa and methods for the production and use thereof.
Coagulation is the end result of a complex series of reactions in which the end product of each reaction initiates the next reaction. Following vascular injury, there is a rapid activation of an otherwise largely passive process to produce a response at the site of the injury. There are a variety of modulating events that regulate the process in a positive or negative fashion to maintain vascular integrity while retaining general plasma fluidity. As recently summarized by Kenneth G. Mann, in "Membrane-Bound Complexes in Blood Coagulation", Progress in Hemostasis and Thrombosis, edited by T. H. Spaet, pages 1-23 (Grune & Stratton 1984), the extensive network of interdependent protein interactions can be represented as a collection of four reaction complexes: ##STR1##
Factor II (prothrombin), Factor X, Factor IX, and Factor VII are vitamin K-dependent proteins which normally circulate as zymogens that are activated to trypsin-like enzymes with active site serine and histidine. In blood, prothrombin is present at about micromolar concentrations; Factor V and Factor X are present at about one-tenth that amount (10.sup.-7 M). The reaction rate converting Factor II to IIa (thrombin) is about 278,000 fold greater when all components of the complex interact (i.e., Factor II, Factor V, Ca++, and the phospholipid or membrane surface)(Nesheim, et al., J. Biol. Chem. 254, 10952-10062 (1979). Deletion of any one component in the complex leads to a drastic reduction in reaction rate.
Although the mechanisms for regulating coagulation and preventing thrombosis ordinarily are remarkably efficient, they can be disrupted by disease, congenital defect or dysfunction, or an inflammatory stimulus which elicits the release of inflammatory mediators such as the monokines tumor necrosis factor (TNF) and interleukin 1 (IL-1).
A wide variety of pathological conditions, including sepsis, especially gram-negative septic shock, beta Streptococcus and Staphylococcus aureus septicemia, and injuries involving substantial tissue damage, such as burns and crush injuries, can cause the release of inflammatory mediators. Inflammatory mediators are also released in adult respiratory distress syndrome and reperfusion inflammatory syndrome.
In septic shock, the response of the endothelium to the inflammatory stimuli involves both coagulopathy and abnormal permeability. The stimulus activates circulating monocytes and the fixed tissue macrophages in the liver and the lungs. After a lag period of about two to four hours, these blood cells release the monokine inflammatory mediators tumor necrosis factor (TNF) and interleukin 1 (IL-1). These inflammatory stimuli lead to the conversion of endothelial cell surfaces from an anti-coagulant to a procoagulant state, causing intravascular coagulation. When this dysfunction is systemic, it is referred to as disseminated intravascular coagulopathy (DIC). In DIC, the endothelial cell loses its ability to selectively control porosity. The endothelial cells swell and fluid begins leaking into the surrounding tissues, causing anoxia and parenchymal damage. This is accompanied by increased peripheral resistance, decreased venous return and, in many instances, death due to shock.
Septic shock can be simulated in a clinical model by infusion of a lethal dose of Escherichia coli in a baboon, F. B. Taylor, "Baboon model of E. coli Septic shock staging and observations on the role of the vascular endothelium", chapter 13 Critical Care State of the Art, B. F. Fuhrman and W. C. Schoemaker, editors volume 10 (Soc. Critical Care Medicine 1989). The clinical course is characterized as a four stage process. Stage I begins with the inflammatory stimulus, for example, a lethal infusion of Escherichia coli, and continues for about 120 minutes. In this stage, the scavenger cells (monocytes and macrophages) and PMNLs are activated and the inflammatory mediators (TNF, IL-1, free hydroxyl radicals, elastase and others) are released. Stage II begins next and continues for about four hours, or from two to six hours after the insult. During this stage, the mediators cause the endothelial cells to become inflamed or perturbed, converting them from an anticoagulant to a procoagulant state. Fibrinogen levels fall and fibrin degradation products increase. The fibrinolytic activity of whole blood increases markedly by one or two hours and then decreases almost immediately at three hours after the insult. Stage III occurs at about six hours following the insult and continues for about four hours. In this stage, the endothelial lose their ability to selectively control permeability and fluid begins to leak into the tissues injuring target organs. In the fourth and final stage, the parenchymal edema produces shunting, peripheral and eventually central anoxia, and decreased mean systemic arterial pressure. Death occurs typically about 24 to 32 hours after the insult.
Treatment for these disorders involving inflammatory mediators usually involves the administration of anticoagulants. Anticoagulants are also used in the prevention of reocclusion following angioplasty, acute organ rejection, and deep vein thrombosis following surgery, and in the treatment of unstable angina, mural thrombosis, stroke, myocardial infarction, and pulmonary embolism.
There are currently a number of known and medically accepted anticoagulant agents. Heparin and vitamin K antagonists such as Coumarin and aspirin are the most widely used anticoagulants. Heparin works in conjunction with another plasma protein, antithrombin III, to inhibit coagulation. Heparin treatment is not effective in all cases since antithrombin III is often at low levels in severe shock and other consumptive coagulation processes. In addition, patients may have adverse reactions to heparin, including heparin induced thrombocytopenia. Further, maintenance of an effective heparin dose in vivo has proven difficult.
Coumarin drugs are slow to be effective, taking several days before antithrombotic effects are observed. Bleeding is a common complication of vitamin K antagonist treatment and, as with heparin, the effective dose is hard to monitor since the required dose is impacted by both diet and other drugs that are present in the patient especially antibiotics. In addition, there is a rare complication that leads to skin necrosis and potential loss of limbs or, in exceptional cases, the individual's life (Esmon, C. T., et al., "Anticoagulant Proteins C and S". in New Dimensions of Warfarin Prophylaxis. Wessler, et al., eds., Plemum Publishing Corp., New York, 47-54, 1987). Aspirin is limited to inhibition of platelet aggregation and is of little efficacy in treating patients at severe risk of thrombosis.
An alternative method for the suppression of clotting which has been proposed is the direct administration of compounds which inhibit thrombin. Many studies have been done wherein the active site of the coagulation enzymes is inhibited in vitro using a variety of different types of inhibitors, especially low molecular weight serine esterase inhibitors such as diisopropylfluorophosphate (DFP) or phenylmethanesulfonyl fluoride (PMSF). For example, Skogen, et al., in J. Biol. Chem. 259(4), 2306-2310 (1984), used the serine esterase inhibitor (p-amidinophenyl)methanesulfonyl fluoride (pPMSF) to inhibit the active site of factor Xa, both in its native form and in modified form, to determine the role of factor Va binding to factor Xa in formation of the prothrombinase complex.
Hanson, et al., Proc. Natl. Acad. Sci. USA, 85:3184-3188, (1988), used a synthetic antithrombin, D-phenylalanyl-L-prolyl-L-arginyl chloromethyl ketone, PPACK, to block arterial thrombosis by specific interaction with thrombin. A disadvantage to this approach, however, is the requirement for administration of high levels of reactive compounds having unknown potential side effects. Further these small compounds are cleared rapidly form the blood stream and the monitoring of a maintenance dose is complex.
There are many problems in trying to extrapolate from in vitro studies involving inhibition of coagulation factors to in vivo uses of an inhibited protein as an anticoagulant. Modified proteins, such as an inactivated coagulation factor, may be cleared much more rapidly from the blood by cells of the reticuloendothelial system than the unmodified proteins. The coagulation process is complex, involving interactions between proteins in the intrinsic and extrinsic pathways and cell surface receptors. In vitro, the proteins are usually present in purified form and there are generally no cell surface receptors or other plasma protein components present which may alter interactions with the inhibited protein. For example, in vivo studies using thrombin with the active site inactivated demonstrated that infusion of this proteolytically inactive protein actually induced a procoagulant state, possibly because of binding with endogenous inhibitors of coagulation, such as thrombomodulin.
A rapid and effective anticoagulant having a relatively long half-life in vivo and free from the side effects and shortcomings of the ones presently in use would have utility in the prevention of reocclusion following angioplasty, acute organ rejection, and deep vein thrombosis following surgery, and in the treatment of unstable angina, mural thrombosis, stroke, myocardial infarction, and pulmonary embolism.
It is therefore an object of the present invention to provide a safe, effective, and fast acting anticoagulant composition for use in vivo.
It is a further object of the present invention to provide a method for the production of anticoagulant compositions for in vivo application.
It is a still further object of the present invention to provide a method of treating individuals in need of such treatment with an effective amount of an anticoagulant composition.