Modified factor VII

FIELD OF THE INVENTION
The present invention relates to novel methods of treatment and novel uses of modified forms of Factor VII that inhibit thrombus formation, maintain or improve vascular patency, improve regional myocardial blood flow, and modulates myocardial injury during post-ischemic reperfusion.
BACKGROUND OF THE INVENTION
Blood coagulation is a process consisting of a complex interaction of various blood components, or factors, which eventually gives rise to a fibrin clot. Generally, the blood components which participate in what has been referred to as the coagulation "cascade" are proenzymes or zymogens, enzymatically inactive proteins which are converted to proteolytic enzymes by the action of an activator, itself an activated clotting factor. Coagulation factors that have undergone such a conversion and generally referred to as "active factors," and are designated by the addition of a lower case "a" suffix (e.g., Factor VIIa).
Activated Factor X ("Xa") is required to convert prothrombin to thrombin, which then converts fibrinogen to fibrin as a final stage in forming a fibrin clot. There are two systems, or pathways, that promote the activation of Factor X. The "intrinsic pathway" refers to those reactions that lead to thrombin formation through utilization of factors present only in plasma. A series of protease-mediated activations ultimately generates Factor IXa which, in conjunction with Factor VIIIa, cleaves Factor X into Xa. An identical proteolysis is effected by Factor VIIa and its co-factor, tissue factor, in the "extrinsic pathway" of blood coagulation. Tissue factor is a membrane bound protein and does not normally circulate in plasma. Upon vessel disruption, however, it can complex with Factor VIIa to catalyze Factor X activation or Factor IX activation in the presence of Ca.sup.++ and phospholipid (Nemerson and Gentry, Biochem. 25:4020-4033 (1986)). While the relative importance of the two coagulation pathways in hemostasis is unclear, in recent years Factor VII and tissue factor have been found to play a pivotal role in the regulation of blood coagulation.
Factor VII is a trace plasma glycoprotein that circulates in blood as a single-chain zymogen. The zymogen is catalytically inactive (Williams et al., J. Biol. Chem. 264:7536-7543 (1989); Rao et al., Proc. NatI. Acad. Sci. USA. 85:6687-6691 (1988)). Single-chain Factor VII may be converted to two-chain Factor VIIa by Factor Xa, Factor Xlla, Factor IXa or thrombin in vitro. Factor Xa is believed to be the major physiological activator of Factor VII. Like several other plasma proteins involved in hemostasis, Factor VII is dependent on vitamin K for its activity, which is required for the g-carboxylation of multiple glutamic acid residues that are clustered in the amino terminus of the protein. These g-carboxylated glutamic acids are required for the metal-associated interaction of Factor VII with phospholipids.
The conversion of zymogen Factor VII into the activated two-chain molecule occurs by cleavage of an internal peptide bond located approximately in the middle of the molecule. In human Factor VII, the activation cleavage site is at Arg.sub.152 -lle.sub.153 (Hagen et al., Proc. Natl. Acad. Sci. USA 83: 2412-2416 (1986); Thim et al., Biochem. 27:7785-7793 (1988) both of which are incorporated herein by references). Bovine factor VII is activated by cleavage at the analogous Arg.sub.152 -lle.sub.153 bond (Takeya et al., J. Biol. Chem. 263: 14868-14877, 1988). In the presence of tissue factor, phospholipids and calcium ions, the two-chain Factor VIIa rapidly activates Factor X or Factor IX by limited proteolysis.
It is often necessary to selectively block the coagulation cascade in a patient. Anticoagulants such as heparin, coumarin, derivatives of coumarin, indandione derivatives, or other agents may be used, for example, during kidney dialysis, or to treat deep vein thrombosis, disseminated intravascular coagulation (DIC), and a host of other medical disorders. For example, heparin treatment or extracorporeal treatment with citrate ion (U.S. Pat. No. 4,500,309) may be used in dialysis to prevent coagulation during the course of treatment. Heparin is also used in preventing deep vein thrombosis in patients undergoing surgery.
Treatment with heparin and other anticoagulants may, however, have undesirable side effects. Available anticoagulants generally act throughout the body, rather than acting specifically at a clot site. Heparin, for example, may cause heavy bleeding. Furthermore, with a half-life of approximately 80 minutes, heparin is rapidly cleared from the blood, necessitating frequent administration. Because heparin acts as a cofactor for antithrombin III (AT III), and AT III is rapidly depleted in DIC treatment, it is often difficult to maintain the proper heparin dosage, necessitating continuous monitoring of AT III and heparin levels. Heparin is also ineffective if AT III depletion is extreme. Further, prolonged use of heparin may also increase platelet aggregation and reduce platelet count, and has been implicated in the development of osteoporosis. Indandione derivatives may also have toxic side effects.
In addition to the anticoagulants briefly described above, several naturally occurring proteins have been found to have anticoagulant activity. For example, Reutelingsperger (U.S. Pat. No. 4,736,018) isolated anticoagulant proteins from bovine aorta and human umbilical vein arteries. Maki et al. (U.S. Pat. No. 4,732,891) disclose human placenta-derived anticoagulant proteins. In addition, AT III has been proposed as a therapeutic anticoagulant (Schipper et al., Lancet 1 (8069): 854-856 (1978); Jordan, U.S. Pat. No. 4,386,025; Bock et al., U.S. Pat. No. 4,517,294).
Proliferation of smooth muscle cells (SMCs) in the vessel wall is an important event in the formation of vascular lesions in atherosclerosis, after vascular reconstruction or in response to other vascular injury. For example, treatment of atherosclerosis frequently includes the clearing of blocked vessels by angioplasty, endarterectomy or reduction atherectomy, or by bypass grafting, surgical procedures in which atherosclerotic plaques are compressed or removed through catheterization (angioplasty), stripped away from the arterial wall through an incision (endarterectomy) or bypassed with natural or synthetic grafts. These procedures remove the vascular endothelium, disturb the underlying intimal layer, and result in the death of medial SMCs. This injury is followed by medial SMC proliferation and migration into the intima, which characteristically occurs within the first few weeks and up to six months after injury and stops when the overlying endothelial layer is reestablished. In humans, these lesions are composed of about 20% cells and 80% extracellular matrix.
In about 30% or more of patients treated by angioplasty, endarterectomy or bypass grafts, thrombosis and/or SMC proliferation in the intima causes re-occlusion of the vessel and consequent failure of the reconstructive surgery. This closure of the vessel subsequent to surgery is known as restenosis.
There is still a need in the art for improved compositions having anticoagulant activity which can be administered at relatively low doses and do not produce the undesirable side effects associated with traditional anticoagulant compositions. The present invention fulfills this need by providing anticoagulants that act specifically at sites of injury, and further provides other related advantages. The modified Factor VII molecules are particularly useful for administration to humans to treat a variety of conditions involving intravascular coagulation. For example, although deep vein thrombosis and pulmonary embolism can be treated with conventional anticoagulants, the modified Factor VII described herein may be used to prevent the occurrence of thromboembolic complications in identified high risk patients, such as those undergoing surgery or those with congestive heart failure. In addition, modified Factor VII may act as an antagonist for tissue factor-mediated induction of coagulation, thus blocking the production of thrombin and the subsequent deposition of fibrin. As such, modified Factor VII may be useful for inhibiting tissue factor activity resulting in, for example, the inhibition of blood coagulation, thrombosis or platelet deposition.
The modified Factor VII molecules may be particularly useful in the treatment of intimal hyperplasia or restenosis due to acute vascular injury. Acute vascular injuries are those which occur rapidly (i.e. over days to months), in contrast to chronic vascular injuries (e.g. atherosclerosis) which develop over a lifetime. Acute vascular injuries often result from surgical procedures such as vascular reconstruction, wherein the techniques of angioplasty, endarterectomy, atherectomy, vascular graft emplacement or the like are employed. Hyperplasia may also occur as a delayed response in response to, e.g., graft emplacement or organ transplantation. Since modified Factor VII is more selective than heparin, generally binding only tissue factor which has been exposed at sites of injury, and because modified Factor VII does not destroy other coagulation proteins, it will be more effective and less likely to cause bleeding complications than heparin when used prophylactically for the prevention of deep vein thrombosis.
Recent advances in the treatment of coronary vascular disease include the use of mechanical interventions to either remove or displace offending plaque material in order to re-establish adequate blood flow through the coronary arteries. Despite the use of multiple forms of mechanical interventions, including balloon angioplasty, reduction atherectomy, placement of vascular stents, laser therapy, or rotoblator, the effectiveness of these techniques remains limited by an approximately 40% restenosis rate within 6 months after treatment.
Restenosis is thought to result from a complex interaction of biological processes including platelet deposition and thrombus formation, release of chemotactic and mitogenic factors, and the migration and proliferation of vascular smooth muscle cells into the intima of the dilated arterial segment.
The inhibition of platelet accumulation at sites of mechanical injury can limit the rate of restenosis in human subjects. Therapeutic use of a monoclonal antibody to platelet GpIIb/IIIa is able to limit the level of restenosis in human subjects (Califf et al., N. Engl. J. Med., 330:956-961 (1994)). The antibody is able to bind to the GpIIb/IIa receptor on the surfaces of platelets and thereby inhibit platelet accumulation. This data suggests that inhibition of platelet accumulation at the site of mechanical injury in human coronary arteries is beneficial for the ultimate healing response that occurs. While platelet accumulation occurs at sites of acute vascular injuries, the generation of thrombin at these sites may be responsible for the activation of the platelets and their subsequent accumulation.
As shown in the examples that follow, the modified Factor VII of the present invention is able to bind to cell-surface tissue factor. For example, DEGR-Factor VIIa binds cell-surface tissue factor with an equivalent or higher affinity than wild-type Factor VIIa. DEGR-Factor VIIa, however, has no enzymatic activity, yet it binds to tissue factor and acts as a competitive antagonist for wild-type Factor VIIa, thereby inhibiting the subsequent steps in the extrinsic pathway of coagulation leading to the generation of thrombin.
Modified Factor VII molecules which maintain tissue factor binding inhibit platelet accumulation at the site of vascular injury by blocking the production of thrombin and the subsequent deposition of fibrin.
Due to the ability of DEGR-Factor VII to block thrombin generation and limit platelet deposition at sites of acute vascular injury, modified Factor VII molecules which maintain tissue factor binding activity but lack Factor VIIa enzymatic activity can be used to inhibit vascular restenosis.
Compositions comprising modified Factor VII are particularly useful in methods for treating patients when formulated into pharmaceutical compositions, where they may be given to individuals suffering from a variety of disease states to treat coagulation-related conditions. Such modified Factor VII molecules, capable of binding tissue factor but having a substantially reduced ability to catalyze activation of other factors in the clotting cascade, may possess a longer plasma half-life and thus a correspondingly longer period of anticoagulative activity when compared to other anticoagulants. Among the medical indications for the subject compositions are those commonly treated with anticoagulants, such as, for example, deep vein thrombosis, pulmonary embolism, stroke, disseminated intravascular coagulation (DIC), fibrin deposition in lungs and kidneys associated with gram-negative endotoxemia, and myocardial infarction. The compositions can be used to inhibit vascular restenosis as occurs following mechanical vascular injury, such as injury caused by balloon angioplasty, endarterectomy, reductive atherectomy, stent placement, laser therapy or rotablation, or as occurs secondary to vascular grafts, stents, bypass grafts or organ transplants. The compositions can thus be used to inhibit platelet deposition and associated disorders. Thus, a method of inhibiting coagulation, vascular restenosis or platelet deposition, for example, comprises administering to a patient a composition comprising modified Factor VII, such as that having at least one amino acid substitution in a catalytic triad of Seru.sub.344, Asp.sub.242 and His.sub.193, in an amount sufficient to effectively inhibit coagulation, vascular restenosis or platelet deposition. The methods also find use in the treatment of acute closure of a coronary artery in an individual (e.g. acute myocardial infarction), which comprises administering the modified Factor VII, which includes DEGR-Factor VII and FFR-Factor VII, in conjunction with tissue plasminogen activator or streptokinase, and can accelerate tPA induced thrombolysis. The modified Factor VII is given prior to, in conjunction with, or shortly following administration of a thrombolytic agent, such as tissue plasminogen activator.
International Application No. WO 92/15686 relates to modified Factor VIIa, polynucleic acid and mammalian cell lines for the production of modified Factor VIIa, and compositions comprising modified Factor VI la for inhibiting blood coagulation.
International Application No. WO 94/27631 relates to methods for inhibiting vascular restenosis, tissue factor activity, and platelet deposition.
International Application No. WO 96/12800 relates to a method for treatment of acute closure of a coronary artery comprising to the individual a composition which comprises modified Factor VIIa in conjunction with tissue plasminogen activator or streptokinase.
SUMMARY OF THE INVENTION
The present invention relates to a method for inhibiting thrombus formation in a patient comprising administering topically to a vascular site susceptible to thrombus formation in the patient a therapeutically effective dose of a composition comprising Factor VII having at least one modification in its catalytic center, which modification substantially inhibits the ability of the modified Factor VII to activate plasma Factor X or IX. The site of thrombus formation may be associated with surgery, microsurgery, angioplasty or trauma.
The invention further relates to a method for maintaining or improving vascular patency in a patient comprising administering locally to a vascular site susceptible to decreased patency a therapeutically effective dose of a composition comprising Factor VII having at least one modification in its catalytic center, which modification substantially inhibits the ability of the modified Factor VII to activate plasma Factor X or IX. The site of thrombus formation may be associated with surgery, microsurgery, angioplasty or trauma.
The invention further relates to a method for preventing or minimizing myocardial injury associated with post-ischemic reperfusion in an individual, comprising administering to the individual a composition which comprises a pharmacologically acceptable Factor VII having at least one modification in its catalytic center, which modification substantially inhibits the ability of the modified Factor VII to activate plasma Factor X or IX.
The invention further relates to a method for improving regional myocardial blood flow during post-ischemic reperfusion in an individual, comprising administering to the individual a composition which comprises a pharmacologically acceptable Factor VII having at least one modification in its catalytic center, which modification substantially inhibits the ability of the modified Factor VII to activate plasma Factor X or IX.
In a preferred embodiment the modification of Factor VII comprises reaction of the Factor VII with a serine protease inhibitor. In a more preferred aspect the protease inhibitor is an organophosphor compound, a sulfanyl fluoride, a peptide halomethyl ketone, or an azapeptide. In an even more preferred aspect the protease inhibitor is a peptide halomethyl ketone selected from Dansyl-Phe-Pro-Arg chloromethyl ketone, Dansyl-Glu-Gly-Arg chloromethyl ketone, Dansyl-Phe-Phe-Arg chloromethyl ketone and Phe-Phe-Arg chloromethylketone, Phe-Phe-Arg chloromethylketone being the most preferred.
The invention further relates to the use of Factor VII having at least one modification in its catalytic center, which modification substantially inhibits the ability of the modified Factor VII to activate plasma Factor X or IX, for the manufacture of a composition for preventing or minimizing myocardial injury associated with post-ischemic reperfusion. The invention further relates to the use of Factor VII having at least one modification in its catalytic center, which modification substantially inhibits the ability of the modified Factor VII to activate plasma Factor X or IX, for the manufacture of a composition for improving regional myocardial blood flow during post-ischemic reperfusion.
The present invention provides methods and compositions to inhibit deleterious events associated with ischemic reperfusion. Severe ischemia to a tissue, organ or limb may be due to a decrease in blood flow and may be associated with trauma, surgical manipulation, or lowered blood pressure. One of the complications associated with severe ischemia is the up-regulation of tissue factor in the arterial system. This increased expression of tissue factor is believed to stimulate a procoagulant response, primarily in the capillary bed, thus initiating and/or sustaining intravascular thrombus formation. Furthermore, the de novo synthesis of TF during reperfusion of post-ischemic hearts by endothelial cells within the coronary vasculature may lead to a decrease in coronary blood flow during reperfusion and thus influencing the fate of the ischemic myocardium that will ultimately undergo necrosis. TF antigen and procoagulant activity is increased in atherectomy specimens obtained from patients with unstable angina, as compared to patients with stable angina. Thus, it is believed that in these patients unstable angina may be precipitated by exposure of TF in the subendothelial tissue of a large epicardial coronary artery as a result of plaque damage. This will eventually promote an intracoronary thrombus formation with a consequent absolute reduction in coronary flow.
TF may also effect coronary flow in a different way. Following reperfusion to the ischemic tissue, thrombi can be generated which may be either occlusive or non-occlusive. The generation of thrombi in the arterial bed, and the deposition of platelets along the thrombus, lead to the secondary generation of ischemia to the tissue. The generation of the thrombi and the presence of platelets can then cause the generation and release of multiple bioactive factors, including those generated from the coagulation pathway, such as thrombin and Factor X, as well as factors released from activated platelets. In turn, these factors may induce the generation of additional factors by the underlying endothelial and smooth muscle cells, or by adjacent mononuclear cells, such as TNF-alpha and IL-1. These factors, in turn, can then activate the endothelial cells leading to the up-regulation of various adhesion molecules associated with monocyte and neutrophil binding. Normally, endothelial cells, being in contact with circulating blood, do not express significant TF activity. Under certain circumstances endothelial cells may actively promote coagulation by expressing TF-like procoagulant activity. In particular, both exogenous and endogenously generated oxygen free radicals (OFRs) can stimulate endothelial cells within the coronary vasculature to synthesize and express significant amounts of TF. OFRs are highly reactive molecular species that may attack various cell constituents. A burst of OFR generation follows restoration of flow after a period of ischemia, and these oxidant species might be responsible for a specific form of reperfusion-mediated tissue injury, secondary to lipid peroxidation and other irreversible alterations of cell constituents. OFRs also dramatically decrease the activity of tissue factor pathway inhibitor (TFPI), a Kunitz-type protein synthesized by endothelial cells, which inhibits the extrinsic coagulation pathway. This double effect of OFRs (TF expression by endothelial cells and decrease in TFPI activity) may shift the natural anticoagulant properties of the normal endothelium toward a procoagulant state, thus favouring an unwanted intravascular activation of the coagulation. Thus, OFR-mediated TF expression within the coronary circulation results in a significant reduction in coronary blood flow during post-ischemic reperfusion. This OFR-mediated expression of TF, with its attendant activation of the extrinsic coagulation pathway, has important consequences, as this phenomenon impacts on the pathophysiology of post-ischemic reperfusion, particularly in patients with acute myocardial infarction undergoing coronary thrombolysis.
The no-reflow phenomenon, that is, lack of uniform perfusion to the microvasculature of a previously ischemic tissue has been described for the first time by Krug et al., (Circ. Res. 1966; 19:57-62). The most important determinants that may influence the fate of ischemic myocardium are believed to be the amount of collateral flow during ischemia, the size of the area at risk and the myocardial oxygen demand.
Over the past decade there has been intense interest in the concept of treating patients with acute myocardial infarction with reperfusion strategies, including coronary thrombolysis, primary angioplastry, or both, However, nor all studies have demonstrated an improvement in left ventricular function after recanalization of the infarct-related artery. At the moment, a substantial number of patients exhibit a "low-flow" condition in the infarct-related coronary bed. This condition is related with an almost complete lack of benefits at least in the term of mortality. These low-flow conditions is believed to be caused, at least in part, by the inability of blood to re-enter all of the vasculature of the previously ischemic myocardium. It has now surprisingly been shown that FVIIai effects, and increases, the regional myocardial blood flow during reperfusion. It has also surprisingly been shown that FVIIai results in a significant reduction in the area of no-reflow,
The binding and transmigration of monocytes and neutrophils, the release of bioactive compounds by these cells, including the generation of free-oxygen radicals, can exacerbate the level of endothelial cell activation and damage. Ultimately, if the cascade of events goes unchecked, this can lead to systemic complications and the potential to stimulate multiple organ failure. By blocking tissue factor by administering a specific inhibitor for tissue factor/Factor VII binding (e.g., FFR-FVIIa), and thereby blocking the initiation of the extrinsic pathway of coagulation, the initiation of the cascade of events may be prevented, thereby modulating the extent deleterious events associated with ischemia/reperfusion, such as, for example, eliminating, or minimizing the myocardial injury or necrosis.

BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 illustrates the construction of an expression vector for a Ser.sub.344 .RTM.Ala modified Factor VII DNA sequence. Symbols used include 0-1, the 0-1 map unit sequence from adenovirus 5; E, the SV40 enhancer; MLP, the adenovirus 2 major late promotor; SS, a set of splice sites; and pA, the polyadenylation signal from SV40 in the late orientation.
FIG. 2 shows the effect of bolus injection of DEGR-Factor VIIa on thrombus formation (platelet deposition) on endarterectomized baboon aorta when compared to saline-treated controls. The arteries were measured over 60 minutes. The DEGR-Factor VIIa significantly inhibited the development of platelet-rich thrombi in this primate model of acute vascular injury.
FIG. 3 shows results obtained when baboon smooth muscle cells were incubated with increasing concentrations of either FVIIa (open box), or DEGR-FVIIa in the presence of a constant amount of FVIIa (5 nM) (closed box). The level of FX activation was subsequently determined using the chromogenic substrate S-2222. The data are presented as the amidolytic activity as a percentage of the activity generated in the presence of 5 nM FVIIa alone.
FIG. 4 depicts the size of the intimal area of baboons following carotid artery endarterectomy and treatment with DEGR-Factor VIIa for 7 or 30 days, compared to control animals.
FIG. 5 illustrates the ratio of the intimal area to the intimal+media area of baboon femoral artery following balloon injury and treatment with DEGR-Factor VIIa, where the control group included 5 vessels, 7 day treatment examined 11 vessels, and 30 day treatment examined 2 vessels (n=number of vessels examined).
FIG. 6 illustrates the experimental protocol for measuring infarct size (IS), no-reflow (NR), area at risk (AR), prothrombin time (PT) and activated partial thromboplastin time (aPTT).
FIG. 7 shows a plot of infarct size at the end of the reperfusion period (IS) expressed as a percent of the area at risk of infarcting in the three treatment groups, the three treatment groups being animals treated with FFR-Factor VIIa, Factor VIIa and saline, respectively. Each bar represents the mean of eight animals .+-.SD.
FIG. 8 shows a plot of the area of no-reflow (NR) at the end of the reperfusion period expressed as a percent of the area at risk of infarcting. (animals treated with FFR-Factor VIIa, Factor VIIa and saline, respectively.) Each bar represents the mean of eight animals .+-.SD.
FIG. 9 shows the relationship between expected no-reflow calculated (as a percent of left ventricle, LV) for each animal by the multiple linear regression equation and the no-reflow actually observed (as percent of LV).
FIG. 10 shows a plot of regional myocardial blood flow (RMBF) for ischemic myocardium assesed at 20 min of ischemia, and after 10 min and 2 hrs of reperfusion.
FIG. 11 shows the effect of FFR-Factor VIIa and Factor VIIa on prothrombin time (PT) and activated partial thromboplastin time (aPTT).

DESCRIPTION OF THE SPECIFIC EMBODIMENTS
Modified Factor VII can be in the form of the zymogen (i.e., a single-chain molecule) or can be cleaved at its activation site. Thus, by "modified Factor VII" is meant to include modified Factor VII and modified Factor VIIa molecules that bind tissue factor and inhibit the activation of Factor IX to IXa and Factor X to Xa. The Factor VII sequence has at least one amino acid modification, where the modification is selected so as to substantially reduce the ability of activated Factor VII to catalyze the activation of plasma Factors X or IX, and thus is capable of inhibiting clotting activity. The modified Factor VII has an active site modified by at least one amino acid substitution, and in its modified form is capable of binding tissue factor. The modified Factor VII compositions are typically in substantially pure form.
In preferred embodiments of human and bovine Factor VII, the active site residue Ser.sub.344 is modified, replaced with Gly, Met, Thr, or more preferably, Ala. Such substitution could be made separately or in combination with substitution(s) at other sites in the catalytic triad, which includes His.sub.193 and Asp.sub.242.
Compositions of the modified Factor VII are suitable for administration to a variety of mammals, particularly humans, to inhibit the coagulation cascade. Modified Factor VII may be administered to a patient in conjunction with or in place of other anticoagulant compounds. Typically, for administration to humans the pharmaceutical compositions will comprise modified human Factor VII protein and pharmaceutically-acceptable carriers and buffers.
Factor VII plays an important role in the coagulation cascade, particularly that involving the extrinsic pathway. Present in the circulating plasma as an inactive single chain zymogen protein, once activated, Factor VIIa, in combination with tissue factor and calcium ions, activates Factor X to Xa and activates Factor IX to IXa, with the eventual formation of a fibrin clot.
Factor VII proteins have a catalytic site which is modified to decrease the catalytic activity of Factor VIIa, while the molecule retains the ability to bind to tissue factor. The modified Factor VII molecules compete with native Factor VII and/or VIIa for binding to tissue factor. As a result, the activation of Factors X and IX is inhibited.
Modified Factor VII may be encoded by a polynucleotide molecule comprising two operatively linked sequence coding regions encoding, respectively, a pre-pro peptide and a gla domain of a vitamin K-dependent plasma protein, and a gla domain-less Factor VII protein, wherein upon expression said polynucleotide encodes a modified Factor VII molecule which does not significantly activate plasma Factors X or IX, and is capable of binding tissue factor. The modified Factor VII molecule expressed by this polynucleotide is a biologically active anticoagulant, that is, it is capable of inhibiting the coagulation cascade and thus the formation of a fibrin deposit or clot. To express the modified Factor VII the polynucleotide molecule is transfected into mammalian cell lines, such as, for example, BHK, BHK 570 or 293 cell lines.
The catalytic activity of Factor VIIa can be inhibited by chemical derivatization of the catalytic center, or triad. Derivatization may be accomplished by reacting Factor VII with an irreversible inhibitor such as an organophosphor compound, a sulfonyl fluoride, a peptide halomethyl ketone or an azapeptide, or by acylation, for example. Preferred peptide halomethyl ketones include PPACK (D-Phe-Pro-Arg chloromethyl-ketone; (see U.S. Pat. No. 4,318,904, incorporated herein by reference), D-Phe-Phe-Arg and Phe-Phe-Arg chloromethylketone (FFR-cmk); and DEGRck (dansyl-Glu-Gly-Arg chloromethylketone).
The catalytic activity of Factor VIIa can also be inhibited by substituting, inserting or deleting amino acids. In preferred embodiments amino acid substitutions are made in the amino acid sequence of the Factor VII catalytic triad, defined herein as the regions which contain the amino acids which contribute to the Factor VIIa catalytic site. The substitutions, insertions or deletions in the catalytic triad are generally at or adjacent to the amino acids which form the catalytic site. In the human and bovine Factor VII proteins, the amino acids which form a catalytic "triad" are Ser.sub.344, Asp.sub.242, and His.sub.193 (subscript numbering indicating position in the sequence). The catalytic sites in Factor VII from other mammalian species may be determined using presently available techniques including, among others, protein isolation and amino acid sequence analysis. Catalytic sites may also be determined by aligning a sequence with the sequence of other serine proteases, particularly chymotrypsin, whose active site has been previously determined (Sigler et al., J. Mol. Biol., 35:143-164 (1968), incorporated herein by reference), and therefrom determining from said alignment the analogous active site residues.
The amino acid substitutions, insertions or deletions are made so as to prevent or otherwise inhibit activation by the Factor VIIa of Factors X and/or IX. The Factor VII so modified should, however, also retain the ability to compete with authentic Factor VII and/or Factor VIIa for binding to tissue factor in the coagulation cascade. Such competition may readily be determined by means of, e.g., a clotting assay as described herein, or a competition binding assay using, e.g., a cell line having cell-surface tissue factor, such as the human bladder carcinoma cell line J82 (Sakai et al. J. Biol. Chem. 264: 9980-9988 (1989), incorporated by reference herein.)
The amino acids which form the catalytic site in Factor VII, such as Ser.sub.344, Asp.sub.242, and His.sub.193 in human and bovine Factor VII, may either be substituted or deleted. Within the present invention, it is preferred to change only a single amino acid, thus minimizing the likelihood of increasing the antigenicity of the molecule or inhibiting its ability to bind tissue factor, however two or more amino acid changes (substitutions, additions or deletions) may be made and combinations of substitution(s), addition(s) and deletion(s) may also be made. In a preferred embodiment for human and bovine Factor VII, Ser.sub.344 is preferably substituted with Ala, but Gly, Met, Thr or other amino acids can be substituted. It is preferred to replace Asp with Glu and to replace His with Lys or Arg. In general, substitutions are chosen to disrupt the tertiary protein structure as little as possible. The model of Dayhoff et al. (in Atlas of Protein Structure 1978, Nat'l Biomed. Res. Found., Washington, D.C.), incorporated herein by reference, may be used as a guide in selecting other amino acid substitutions. One may introduce residue alterations as described above in the catalytic site of appropriate Factor VII sequence of human, bovine or other species and test the resulting protein for a desired level of inhibition of catalytic activity and resulting anticoagulant activity as described herein. For the modified Factor VII the catalytic activity will be substantially inhibited, generally less than about 5% of the catalytic activity of wild-type Factor VII of the corresponding species, more preferably less than about 1%.
The modified Factor VII may be produced through the use of recombinant DNA techniques. In general, a cloned wild-type Factor VII DNA sequence is modified to encode the desired protein. This modified sequence is then inserted into an expression vector, which is in turn transformed or transfected into host cells. Higher eukaryotic cells, in particular cultured mammalian cells, are preferred as host cells. The complete nucleotide and amino acid sequences for human Factor VII are known. See U.S. Pat. No. 4,784,950, which is incorporated herein by reference, where the cloning and expression of recombinant human Factor VII is described. The bovine Factor VII sequence is described in Takeya et al., J. Biol. Chem. 263:14868-14872 (1988), which is incorporated by reference herein.
The amino acid sequence alterations may be accomplished by a variety of techniques. Modification of the DNA sequence may be by site-specific mutagenesis. Techniques for site-specific mutagenesis are well known in the art and are described by, for example, Zoller and Smith (DNA 3:479-488, 1984). Thus, using the nucleotide and amino acid sequences of Factor VII, one may introduce the alteration(s) of choice.
The Factor VII modified accordingly includes those proteins that have the amino-terminal portion (gla domain) substituted with a gla domain of one of the vitamin K-dependent plasma proteins Factor IX, Factor X, prothrombin, protein C, protein S or protein Z. The gla domains of the vitamin K-dependent plasma proteins are characterized by the presence of gamma-carboxy glutamic acid residues and are generally from about 30 to about 40 amino acids in length with C-termini corresponding to the positions of exon-intron boundaries in the respective genes. Methods for producing Factor VII with a heterologous gla domain are disclosed in U.S. Pat. No. 4,784,950, incorporated by reference herein.
DNA sequences for use in producing modified Factor VII will typically encode a pre-pro peptide at the amino-terminus of the Factor VII protein to obtain proper post-translational processing (e.g. gamma-carboxylation of glutamic acid residues) and secretion from the host cell. The pre-pro peptide may be that of Factor VII or another vitamin K-dependent plasma protein, such as Factor IX, Factor X, prothrombin, protein C or protein S. As will be appreciated by those skilled in the art, additional modifications can be made in the amino acid sequence of the modified Factor VII where those modifications do not significantly impair the ability of the protein to act as an anticoagulant. For example, the Factor VII modified in the catalytic triad can also be modified in the activation cleavage site to inhibit the conversion of zymogen Factor VII into its activated two-chain form, as generally described in U.S. Pat. No. 5,288,629, incorporated herein by reference.
Expression vectors for use in expressing modified Factor VIIa will comprise a promoter capable of directing the transcription of a cloned gene or cDNA. Preferred promoters for use in cultured mammalian cells include viral promoters and cellular promoters. Viral promoters include the SV40 promoter (Subramani et al., Mol. Cell. Biol. 1:854-864, 1981) and the CMV promoter (Boshart et al., Cell 41:521-530, 1985). A particularly preferred viral promoter is the major late promoter from adenovirus (Kaufman and Sharp, Mol. Cell. Biol. 2:1304-1319, 1982). Cellular promoters include the mouse kappa gene promoter (Bergman et al., Proc. Natl. Acad. Sci. USA 81:7041-7045, 1983) and the mouse V.sub.H promoter (Loh et al., Cell 33:85-93, 1983). A particularly preferred cellular promoter is the mouse metallothionein-l promoter (Palmiter et al., Science 222:809-814, 1983). Expression vectors may also contain a set of RNA splice sites located downstream from the promoter and upstream from the insertion site for the Factor VII sequence itself. Preferred RNA splice sites may be obtained from adenovirus and/or immunoglobulin genes. Also contained in the expression vectors is a polyadenylation signal located downstream of the insertion site. Particularly preferred polyadenylation signals include the early or late polyadenylation signal from SV40 (Kaufman and Sharp, ibid.), the polyadenylation signal from the adenovirus 5 Elb region, the human growth hormone gene terminator (DeNoto et al. Nuc. Acids Res. 9:3719-3730, 1981) or the polyadenylation signal from the human Factor VII gene or the bovine Factor VII gene. The expression vectors may also include a noncoding viral leader sequence, such as the adenovirus 2 tripartite leader, located between the promoter and the RNA splice sites; and enhancer sequences, such as the SV40 enhancer.
Cloned DNA sequences are introduced into cultured mammalian cells by, for example, calcium phosphate-mediated transfection (Wigler et al., Cell 14:725-732, 1978; Corsaro and Pearson, Somatic Cell Genetics 7:603-616, 1981; Graham and Van der Eb, Virology 52d:456-467, 1973) or electroporation (Neumann et al., EMBO J. 1:841-845, 1982). To identify and select cells that express the exogenous DNA, a gene that confers a selectable phenotype (a selectable marker) is generally introduced into cells along with the gene or cDNA of interest. Preferred selectable markers include genes that confer resistance to drugs such as neomycin, hygromycin, and methotrexate. The selectable marker may be an amplifiable selectable marker. A preferred amplifiable selectable marker is a dihydrofolate reductase (DHFR) sequence. Selectable markers are reviewed by Thilly (Mammalian Cell Technology, Butterworth Publishers, Stoneham, Mass., incorporated herein by reference). The choice of selectable markers is well within the level of ordinary skill in the art.
Selectable markers may be introduced into the cell on a separate plasmid at the same time as the gene of interest, or they may be introduced on the same plasmid. If on the same plasmid, the selectable marker and the gene of interest may be under the control of different promoters or the same promoter, the latter arrangement producing a dicistronic message. Constructs of this type are known in the art (for example, Levinson and Simonsen, U.S. Pat. No. 4,713,339). It may also be advantageous to add additional DNA, known as "carrier DNA," to the mixture that is introduced into the cells.
After the cells have taken up the DNA, they are grown in an appropriate growth medium, typically 1-2 days, to begin expressing the gene of interest. As used herein the term "appropriate growth medium" means a medium containing nutrients and other components required for the growth of cells and the expression of the modified Factor VII gene. Media generally include a carbon source, a nitrogen source, essential amino acids, essential sugars, vitamins, salts, phospholipids, protein and growth factors. For production of gamma-carboxylated modified Factor VII, the medium will contain vitamin K, preferably at a concentration of about 0.1 mg/ml to about 5 mg/ml. Drug selection is then applied to select for the growth of cells that are expressing the selectable marker in a stable fashion. For cells that have been transfected with an amplifiable selectable marker the drug concentration may be increased to select for an increased copy number of the cloned sequences, thereby increasing expression levels. Clones of stably transfected cells are then screened for expression of modified Factor VII.
Preferred mammalian cell lines include the COS-1 (ATCC CRL 1650), baby hamster kidney (BHK) and 293 (ATCC CRL 1573; Graham et al., J. Gen. Virol. 36:59-72, 1977) cell lines. A preferred BHK cell line is the tk.sup.- ts13 BHK cell line (Waechter and Baserga, Proc. Natl. Acad. Sci. USA 79:1106-1110, 1982, incorporated herein by reference), hereinafter referred to as BHK 570 cells. The BHK 570 cell line has been deposited with the American Type Culture Collection, 12301 Parklawn Dr., Rockville, Md. 20852, under ATCC accession number CRL 10314. A tk.sup.- ts13 BHK cell line is also available from the ATCC under accession number CRL 1632. In addition, a number of other cell lines may be used, including Rat Hep I (Rat hepatoma; ATCC CRL 1600), Rat Hep II (Rat hepatoma; ATCC CRL 1548), TCMK (ATCC CCL 139), Human lung (ATCC HB 8065), NCTC 1469 (ATCC CCL 9.1), CHO (ATCC CCL 61) and DUKX cells (Urlaub and Chasin, Proc. Natl. Acad. Sci. USA 77:4216-4220, 1980).
Transgenic animal technology may be employed to produce modified Factor VII. It is preferred to produce the proteins within the mammary glands of a host female mammal. Expression in the mammary gland and subsequent secretion of the protein of interest into the milk overcomes many difficulties encountered in isolating proteins from other sources. Milk is readily collected, available in large quantities, and well characterized biochemically. Furthermore, the major milk proteins are present in milk at high concentrations (typically from about 1 to 15 g/l).
From a commercial point of view, it is clearly preferable to use as the host a species that has a large milk yield. While smaller animals such as mice and rats can be used (and are preferred at the proof of principle stage), it is preferred to use livestock mammals including, but not limited to, pigs, goats, sheep and cattle. Sheep are particularly preferred due to such factors as the previous history of transgenesis in this species, milk yield, cost and the ready availability of equipment for collecting sheep milk. See WIPO Publication WO 88/00239 for a comparison of factors influencing the choice of host species. It is generally desirable to select a breed of host animal that has been bred for dairy use, such as East Friesland sheep, or to introduce dairy stock by breeding of the transgenic line at a later date. In any event, animals of known, good health status should be used.
To obtain expression in the mammary gland, a transcription promoter from a milk protein gene is used. Milk protein genes include those genes encoding caseins (see U.S. Pat. No. 5,304,489, incorporated herein by reference), beta-lactoglobulin, a-lactalbumin, and whey acidic protein. The beta-lactoglobulin (BLG) promoter is preferred. In the case of the ovine beta-lactoglobulin gene, a region of at least the proximal 406 bp of 5' flanking sequence of the gene will generally be used, although larger portions of the 5' flanking sequence, up to about 5 kbp, are preferred, such as a .about.4.25 kbp DNA segment encompassing the 5' flanking promoter and non-coding portion of the beta-lactoglobulin gene. See Whitelaw et al., Biochem J. 286: 31-39 (1992). Similar fragments of promoter DNA from other species are also suitable.
Other regions of the beta-lactoglobulin gene may also be incorporated in constructs, as may genomic regions of the gene to be expressed. It is generally accepted in the art that constructs lacking introns, for example, express poorly in comparison with those that contain such DNA sequences (see Brinster et al., Proc. Natl. Acad. Sci. USA 85: 836-840 (1988); Palmiter et al., Proc. Natl. Acad. Sci. USA 88: 478-482 (1991); Whitelaw et al., Transgenic Res. 1: 3-13 (1991); WO 89/01343; and WO 91/02318, each of which is incorporated herein by reference). In this regard, it is generally preferred, where possible, to use genomic sequences containing all or some of the native introns of a gene encoding the protein or polypeptide of interest, thus the further inclusion of at least some introns from, e.g, the beta-lactoglobulin gene, is preferred. One such region is a DNA segment which provides for intron splicing and RNA polyadenylation from the 3' non-coding region of the ovine beta-lactoglobulin gene. When substituted for the natural 3' non-coding sequences of a gene, this ovine beta-lactoglobulin segment can both enhance and stabilize expression levels of the protein or polypeptide of interest. Within other embodiments, the region surrounding the initiation ATG of the modified Factor VII sequence is replaced with corresponding sequences from a milk specific protein gene. Such replacement provides a putative tissue-specific initiation environment to enhance expression. It is convenient to replace the entire modified Factor VII pre-pro and 5' non-coding sequences with those of, for example, the BLG gene, although smaller regions may be replaced.
For expression of modified Factor VII in transgenic animals, a DNA segment encoding modified Factor VII is operably linked to additional DNA segments required for its expression to produce expression units. Such additional segments include the above-mentioned promoter, as well as sequences which provide for termination of transcription and polyadenylation of mRNA. The expression units will further include a DNA segment encoding a secretory signal sequence operably linked to the segment encoding modified Factor VII. The secretory signal sequence may be a native Factor VII secretory signal sequence or may be that of another protein, such as a milk protein. See, for example, von Heinje, Nuc. Acids Res. 14: 4683-4690 (1986); and Meade et al., U.S. Pat. No. 4,873,316, which are incorporated herein by reference.
Construction of expression units for use in transgenic animals is conveniently carried out by inserting a modified Factor VII sequence into a plasmid or phage vector containing the additional DNA segments, although the expression unit may be constructed by essentially any sequence of ligations. It is particularly convenient to provide a vector containing a DNA segment encoding a milk protein and to replace the coding sequence for the milk protein with that of a modified Factor VII polypeptide, thereby creating a gene fusion that includes the expression control sequences of the milk protein gene. In any event, cloning of the expression units in plasmids or other vectors facilitates the amplification of the modified Factor VII sequence. Amplification is conveniently carried out in bacterial (e.g. E. coli host cells, thus the vectors will typically include an origin of replication and a selectable marker functional in bacterial host cells.
The expression unit is then introduced into fertilized eggs (including early-stage embryos) of the chosen host species. Introduction of heterologous DNA can be accomplished by one of several routes, including microinjection (e.g. U.S. Pat. No. 4,873,191), retroviral infection (Jaenisch, Science 240: 1468-1474 (1988)) or site-directed integration using embryonic stem (ES) cells (reviewed by Bradley et al., Bio/Technology 10: 534-539 (1992)). The eggs are then implanted into the oviducts or uteri of pseudopregnant females and allowed to develop to term. Offspring carrying the introduced DNA in their germ line can pass the DNA on to their progeny in the normal, Mendelian fashion, allowing the development of transgenic herds.
General procedures for producing transgenic animals are known in the art. See, for example, Hogan et al., Manipulating the Mouse Embryo: A Laboratory Manual, Cold Spring Harbor Laboratory, 1986; Simons et al., Bio/Technology 6: 179-183 (1988); Wall et al., Biol. Reprod. 32: 645-651 (1985); Buhler et al., Bio/Technology 8: 140-143 (1990); Ebert et al., Bio/Technology 9: 835-838 (1991); Krimpenfort et al., Bio/Technology 9: 844-847 (1991); Wall et al., J. Cell. Biochem. 49: 113-120 (1992); U.S. Pat. Nos. 4,873,191 and 4,873,316; WIPO publications WO 88/00239, WO 90/05188, WO 92/11757; and GB 87/00458, which are incorporated herein by reference. Techniques for introducing foreign DNA sequences into mammals and their germ cells were originally developed in the mouse. See, e.g., Gordon et al., Proc. NatI. Acad. Sci. USA 77: 7380-7384 (1980); Gordon and Ruddle, Science 214: 1244-1246 (1981); Palmiter and Brinster, Cell 41: 343-345 (1985); Brinster et al., Proc. NatI. Acad. Sci. USA 82: 4438-4442 (1985); and Hogan et al. (ibid.). These techniques were subsequently adapted for use with larger animals, including livestock species (see e.g., WIPO publications WO 88/00239, WO 90/05188, and WO 92/11757; and
Simons et al., Bio/Technology 6: 179-183 (1988). To summarize, in the most efficient route used to date in the generation of transgenic mice or livestock, several hundred linear molecules of the DNA of interest are injected into one of the pro-nuclei of a fertilized egg according to established techniques. Injection of DNA into the cytoplasm of a zygote can also be employed.
Production in transgenic plants may also be employed. Expression may be generalized or directed to a particular organ, such as a tuber. See, Hiatt, Nature 344:469-479 (1990); Edelbaum et al., J. Interferon Res. 12:449-453 (1992); Sijmons et al., Bio/Technology 8:217-221 (1990); and European Patent Office Publication EP 255,378.
Modified Factor VII may be purified by affinity chromatography on an anti-Factor VII antibody column. The use of calcium-dependent monoclonal antibodies, as described by Wakabayashi et al., J. Biol. Chem. 261:11097-11108, (1986) and Thim et al., Biochem. 27: 7785-7793, (1988), incorporated by reference herein, is particularly preferred. Additional purification may be achieved by conventional chemical purification means, such as high performance liquid chromatography. Other methods of purification, including barium citrate precipitation, are known in the art, and may be applied to the purification of the novel modified Factor VII described herein (see, generally, Scopes, R., Protein Purification, Springer-Verlag, N.Y., 1982). Substantially pure modified Factor VII of at least about 90 to 95% homogeneity is preferred, and 98 to 99% or more homogeneity most preferred, for pharmaceutical uses. Once purified, partially or to homogeneity as desired, the modified Factor VII may then be used therapeutically.
The modified Factor VII is cleaved at its activation site to convert it to its two-chain form. Activation may be carried out according to procedures known in the art, such as those disclosed by Osterud, et al., Biochemistry 11:2853-2857 (1972); Thomas, U.S. Pat. No. 4,456,591; Hedner and Kisiel, J. Clin. Invest. 71:1836-1841 (1983); or Kisiel and Fujikawa, Behring Inst. Mitt. 73:2942 (1983), which are incorporated herein by reference. The resulting molecule is then formulated and administered as described below.
Compositions
The compounds will typically be administered within about 24 hours prior to performing the intervention, and for as much as 7 days or more thereafter. Administration for preventing or minimizing myocardial injury can be by a variety of routes as further described herein. The compounds can also be administered locally at vascular sites susceptible of thrombus formation, for example, at sites of anastomosis, or locally at vascular sites susceptible to decreased patency.
In the prevention of or treatment of myocardial injury, the dose of modified Factor VII ranges from about 50 mg to 500 mg/day, more typically 1 mg to 200 mg/day, and more preferably 10 mg to about 175 mg/day for a 70 kg patient as loading and maintenance doses, depending on the weight of the patient and the severity of the condition.
The pharmaceutical compositions for treatment of myocardial injuries are intended for parenteral administration for prophylactic and/or therapeutic treatment. Preferably, the pharmaceutical compositions are administered parenterally, i.e., intravenously, subcutaneously, or intramuscularly. The compositions for parenteral administration comprise a solution of the modified Factor VII molecules dissolved in an acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers may be used, e.g., water, buffered water, 0.4% saline, 0.3% glycine and the like. The modified Factor VII molecules can also be formulated into liposome preparations for delivery or targeting to sites of injury. Liposome preparations are generally described in, e.g., U.S. Pat. No. 4,837,028, U.S. Pat. No. 4,501,728, and U.S. Pat. No. 4,975,282, incorporated herein by reference. The compositions may be sterilized by conventional, well known sterilization techniques. The resulting aqueous solutions may be packaged for use or filtered under aseptic conditions and lyophilized, the lyophilized preparation being combined with a sterile aqueous solution prior to administration. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, etc. The concentration of modified Factor VII in these formulations can vary widely, i.e., from less than about 0.5%, usually at or at least about 1% to as much as 15 or 20% by weight and will be selected primarily by fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected.
Thus, a typical pharmaceutical composition for intravenous infusion could be made up to contain 250 ml of sterile Ringer's solution, and 10 mg of modified Factor VII. Actual methods for preparing parenterally administrable compounds will be known or apparent to those skilled in the art and are described in more detail in for example, Remington's Pharmaceutical Science, 16th ed., Mack Publishing Company, Easton, Pa. (1982), which is incorporated herein by reference.
The compositions containing the modified Factor VII molecules can be administered for prophylactic and/or therapeutic treatments. In therapeutic applications, compositions are administered to a patient already suffering from a disease, as described above, in an amount sufficient to cure or at least partially arrest the disease and its complications. An amount adequate to accomplish this is defined as "therapeutically effective dose." Amounts effective for this use will depend on the severity of the disease or injury and the weight and general state of the patient, but generally range from about 0.05 mg up to about 500 mg of modified Factor VII per day for a 70 kg patient, with dosages of from about 1.0 mg to about 200 mg of modified Factor VII per day being more commonly used. It must be kept in mind that the materials of the present invention may generally be employed in serious disease or injury states, that is, life-threatening or potentially life threatening situations. In such cases, in view of the minimization of extraneous substances and general lack of immunogenicity of modified human Factor VII in humans, it is possible and may be felt desirable by the treating physician to administer substantial excesses of these modified Factor VII compositions.
In prophylactic applications, compositions containing the modified Factor VII are administered to a patient susceptible to or otherwise at risk of a disease state or injury to enhance the patient's own anticoagulative capabilities. Such an amount is defined to be a "prophylactically effective dose." In this use, the precise amounts again depend on the patient's state of health and weight, but generally range from about 0.05 mg to about 500 mg per 70 kilogram patient, more commonly from about 1.0 mg to about 200 mg per 70 kg of body weight.
Single or multiple administrations of the compositions can be carried out with dose levels and pattern being selected by the treating physician. For ambulatory patients requiring daily maintenance levels, the modified Factor VII may be administered by continuous infusion using a portable pump system, for example.
Local delivery of the modified Factor VII such as, for example topical application of modified Factor VII at vascular sites susceptible to thrombus formation, (e.g. sites of anastomosis) or at vascular sites susceptible to decreased patency may be carried out, for example, by way of spray, perfusion, double balloon catheters, stent, incorporated into vascular grafts or stents, hydrogels used to coat balloon catheters, or other well established methods. In any event, the pharmaceutical formulations should provide a quantity of modified Factor VII of this invention sufficient to effectively treat the patient.
The following examples are offered by way of illustration, not by way of limitation.
EXAMPLES
Example I
Expression of Ser.sub.344 Ala.sub.344 Factor VII
To generate the Ser.sub.344 .RTM.Ala Factor VII active site mutant, plasmid FVII(565+2463)/pDX (U.S. Pat. No. 4,784,950 incorporated herein by reference; deposited with the American Type Culture Collection under accession number 40205) was digested with Xba I and Kpn I, and the resulting 0.6 kb fragment, comprising the coding region for serine 344, was recovered. This fragment was cloned into Xba I, Kpn I-digested M13mp19 as shown in the Figure. This manipulation and subsequent steps described below were generally performed according to standard protocols (as described, for example, by Maniatis et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1982) incorporated herein by reference).
Mutagenesis was carried out on the M13 template according to the methods of Zoller and Smith, supra, using the mutagenic oligonucleotide ZC1656 (5' TGG GCC TCC GGC GTC CCC CTT 3'(SEQ ID NO:3)) and the "universal" second primer ZC87 (5' TCC CAG TCA CGA CGT 3'(SEQ ID NO:4)). Reaction products were screened using kinased ZC1656. Positive plaques were picked, and template DNA was prepared and sequenced from the Pst I site at 1077 to the Kpn I site at 1213. Sequence analysis confirmed the presence of the desired mutation. The mutant clone was designated 1656.
An expression vector was then constructed using the 1656 clone. The mutagenized sequence was isolated from the M13 vector as a 18 0.14 kb Pst I-Kpn I fragment. This fragment was ligated to the 1.7 kb Hind III-Xba I fragment from FVII(565+2463)/pDX, the 0.5 kb Xba l-Pst I fragment from FVII(565+2463)/pDX, and the 4.3 kb Kpn I-Hind IlIl fragment from FVII(565+2463)/pDX, as shown in the Figure. The presence of the desired mutant sequence was confirmed by digesting mutant and wild-type clones with Pst I, and a mutant Factor VII insert in M13 with Kpn I and Xba I, preparing Southern blots of the digested DNA, and probing the blots with radiolabeled ZC1656.
The baby hamster kidney cell line BHK 570 (deposited with the American Type Culture Collection under accession number 10314) was transfected with two isolates (designated #544 and #545) of the 1656 expression vector. The cells were prepared by diluting a confluent 10 cm plate of BHK 570 cells 1:10 into five 10 cm plates in non-selective medium (Dulbecco's modified Eagle's medium [DMEM] containing 10% fetal bovine serum and 1% PSN antibiotic mix [GIBCO Life Technologies, Gaithersburg, Md.]). After 24 hours, when the cells had reached 20-30% confluency, they were co-transfected with one isolate of the expression vector encoding the 1656 mutation, plasmid p486 (comprising the Adenovirus 5 ori, SV40 enhancer, Adenovirus 2 major late promotor, Adenovirus 2 tripartite leader, 5' and 3' splice sites, the DHFRr cDNA and SV40 polyadenylation signal in pML-1 (Lusky and Botchan, Nature 293: 79-81, (1981)) and 10 mg of carrier DNA (sonicated salmon sperm DNA) as shown in Table 1. The DNA was added to a 15 ml tube, then 0.5 ml of 2X Hepes (25 g Hepes, 40 g NaCl, 1.8 g KCl, 0.75 g Na.sub.2 HPO.sub.4 2H.sub.2 O, 5 g dextrose diluted to 2.5 l with distilled water and pH adjusted to pH 6.95-7.0) was added and the tubes were mixed. The DNA in each tube was precipitated by the addition of 0.5 ml of 0.25M CaCl.sub.2 while air was bubbled through the DNA/Hepes solution with a pasteur pipet. The tubes were then vortexed, incubated at room temperature for 15 minutes, and vortexed again. The DNA mixtures were then added dropwise onto the plates of cells with a pipette. The plates were swirled and incubated at 37.degree. C. for 4-6 hours. After incubation, 2 ml of 20% glycerol diluted in Tris-saline (0.375 g KCl, 0.71 g Na.sub.2 HPO.sub.4, 8.1 g NaCl, 3.0 g Tris-HCl, 0.5 g sucrose, diluted in a total of 1 liter and pH adjusted to pH 7.9) was then added to each plate. The plates were swirled and left at room temperature for two minutes. The medium was then removed from the plates and replaced with 2 ml of Tris-saline. The plates were left at room temperature for 2 minutes, then the Tris-saline was removed and replaced with 10 ml of nonselective medium. The plates were incubated at 37.degree. C. for two days.
TABLE 1______________________________________Transfection*Plasmid Name 544 545 544 Control 545 Control______________________________________Clone 544 15 mml -- 15 ml --Clone 545 -- 30 ml -- 30 mlp486 1.5 ml 1.5 ml -- --Carrier DNA 1.6 ml 1.6 ml 1.6 ml 1.6 ml______________________________________ *DNA concentrations used were: clone 544: 0.7 mg/ml; clone 545: 0.3 mg/ml p486: 1.49 mg/ml.
After the two day incubation, the cells were diluted in selection medium (DMEM containing 10% dialyzed fetal bovine serum, 1% PSN antibiotic mix and 150 nM methotrexate) and plated at dilutions of 1:100, 1:250 and 1:500 in maxi plates. The plates were incubated at 37.degree. C. for one week. After one week, the medium was changed and replaced with selection medium, and the plates were checked for colony formation.
Eight days later, after colony formation, twelve colonies were randomly chosen from the 1:500 dilution plates of the #544 and #545 transfection plates. Each clone was plated into one well of a 6-well plate and grown in selection medium. After seven days, the plates were confluent, and the clones were each split into 10 cm plates in selection medium.
The clones described above and control cells transfected to express wild-type factor VII were metabolically labeled with .sup.35 S-Methionine-Cysteine Protein Labeling Mix (NEN DuPont Biotechnology Systems, Wilmington, Del.). The clones were grown and prepared for a pulse label experiment in selective medium. The cells were rinsed with phosphate buffered saline (Sigma, St. Louis, Mo.) and pulsed for four hours in 20 mCi/ml .sup.35 S-Cys-.sup.35 SMet. After four hours, supernatants and cells were harvested. The cells were lysed essentially as described by Lenk and Penman (Cell 16: 289-302, (1979)) and 400 ml of each lysate and precleared with 50 ml of staph A (Sigma, St. Louis, Mo.).
Samples from the metabolically labeled cells were radioimmunoprecipitated (RIP) by first incubating the samples with 6 ml of anti-Factor VII polyclonal antisera for four hours. Sixty microliters of washed staphylococcal protein A was added to each sample, and the samples were rocked for 1.5 hours at 4.degree. C. The samples were centrifuged, and the supernatant was removed. The pellets were washed twice in 0.7M RIPA buffer (10 mM Tris, pH 7.4, 1% deoxycholic acid [Calbiochem Corp., La Jolla, Calif.], 1% Triton X-100, 0.1% SDS, 5 mM EDTA, 0.7M NaCl) and once in 0.15M RIPA buffer (10 mM Tris, pH 7.4, 1% deoxycholic acid [Calbiochem Corp., La Jolla, Calif.], 1% Triton X-100, 0.1 SDS, 5 mM EDTA, 0.15M NaCl). One hundred microliters of 1x SDS dye (50 mM Tris-HCl, pH 6.8, 100 mM dithiothreitol, 2% SDS, 0.1% bromphenol blue, 10% glycerol) was added to each sample, and the samples were boiled for 5 minutes followed by centrifugation to remove the protein A. Fifty microliters of each sample was run on a 10% polyacrylamide gel. Results showed that 9 of 10 clones secreted modified Factor VII.
Example II
ANTICOAGULANT ACTIVITY OF MODIFIED FACTOR VII
The ability of the modified Factor VII protein to inhibit clotting was measured in a one-stage clotting assay using wild-type Factor VII as a control. Recombinant proteins were prepared essentially as described above from cells cultured in media containing 5 mg/ml vitamin K. Varying amounts of the modified Factor VII (from clone 544) or recombinant wild-type Factor VII were diluted in 50 mM Tris pH 7.5, 0.1% BSA to 100 ml. The mixtures were incubated with 100 ml of Factor VII-deficient plasma (George King Bio-Medical Inc., Overland Park, Kans.) and 200 ml of thromboplastin C (Dade, Miami, Fla.; contains rabbit brain thromboplastin and 11.8 mM Ca.sup.++). The clotting assay was performed in an automatic coagulation timer (MLA Electra 800, Medical Laboratory Automation Inc., Pleasantville, N.Y.), and clotting times were converted to units of Factor VII activity using a standard curve constructed with 1:5 to 1:640 dilutions of normal pooled human plasma (assumed to contain one unit per ml Factor VII activity; prepared by pooling citrated serum from healthy donors). Using this assay the preparations of modified Factor VII exhibited no detectable coagulant activity. Table 2 shows results of the assay in terms of clotting times for control (untransfected) BHK cell-conditioned media (+/- vitamin K), wild-type Factor VII and two isolates of cells expressing the modified Factor VII. Factor VII activity is seen as a reduction in clotting time over control samples.
TABLE 2______________________________________Sample Dilution Clotting Time (sec.)______________________________________Control +K 1:5 33.1 1:10 33.4Control -K 1:5 34.3 1:10 33.2Wild-type Factor VII 1:20 19.0 1.40 21.5 1:80 23.3Modified Factor VII (#6) 1:1 33.5Modified Factor VII (#10) 1:1 32.5______________________________________
To determine the effect of the modified Factor VII on plasma factor substrates, preparations of modified Factor VII and recombinant wild-type or native Factor VII are incubated with either Factor X or Factor IX and the activation thereof monitored by clotting assays or polyacrylamide gel electrophoresis.
Example Ill
Ability of Modified Factor VII to Bind Tissue Factor
The ability of the modified Factor VII to compete with wild-type Factor VII for tissue factor and inhibit its clotting activity was assessed in a one-step clotting assay in the presence of a limiting amount of tissue factor (thromboplastin).
Clotting times were determined in a one-step assay similar to that described in Example II. A limited amount of tissue factor, a constant amount of wild type Factor VII, and increasing amounts of variant Factor VII were used in the mixing experiments. An inhibition of Factor VII/VIIa procoagulant activity would be seen as an increase in clotting time in assays containing increasing amounts of variant Factor VII.
The amount of Factor VII activity in the test samples was calculated as a percentage of a standard curve that measured Factor VII activity in normal pooled plasma. The standard curve for Factor VII activity was generated using serial dilutions of normal pooled plasma in phosphate buffered solution (PBS) that ranged from 1:5 to 1:640. For this purpose it was assumed that normal plasma contains approximately 500 ng/ml of Factor VII and this was considered to be one unit of activity. A mixture of 100 ml Factor VII-deficient plasma, 100 ml plasma dilution and 200 ml of thromboplastin-C (Dade, Miami, Fla.) was used to measure clotting time on a MLA Electra 800 automatic timer. To establish the standard curve, the results were graphed as percentage of activity (1:5=100% activity) versus clotting time in seconds.
The assay required that the medium containing the wild type and variant Factor VII be composed of less than one percent serum. The dilutions were made in PBS so that clotting times would fall along the standard curve. A minimum dilution of 1:2 was typical. The final volume was 100 ml. Two different human Factor VII Ser.sub.344 Ala variants, designated clones "#10" and "#6" were tested in the experiments. The results, set forth in the Table below, show that as the amount of Factor VII variant increased, the percent of Factor VIIa activity decreased.
TABLE 3______________________________________Results of mixing assay with Ser344 -> AlaVariants (B4A1 (wild type) medium was used as 100% activity at10 .mu.l/reaction) Variant B4A1Ser344 -> Ala medium medium Percent FVIIaClone No. amount amount BHK control* Activity______________________________________#10 10 .mu.l 10 .mu.l 0 70#10 20 .mu.l 10 .mu.l 0 51#10 30 .mu.l 10 .mu.l 0 43#10 40 .mu.l 10 .mu.l 0 34#10 50 .mu.l 10 .mu.l 0 28#10 (-K).sup..sctn. 20 .mu.l 10 .mu.l 0 78#6 10 .mu.l 10 .mu.l 0 74#6 20 .mu.l 10 .mu.l 0 56#6 30 .mu.l 10 .mu.l 0 46#6 40 .mu.l 10 .mu.l 0 41#6 50 .mu.l 10 .mu.l 0 32#6 20 .mu.l 10 .mu.l 0 85BHK control 0 10 .mu.l 20 .mu.l 91BHK control 0 10 .mu.l 20 .mu.l 107(-K)______________________________________ Untransfected conditioned medium .sup..sctn. For expression of the Factor VII variant, cells were grown in the presence of vitamin K, except where noted "(-K)".
These experiments showed that variants of Factor VII having a Ser.sub.344 Ala substitution competed with native Factor VII in a dose dependent fashion and inhibited the procoagulant activity of native Factor VII/VIIa. It can thus be concluded that Ser.sub.344 Ala variant human Factor VII competes with native human Factor VIIa and consequently inhibits activation of Factor X and/or IX in human plasma.
Example IV
Reaction of Factor VII with PPACK
Recombinant Factor VII was produced in transfected baby hamster kidney cells. The protein was purified and activated as disclosed by Thim et al. (Biochemistry 27: 7785-7793, 1988), Brinkous et al. (Proc. Natl. Acad. Sci. USA 86: 1382-1386, 1989) and Bjoern and Thim (Res. Discl. No. 269, 564, 1986), which are incorporated herein by reference. The cell culture medium was recovered, filtered and diluted to reduce salt concentration. The diluted medium was then fractionated by anion exchange chromatography using an elution buffer containing CaCl.sub.2. The Factor VII fraction was recovered and further purified by immunochromatography using a calcium-dependent anti-Factor VII monoclonal antibody. Additional purification was carried out using two anion exchange chromatography steps wherein Factor VII was eluted using CaCl.sub.2 and NaCl, respectively. Factor VIIa was recovered in the final eluate.
Recombinant Factor VIIa (1 mM) in 50 mM Tris-HCl, 100 mM NaCl, 5 mM CaCl.sub.2, ph 7.4 was incubated with 20 mM PPack (D-Phenylalanyl-Prolyl-Arginyl Chloromethyl Ketone; Calbiochem, La Jolla, Calif.) for 5, 20 and 60 minutes. Buffer containing the chromogenic substrate S2288 (H-D-lsoleucine-L-ProlyI-L-Arginine p-nitroanilide; Kabi Vitrum AB, Molndal, Sweden) was then added to obtain a 2.5 fold dilution and a final concentration of 0.3 mM S2288. The generation of p-nitroaniline was measured and compared to results using untreated Factor VIIa as a control. The results indicated that Factor VIIa is fully inactivated after about 60 minutes under these reaction conditions.
Example V
Generation of DEGR-Factor VIIa
Recombinant human Factor VIIa was prepared as described in Example IV. Recombinant human Factor VIIa, in 10 mM glycine buffer, pH 8.0, 10 mM CaCl.sub.2, 50 mM NaCl, was diluted to a concentration of 1.5 mg/ml. A 10-fold molar excess of Dansyl-L-Glu-Gly-Arg-Chloromethyl Ketone, DEGRck, (Calbiochem, La Jolla, Calif. 92037) which had been dissolved with distilled H.sub.2 O was added to the Factor VIIa. After a 2 hr incubation at 37.degree. C., a second 10-fold molar excess of DEGRck was added to the mixture and incubated for an additional 2 hr at 37.degree. C. A third 10-fold molar excess of DEGRck was added to the Factor VIIa and incubated for approximately 16 hours at 4.degree. C. The DEGR-Factor VIIa sample was then extensively dialyzed at 4.degree. C. against Tris buffered saline (0.05M Tris-HCl, 0.1M NaCl, pH 7.5) to remove any free DEGRck.
The final DEGR-Factor VIIa mixture was tested for the presence of free DEGRck in a Factor Xa chromogenic substrate assay. The DEGR-Factor VIIa mixture was added to purified human Factor Xa along with the chromogenic substrate S-2222. This substrate is cleaved specifically by Factor Xa and not by Factor VIIa. Unbound DEGRck in the mixture is able to bind to the Factor Xa and there by inhibit the chromogenic activity of the Factor Xa. Spiking free DEGR-ck into a Factor Xa mixture generated a standard curve to measure the level of free DEGRck in solution versus the inhibition of Factor Xa chromogenic activity. Analysis of the DEGR-Factor VIIa mixture showed that the ratio of free DEGRck:DEGR-Factor VIIa was less than 0.5% following extensive dialysis, thereby ensuring that the inhibition observed by DEGR-Factor VIIa in the various assay systems described below was not due to the presence of free DEGRck.
Example VI
Factor Xa Generation on Rat Smooth Muscle Cells
Vascular smooth muscle cells were analyzed for the presence of cell-surface tissue factor by measuring the ability of the cells to stimulate the conversion of Factor X to Factor Xa using a chromogenic substrate that is specific for Factor Xa.
Rat vascular smooth muscle cells (Clowes et al., J. Clin. Invest. 93:644-651 (1994)) were plated into 96-well culture dishes (American Scientific Products, Chicago, Ill.) at 8,000 cells per well in growth media (Table 4).
TABLE 4______________________________________500 ml Dulbecco's Modified Eagle's Medium (DMEM) (GIBCO-BRL,Gaithersburg, MD.)10% fetal calf serum (Hyclone, Logan, UT.)1 mM sodium pyruvate (Irvine, Santa Ana, CA.)0.29 mg/ml L-glutamine (Hazelton, Lenexa, KS.)1x PSN; (100X is 5 mg/ml penicillin, 5 mg/ml streptomycin,10 mg/ml neomycin) (GIBCO-BRL, Gaithersburg, MD.)______________________________________
After a 48 hour incubation at 37.degree. C. the medium was changed to serum free medium (Table 5).
TABLE 5______________________________________250 ml Dulbecco's Modified Eagle's Medium (DMEM)250 ml Ham's F-12 Medium (Fred Hutchinson Cancer Research Center,Seattle, WA)1 mM sodium pyruvate.29 mg/ml L-glutamine20 mM transferrin (JRH, Lenexa, KS.)5 mM insulin (GIBCO-BRL)16 ng selenium (Aldrich, Milwaukee, WI.)1 mg/ml bovine serum albumin (Sigma, St. Louis, MO)______________________________________
The cells were incubated 72 hours at 37.degree. C. After incubation, either PDGF-BB (10 ng/ml) or 10% fetal calf serum was added to the cells to stimulate tissue factor expression (Taubman et al., J. Clin. Invest. 91:547-552, 1993). A parallel set of cells received neither PDGF nor serum to monitor for intrinsic activity of unstimulated cells. After a 6 hour incubation, recombinant human Factor VIIa was added to the cells at a final concentration of 10 nM. One set of cells did not have Factor VIIa added as a negative control. The cells were incubated for 2 hours at 37.degree. C. and washed with HEPES buffer (10 mM HEPES, 137 mM NaCl, 4 mM KCl, 5 mM CaCl.sub.2, 11 mM glucose, 0.1% BSA). After washing, cells were incubated for 5 min with 50 ml per well of 200 nM plasma-purified human Factor X in a Tris-buffered saline supplemented with 5 mM CaCl.sub.2. Twenty-five microliters of 0.5M EDTA and 25 ml of an 800 mM solution of S-2222 chromogenic substrate (Kabi Pharmacia, Franklin, Ohio) were added to each well. The plates were incubated for 40 min at room temperature, then analyzed at 405 nm using a THERMOMAX microplate reader (Molecular Devices, Menlo Park, Calif.).
Table 6 shows an increase in absorbance for the Factor VIIa treated wells as compared to the control wells (no Factor VIIa added). The increase in absorbance is a direct measurement of the level of Factor Xa generated in the wells and its subsequent cleavage of the chromogenic substrate, releasing the chromophore. The data also demonstrate that the level of chromogenic activity in cells pretreated with either PDGF-BB or 10% fetal calf serum was higher than unstimulated cells.
TABLE 6______________________________________ Test Sample OD.sub.405______________________________________ Control 0.043 Intrinsic 0.247 PDGF-BB 0.360 10% FCS 0.342______________________________________
These results clearly show there is a Factor VIIa-dependent activation of Factor X to Factor Xa on the cell surface of rat vascular smooth muscle cells.
Example VII
Inhibition of Cell-Surface ChromogenicActivity By DEGR-Factor VIIa
Rat vascular smooth muscle cells were plated into 96-well culture dishes as described above. The cells were cultured for 72 hours in serum free media as described above and treated with the addition of 10% fetal calf serum for 6 hours to stimulate tissue factor expression. After stimulation, buffer only (control), 10 nM Factor VIIa, or 10 nM Factor VIIa+100 nM DEGR-Factor VIIa was added to each well. The cells were incubated for 2 hours at 37.degree. C., then washed with HEPES buffer. After washing, the cells were incubated for 5 minutes with 50 ml per well of 200 nM Factor X in Tris-buffered saline supplemented with 5 mM CaCl.sub.2. Twenty-five microliters of 0.5M EDTA and 25 ml of S-2222 (800 mM) chromogenic substrate (Kabi Pharmacia) were added to each well. The cells were incubated at room temperature for 40 minutes. Chromogenic activity was analyzed at 405 nm as described above.
Table 7 shows stimulation of chromogenic activity in the wells treated with Factor VIIa only, and inhibition of stimulation when DEGR-Factor VIIa was co-incubated with the Factor VIIa. These results demonstrate that DEGR-Factor VIIa acts as a competitive antagonist for Factor VIIa binding, thereby inhibiting the activation of Factor X to Factor Xa and the subsequent cleavage of the S-2222 chromogen.
TABLE 7______________________________________Test Sample OD.sub.405______________________________________Control 0.035Factor VIIa 0.342Factor VIIa + 0.073DEGR-Factor VIIa______________________________________
Example VIII
Dose Dependent Inhibition by DEGR-Factor VIIa of Cell Surface Chromogenic Activity on Rat Smooth Muscle Cells
Rat vascular smooth muscle cells were plated into 96-well culture dishes at 4,000 cells per well in growth medium supplemented with 1% fetal calf serum (as in Table 4 without 10% fetal calf serum). After 5 days the medium was removed, and either increasing concentrations of Factor VIIa alone or 10 nM Factor VIIa with increasing concentrations of DEGR-Factor VIIa were added to the cells. The cells were incubated with the Factor VII mixtures for 2 hours at 37.degree. C. After incubation, the cells were washed and incubated with 50 ml of 200 nM Factor X in tris buffered saline for 5 minutes at room temperature. Each well had 25 ml of 0.5M EDTA and 25 ml of 800 mM S-2222 (Kabi Pharmacia) added to it, and the plates were incubated for 40 minutes at room temperature. Chromogenic activity was analyzed at 405 nm in a microplate reader as described above.
Table 8 shows a dose-dependent increase in chromogenic activity with increasing amounts of Factor VIIa added to the wells. When the mixture of DEGR-Factor VIIa with 100 nM Factor VIIa was added to the cells (Table 9) there was a dose dependent inhibition in chromogenic activity. A 1:1 molar ratio of DEGR-Factor VIIa:Factor VIIa inhibited approximately 95% of the chromogenic activity. These data suggest that in this experimental design DEGR-Factor VIIa has a significantly higher affinity for cell-surface tissue factor than native Factor VIIa on smooth muscle cells in culture. If DEGR-Factor VIIa and Factor VIIa had equal affinity for binding tissue factor then the level of inhibition observed when the two molecules were added to the cells in an equal molar ratio would not have been as high.
TABLE 8______________________________________Factor VIIa Conc.(nM) OD.sub.405______________________________________0.10 0.0050.39 0.0251.56 0.0556.25 0.11125.00 0.154100.00 0.208______________________________________
Table 9 shows the dose dependent inhibition of Factor Xa chromogenic activity on rat smooth muscle cells by DEGR-Factor VIIa. Increasing concentrations of DEGR-Factor VIIa were co-incubated with 100 nM Factor VIIa, and the Factor Xa chromogenic activity determined using chromogenic substrate S-2222.
TABLE 9______________________________________DEGR-Factor VIIaConc. (nM) OD.sub.405______________________________________0.10 0.2080.39 0.1761.56 0.1166.25 0.07325.00 0.026100.00 0.014______________________________________
Example IX
Inhibition of Factor Xa generation by DEGR-Factor VIIa in a soluble tissue factor assay
The conversion of Factor X to Factor Xa using purified recombinant soluble tissue factor was established using a chromogenic assay. Tissue factor was expressed and purified from Saccharomyces cerevisiae (Shigematsu et al., J. Biol. Chem. 267:21329-21337, 1992). Soluble tissue factor was purified and characterized by Dr. W. Kisiel (University of New Mexico). A reaction mixture containing 65.9 ml of soluble tissue factor (2.2 mM), 29.0 ml of PCPS (1 mM, Sigma, St. Louis, Mo.), 29.5 ml human Factor X (4.1 mM), 2.77 ml Hank's buffer (25 mM Tris, pH 7.4, 150 mM NaCl, 2.7 mM KCl, 5 mM CaCl.sub.2, 0.1% BSA) was prepared. Forty microliter of tissue factor/Factor X mixture, 25 ml Factor VIIa diluted with TBS and 25 ml of DEGR-Factor VIIa diluted with TBS were added to each well of a 96-well microtiter plate. A control using 40 ml of tissue factor/Factor X mixture; 25 ml Factor VIIa diluted with TBS, and 25 ml of TBS only was included. Ten microliters of S-2222 (4 mM) chromogenic substrate was added to the reaction mixture in the wells and incubated at room temperature for 2-10 minutes. Results were analyzed at 405 nm in a microplate reader as described above.
Determination of a standard curve for Factor VIIa activation of Factor X was made using increasing concentrations of Factor VIIa added in the absence of DEGR-Factor VIIa. The results, presented in Table 10, show that there is a dose-dependent increase in chromogenic activity with increasing amounts of Factor VIIa added to the reaction mixture. The simultaneous addition of varying amounts of DEGR-Factor VIIa and 100 nM Factor VIIa led to a dose dependent decrease in chromogenic activity (Table 11). These data demonstrate that DEGR-Factor VIIa acts as a competitive antagonist for native Factor VIIa binding to soluble tissue factor, and thereby inhibits the generation of Factor Xa as measured by the decrease in chromogenic activity towards the chromogenic substrate S-2222.
TABLE 10______________________________________Stimulation of Factor Xa chromogenic activity with increasingconcentrations of Factor VIIa added to soluble tissue factor. Changesin optical density were measured using chromogenic substrate S-2222.Factor VIIa Conc (nM) OD.sub.405______________________________________0.78 0.1681.56 0.2883.12 0.4786.25 0.69412.50 0.76425.00 0.79050.00 0.738100.00 0.770______________________________________
TABLE 11______________________________________Inhibition of Factor Xa chromogenic activity by the addition ofDEGR-Factor VIIa to soluble tissue factor in the presence of nativeFactorVIIa is measured. Changes in optical density were measured using thechromogenic substrate S-2222.DEGR-Factor VIIaConc. (nM) OD.sub.405______________________________________0 0.81050 0.750100 0.609200 0.296400 0.167800 0.0831600 0.055______________________________________
Example X
Inhibition of Coagulation by DEGR-Factor VIIa
Standard clotting assays to monitor the effect of DEGR-Factor VIIa on clotting time were prepared as follows: 100 ml of normal baboon plasma, collected with sodium citrate as anticoagulant, was added to 100 ml of varying concentrations of DEGR-Factor VIIa diluted in TBS (20 mM Tris, pH 7.4,150 mM NaCl). The samples were mixed and briefly incubated at 37.degree. C. The samples were added to an Electra 800 Automatic Coagulation Timer (Medical Laboratories Automation, Pleasantville, N.Y.). After incubation, 200 ml of a tissue factor preparation containing 25 mM CaCl.sub.2 was added to the DEGR-Factor VIIa preparations. A tissue factor preparation was made as a saline extract of baboon brain from freshly frozen brain tissue and characterized for its ability to initiate coagulation in baboon plasma. A concentration of tissue factor that gave a clotting time of about 40 seconds was selected.
The data, presented in Table 12, demonstrates a dose-dependent increase in clotting time due to the addition of DEGR-Factor VIIa. A dose as low as 1 mg/ml of DEGR-Factor VIIa in plasma resulted in a significant increase in clotting time.
TABLE 12______________________________________Dose dependent increase in clotting time due to DEGR-Factor VIIa.DEGR-FactorVIIa Clotting Time(.mu.g/ml plasma) (seconds)______________________________________0 40.70.5 46.21.0 50.82.5 64.55.0 108.110.0 158.4______________________________________
Example XI
Inhibition of Platelet Accumulation With DEGR-Factor VIIa
DEGR-Factor VIIa was analyzed for its ability to inhibit platelet accumulation at sites of arterial thrombosis due to mechanical injury in non-human primates. A model of aortic endarterectomy was utilized in baboons, essentially as described by Lumsden et al. (Blood 81:1762-1770 (1993)). A section of baboon aorta 1-2 cm in length was removed, inverted and scraped to remove the intima of the artery and approximately 50% of the media. The artery was reverted back to its correct orientation, cannulated on both ends and placed into an extracorporeal shunt in a baboon, thereby exposing the mechanically injured artery to baboon blood via the shunt. Just prior to opening of the shunt to the circulating blood, .sup.111 In-labeled autologous platelets were injected intravenously into the animal. The level of platelet accumulation at the site of the injured artery was determined by real-time gamma camera imaging.
Evaluation of DEGR-Factor VIIa for inhibition of platelet accumulation was done using bolus injections of DEGR-Factor VIIa or saline control and were given just prior to the opening of the shunt. The injured arteries were measured continuously for 60 minutes. A dose of 0.005 mg/kg of DEGR-Factor VIIa inhibited platelet accumulation. At a 1.0 mg/kg bolus injection, approximately 90% of platelet accumulation was inhibited at 1 hour post drug administration. These results are shown in FIG. 2.
These data show that inhibition of tissue factor with DEGR-Factor VIIa can significantly inhibit the development of platelet-rich thrombi in a nonhuman primate model of acute vascular injury.
Example XII
DEGR-FVIIa Inhibits Vascular Restenosis Following Balloon Angioplasty in Atherosclerotic Rabbits
DEGR-FVIIa was evaluated for its ability to modulate lesion development following balloon angioplasty in New Zealand White (NZW) atherosclerotic rabbits. This animal model has been well characterized and has proven to be a good model for evaluating anti-thrombotic compounds on vascular lesion development (Gimple et al., Circulation 86:1536-1546 (1992), and Rogosta et al., Circulation 89:1262-1271 (1994)). The animal model used to evaluate DEGR-FVIIa is essentially as described by Ragosta, ibid.
Anesthesia was induced in rabbits with 5 mg/kg xylazine and 35 mg/kg ketamine by intramuscular injection. The proximal femoral arteries were exposed by cutdown below the inguinal ligament with proximal and distal ligatures. The isolated segments were cannulated with 27 gauge needles. A vent was created by needle puncture. The isolated segments were flushed with saline to clear residual blood, and desiccated by air infused at a rate of 80 ml/min for 8 minutes. Following air-drying, the isolated segments were again flushed with saline and the ligatures removed. Hemostasis was maintained with non-occlusive local pressure. The segments were demarcated with metal clips. Local spasm was treated with Xylocaine 1% locally. The day following surgery, the animals were placed on 1% cholesterol and 6% peanut oil diet for one month until balloon angioplasty. Tylenol 10 mg/kg orally was given for postoperative pain relief for 3-5 days. Ambipen 1 cc was given after the surgical procedure during postoperative days 3 to 5.
The test drug delivery for the animals consisted of an initial bolus injection immediately prior to balloon angioplasty, followed by a continuous systemic infusion by osmotic pump via the internal jugular vein. The duration of the drug infusion was 3 days. The control animals received heparin, 150 U/kg IV bolus, prior to balloon angioplasty followed by saline infusion. The DEGR-FVIIa treated animals received a 1 mg/kg bolus injection followed by 50 mg/kg/hr infusion.
For the placement of the osmotic pumps for continuous systemic infusion, anesthesia was induced in the animals as described above, and maintained throughout the procedure with additional IM injections of ketamine and Xylazine. Through a midline neck incision, the right internal jugular vein was isolated by blunt dissection and the distal end ligated. A silastic tube (PE-160) was introduced into the right internal jugular vein. A subcutaneous tunnel was created to pass the silastic tube. This tube was connected with the osmotic pump. The osmotic pump was implanted subcutaneously in the back of the rabbit. The right common carotid artery was isolated by blunt dissection and the distal end ligated. Via an arteriotomy, a 5F introducer was placed and advanced to the junction of the aortic arch. Blood was drawn for determination of hemostatic parameters, drugs and cholesterol levels. Twenty milligrams of xylocain was injected intraarterially. A control aortoiliofemoral angiogram was performed via a 5F Berman catheter positioned above the aortic bifurcation using 34 ml renographin injected over 3 seconds by hand.
After removal of the Berman catheter, a 0.014-inch guidewire was introduced in the descending aorta and positioned above the aortic bifurcation. Under fluoroscopic guidance, an appropriately sized balloon angioplasty catheter of 2.0 to 2.5 mm was introduced and advanced over the guidewire and positioned across the stenosis. The balloon was inflated to 6 atmospheres for 60 seconds with a hand inflator. Three inflations were performed with 60 second intervals between inflations. This procedure was performed in both femoral arteries in each animal.
Following balloon dilatation, the angioplasty catheter was withdrawn and the Berman catheter reintroduced to a position 3 cm above the aortic bifurcation. To minimize spasm 20 mg of lidocaine was given intraarterially. A post procedure angiogram was performed as described above. A 1 cm grid was positioned at the level of the femoral artery to calculate the actual diameter. The catheter was then removed. The right carotid artery was ligated with 3-0 silk and the wound sutured by layers. Ambipen and acetaminophen were given as above.
Prothrombin time and concentration of DEGR-FVIIa in the blood were determined at immediately pre-bolus injection of the test compound, 1 hr post bolus injection, and at 3 days at the end of continuous infusion. One to two mls of citrated plasma was obtained and the prothrombin times and antigen levels determined.
A standard clotting assay was used to monitor the prothrombin time in the control and DEGR-FVIIa-treated animals as follows. Twenty-five microliters of test rabbit plasma, collected with sodium citrate as anticoagulant, was added to 150 ml of TBS (20 mM Tris, pH 7.4, 150 mM NaCl). The samples were mixed and added to an Electra 800 Automated Coagulation Timer (Medical Laboratories Automation, Pleasantville, N.Y.). After incubation, 200 ml of thromboplastin preparation (Sigma Chemical) containing 25 mM CaCl.sub.2 was added to the plasma preparations. A concentration of thromboplastin that gave a clotting time of approximately 20 seconds in the control rabbit plasma was selected.
An ELISA assay was used to determine the concentration of DEGR-FVIIa in plasma samples from the control and DEGR-FVIIa treated rabbits. The assay involved first diluting an anti-human FVII monoclonal antibody (Dr. W. Kisiel, U. of New Mexico) to 2.0 mg/ml in 0.1M carbonate buffer pH 9.6, and adding 100 ml/well to 96-well plates. The plates were then incubated at 4.degree. C. overnight and subsequently washed two times using wash buffer (PBS, pH 7.4, containing 0.05% Tween 20). Blocking of nonspecific binding sites was achieved with 200 ml of blocking buffer per well (PBS, pH 7.4, containing 0.05% Tween 20 and 1% BSA) incubated at 37.degree. C for 2 hr, followed by a wash using the wash buffer.
After blocking, a standard dilution series of DEGR-FVIIa ranging from 20-0.027 ng/ml was added, along with a dilution series of the test rabbit plasma (1:100 to 1:4000 in blocking buffer) applied at 100 ml/well. Non-immune rabbit plasma was used as a negative control. Plates were then incubated for 1 hr at 37.degree. C. followed by four washes with wash buffer.
DEGR-FVIIa was detected by adding 100 ml/well of a 1:1,000 dilution of rabbit anti-human FVII polyclonal antibody (Dr. Kisiel, U. New Mexico) in blocking buffer. Plates were incubated for 1 hr at 37.degree. C., followed by five washes with wash buffer. Specific antibody binding was detected using 100 ml/well of a 1:2,000 dilution of goat anti-rabbit IgG antibody-peroxidase conjugate (Tago, Inc.). Plates were incubated for 1 hr at 37.degree. C. and washed six times with wash buffer. Finally, 100 ml of substrate solution was added (0.42 mg/ml of o-phenylenediamine dihydrochloride [OPD] in 0.2M citrate buffer, pH 5.0, containing 0.3% H.sub.2 O.sub.2). After 1-3 min at room temp. the color reaction was stopped by adding 100 ml/well of 1N H.sub.2 SO.sub.4 and the plates were read at 490 nm on a Microplate spectrophotometer. The concentration of DEGR-FVIIa in the plasma samples was determined by comparing the A490 values of the unknown to those of the DEGR-FVIIa standard curve.
Analysis of plasma samples for prothrombin times and DEGR-FVIIa antigen levels is shown in Table 13 and Table 14, respectively. The data are presented for each individual animal. Table 15 shows a summary of the mean clotting times. In all cases, the DEGR-FVIIa treated animals had elevated prothrombin times at the 1 hr post-bolus injection time point which returned to near pre-treatment levels at the 3-day time point. Analysis of the DEGR-FVIIa antigen levels also showed a high level of DEGR-FVIIa in the plasma at the 1 hr time point, ranging between 2-6 mg/ml in the plasma, with much lower circulating levels at the 3 day time point. The levels of DEGR-FVIIa measured at the 1 hr time period correspond with a predicted increase in prothrombin time, as determined by spiking normal rabbit plasma with DEGR-FVIIa in vitro and determining prothrombin times in a standard dilute thromboplastin assay.
TABLE 13______________________________________MEASUREMENT OF PROTHROMBIN TIMES Clotting Time (seconds)Animal Number Treatment Pretreatment 1 hour 3 days______________________________________73 Control 24.8 22.3 17.874 Control 24.8 27.9 18.675 Control 24.6 N/D 20.576 Control 22 N/D 17.9169 Control 21.2 22.9 22170 Control 24.9 23.5 18.6173 Control 25.9 21 20.8174 Control 25 29.4 20.177 DEGR-FVIIa 22.5 40.1 18.378 DEGR-FVIIa 24.3 34 20.980 DEGR-FVIIa 24.7 50 21.796 DEGR-FVIIa N/A N/A 2197 DEGR-FVIIa 23.6 33.3 21.2171 DEGR-FVIIa 20.6 45.8 21.9172 DEGR-FVIIa 23.5 41.6 22.4______________________________________ N/A = Data Not Available
TABLE 14______________________________________ELISA TO DETECT DEGR-FVIIa IN RABBIT PLASMA FVIIa ELISA (ng/ml)Animal Number Treatment Pretreatment 1 hour 3 days______________________________________73 Control 0 13 074 ControI 36 14 475 Control 0 N/A 976 Control 0 N/A 14169 Control 0 0 1170 Control 0 0 0173 Control 36 31 0174 Control 87 86 16077 DEGR-FVIIa 0 3,210 10278 DEGR-FVIIa 1 4,950 780 DEGR-FVIIa 13 4,543 66196 DEGR-FVIIa 65 4,900 11797 DEGR-FVIIa 4 4,600 502171 DEGR-FVIIa 13 2,145 212172 DEGR-FVIIa 9 2,830 228______________________________________ N/A = Data Not Available
TABLE 15______________________________________Statistical Summary of Plasma Clotting Times.______________________________________PRE-BLEEDUnpaired t-Test XDF: Unpaired t Value: Prob. (2-tail):______________________________________12 1.12 0.2852Group: Count: Mean: Std. Dev.: Std. Error:______________________________________Control 8 24.15 1.64 0.58DEGR-VIIa 6 23.2 1.48 0.60______________________________________1 Hr POST ANGIOUnpaired t-Test XDF: Unpaired t Value: Prob. (2-tail):______________________________________10 -5.44 0.0003Group: Count: Mean: Std. Dev.: Std. Error:______________________________________Control 6 24.5 3.35 1.37DEGR-VIIa 6 40.8 6.53 2.67______________________________________3 Days POST ANGIOUnpaired t-Test XDF: Unpaired t Value: Prob. (2-tail):______________________________________13 -2.04 0.0622Group: Count: Mean: Std. Dev.: Std. Error:______________________________________Control 8 19.54 1.53 0.54DEGR-VIIa 7 21.06 1.33 0.50______________________________________
Three weeks post-angioplasty a follow-up angiogram was repeated as described above via the left carotid artery immediately prior to sacrifice. Through a vertical lower abdominal incision, the distal aorta was isolated, tied off proximally, and the perfusion cannula inserted above the aortic bifurcation. The distal aorta was flushed with 50 ml of saline followed by in vivo fixation with 500 ml of Histochoice (AMRESCO, Solon, Ohio) solution infused over 15 mins at 120 mmHg. Once perfusion was started, the animals were sacrificed with an overdose of nembutal (3 ml sodium pentobarbital IV, 65 mg/ml). A 5 cm segment of femoral artery was excised bilaterally. The tissue was preserved in Histochoice solution for light microscopy.
To determine intimal lesion development at the site of balloon angioplasty, the excised femoral arteries were cut in serial 3 mm sections, embedded in paraffin, and sections cut from multiple regions of each artery. The sections were mounted onto glass slides and the slides stained with hematoxylin and eosin, and Van Giemson stains. Morphometric analysis was performed with Bioquant Program to obtain area measurements for the lumen, the intima and the media. Morphometric analysis of tissue sections from the injured arteries were done measuring the total luminal area; the area of the intima, determined by measuring the area within the internal elastic lamina and subtracting the corresponding luminal area from each tissue section; and the area of the media, determined by measuring the area inside the external elastic lamina and subtracting the area inside the internal elastic lamina. Measurements for intimal lesions in the femoral arteries in control and DEGR-FVIIa treated animals showed that there was a significant decrease in the size of the intima in the DEGR-FVIIa treated animals (Table 16). In contrast, measurement of the medial area showed no significant difference between the two groups.
TABLE 16______________________________________MEASUREMENTS OF THE INTIMA AND MEDIA INBALLOON ANGIOPLASTY TREATED RABBITS______________________________________Group N Intima (mm2) Std. Dev. Prob. (2-tail)______________________________________Control 13 0.819 0.414 0.0138DEGR-FVIIa 10 0.438 0.192______________________________________Group N Media (mm2) Std. Dev. Prob. (2-tail)______________________________________Control 13 0.389 0.098 0.172DEGR-FVIIa 10 0.329 0.105______________________________________
The data from the angiographic measurements are presented in Table 17 as the Mean Luminal Diameter (MLD) +/- standard deviation for the control and DEGR-FVIIa treated animal for all three time points: immediately pre-angioplasty, immediately post-angioplasty, and 21 days post-angioplasty. There was no significant difference in the MLD between the control and DEGR-FVIIa treated animals at either the pre- or immediately post-angioplasty measurements. A significant increase in MLD was observed, however, in the DEGR-FVIIa treated animals at the 21 day post-angioplasty measurement.
TABLE 17______________________________________MEASUREMENT OF MINIMAL LUMINAL DIAMETER (MLD)Group N Mean MLD Std. Dev. Prob. (2-tail)______________________________________Pre-PTCA Measurement of MLDControl 13 1.202 0.24 0.3883DEGR-FVIIa 10 1.283 0.19Post-PTCA Measurement of MLDControl 13 1.492 0.551 0.5326DEGR-FVIIa 10 1.323 0.72521 Day Measurement of MLDControl 13 0.889 0.228 0.0001DEGR-FVIIa 10 1.393 0.242______________________________________
Example XIII
Inhibition of Cell-Surface Factor Xa Generation on Baboon SMCs by DEGR-FVIIa
A cell-surface chromogenic assay was developed, essentially as described in Example VIII above, to measure the efficacy of DEGR-FVIIa to block FVIIa binding to cell-surface tissue factor and the subsequent conversion of Factor X to Factor Xa on monolayers of baboon smooth muscle cells (SMCs). This method is a modification of those described by Sakai et al., J. Bio. Chem. 264:9980-9988 (1989) and Wildgoose et al., Proc. Natl. Acad. Sci. USA. 87:7290-7294 (1990). Baboon SMCs were obtained from the University of Washington, Seattle, Wash., and were cultured from aortic explants. The baboon SMCs were plated into 96-well culture dishes at a concentration of 8,000 cells/well in 200 ml/well DMEM culture media supplemented with 10% fetal calf serum, and maintained in this media for 4 days at 37.degree. C. in 5% CO.sub.2. At the time of assay 110 ml of culture media was removed, and increasing concentrations of FVIIa or FVIIa in combination with DEGR-FVIIa were added to wells. A standard curve for FVIIa concentration was generated, ranging from 5 nM to 0.04 nM. To measure the inhibitory activity of DEGR-FVIIa on FVIIa activity, increasing concentrations of DEGR-FVIIa were added to test wells in the presence of a constant amount of FVIIa (5 nM). Both FVIIa and DEGR-FVIIa were diluted with HEPES buffer (10 mM HEPES, 137 mM NaCl, 4 mM KCl, 5 mM CaCl.sub.2, 11 mM glucose, 0.1% BSA) and 10 ml of 10x stock solutions added to the cells. The cells were incubated with the test compounds for 2 hr at 37.degree. C., then washed 3 times with HEPES buffer. Fifty microliters of a 200 nM solution of Factor X in Tris buffer (25 mM Tris, pH 7.4, 150 mM NaCl, 2.7 mM KCl, 5 mM CaCl.sub.2, 0.1% BSA) was then added to each well. After 4 mins at room temp., 25 ml of 0.5M EDTA was added to stop the Factor X to Xa conversion. Twenty-five microliters per well of 0.8 mM S-2222, a factor Xa-specific chromogenic substrate, in Tris buffer was added and the absorbance at 405 nM read after 60 mins in a Thermomax microplate reader (Molecular Devices Corp., Menlo Park, Calif.).
The results, shown in FIG. 3, demonstrate a dose dependent increase in amidolytic activity for the FVIIa treated wells (open boxes). The increase in absorbance is a direct measure of the level of Factor Xa generated in the wells and its subsequent cleavage of the chromogenic substrate. The addition of increasing amounts of DEGR-FVIIa with a constant amount of FVIIa (5 nM) showed a dose dependent decrease in amidolytic activity with increasing levels of DEGR-FVIIa (closed boxes). An equal molar ratio of DEGR-FVIIa to FVIIa was able to inhibit >90% of the chromogenic activity. Even at a 10-fold lower level of DEGR-FVIIa, there was still a 40% inhibition in the generation of Factor Xa chromogenic activity. These results support the conclusion that DEGR-FVIIa is an extremely potent antagonist of the activation of Factor X to Xa by FVIIa on the surface of intact cell monolayers of SMCs.
Example XIV
Effect of DEGR-Factor VIIa on Vascular Thrombosis Formation and Vascular Lesion Formation in Baboons
Human DEGR-Factor VIIa was tested for the ability to inhibit tissue factor (TF) and activated Factor VII (FVIIa) mediation of vascular lesion formation (VLF) induced by mechanical vascular injury in nonhuman primates.
Beginning immediately prior to creating mechanical vascular injury in baboons, DEGR-Factor VIIa was infused intravenously for 7 days (5 animals) or 30 days (1 animal). Measurements were performed for vascular lesion formation on day 30. The results in 5 treated animals were compared with the findings in 5 concurrent vehicle buffer-infused controls.
Baseline measurements were obtained on study animals for: a) platelet counts, neutrophil counts, monocyte counts and red cell counts; b) plasma fibrinogen level; c) activity levels of plasma coagulation factors VII, VIIa, X and V, together with the antigenic levels of FVII; and d) baseline plasma sample for anti-Factor VIIa antibody level.
Under halothane anesthesia and sterile operating conditions, animals labeled with autologous .sup.111 In-platelets received intravenous infusions of DEGR-FVIIa using the tether system for continuous intravenous administration (initial bolus injection of 1 mg/kg followed by continuous intravenous infusion of 50 mg/kg/hr. The animals received surgical carotid endarterectomy, bilateral brachial artery or bilateral femoral artery Fogarty balloon catheter angioplasties.
The DEGR-FVIIa was administered for 7 or 30 days by continuous infusions via venous catheter using the tether system. Thirty days after surgery the animals were anesthetized with halothane and underwent in situ pressure-perfusion fixation with 4% paraformaldehyde containing 0.1% glutaraldeyde for 30 min. At that time, vascular segments (containing the sites previously injured) were harvested using procedures of Harkeret al., Circulation 83:41-44 (1991) and Hanson et al., Hypertension 18:1170-1176 (1991). The specimens were post-fixed in vitro (4% paraformaldehyde containing 0.1% glutaraldehyde), cryopreserved and processed for morphometric analysis of lesion extent.
Eleven normal mature baboons (Paio anubis) were studied. Six animals received DEGR-FVIIa infusions (50 mg/kg/hr) and the remaining five were control animals that did not receive DEGR-FVIIa. The animals were dewormed and observed to be disease-free for three months prior to use. All procedures were approved by the Institutional Animal Care and Use Committee and were in compliance with procedures and methods outlined by NIH Guide for the Care and Use of Laboratory Animals, as well as the Animal Welfare Act and related institutional policies. Invasive procedures were carried out under halothane anesthesia after induction by ketamine (10 mg/kg intramuscularly) and valium (0.5 mg/kg intravenously). For subsequent short-term immobilization in performing experimental procedures postoperatively, ketamine hydrochloride (5-20 mg/kg intramuscularly) was used.
Carotid endarterectomy was performed through a midline neck incision using the technique of Hanson et al., Hypertension 18:I170-I-176 (1991) and Krupski et al., Circulation 84:1749-1757 (1991), incorporated herein by reference. Endarterectomy was used as a vascular injury model because of its clinical relevance, and because VLF induced by endarterectomy of normal arteries has been shown to be reproducible. In brief, the common carotid artery was dissected free of surrounding tissues from the clavicle proximally to the carotid bifurcation distally. The common carotid artery was cross-clamped using atraumatic vascular clamps placed at each end of the exposed vessel three minutes after a bolus injection of heparin sulfate (100 U/kg intravenously; Elkins-Simm Inc., Cherry Hill, N.J.) and divided 1 cm proximal to the distal crossclamp. The proximal arterial segment was then everted over curved forceps. After maximal eversion was obtained, a pair of polypropylene stay sutures (7-0) was placed on either side proximally and a second pair placed distally in the lumen-exposed segment. The endarterectomy was then performed beginning 1 cm from the divided end of the everted vessel segment and continued for a measured distance of 1 cm. This procedure involves mechanical removal of the normal intima and a partial thickness of media using forceps and a surgical microscope (32X magnification). Following endarterectomy, the vessel was returned to its normal configuration, and an end-to-end anastomosis performed with 7-0 polypropylene suture and continuous technique under 2.5-fold magnification, and the wound closed in layers.
For morphometric analysis of VLF, sections embedded in paraffin and stained for connective tissue components (collagen, elastin) and with hematoxylin-eosin, were evaluated using a Zeiss Photoscope coupled with image analysis system (Thomas Optical Measurement Systems, Columbus, Ga.) consisting of high resolution (580 lines) CCD microscope camera coupled to a high resolution (700 lines) monitor, an IBM 386 chip, 80 MB computer with high resolution graphics digitablet for image acquisition and storage. Quantitative image analysis was performed using a morphometric software driver (Optimas, Bioscan, Inc., Edmonds, Wash.). Arterial cross-sections were analyzed with respect to the total area of neointimal proliferative lesion and corresponding area of arterial media. For statistical analysis, comparisons between groups were made using the Student's t test (two tailed) for paired and unpaired data.
The results showed that the intimal area was significantly decreased in the animals treated with DEGR-Factor VIIa for seven days and studied at 30 days as compared to control animals who had undergone the same vascular injury but who did not obtain any DEGR-Factor VIIa (FIG. 4). A similar result was found in the animal treated with DEGR-Factor VIIa for 30 days and examined at 30 days.
Preliminary studies with a balloon angiographic brachial artery model suggested no measurable benefit of DEGR-Factor VIIa therapy. This model, however, has not been shown in baboons to be a prothrombotic model in which tissue factor plays a key role.
Studies with the femoral artery balloon injury in the baboon did show a statistically significant benefit from DEGR-Factor VIIa as compared to controls, as shown in FIG. 5.
Example XV
Effect of DEGR-Factor VIIa on tPA-Induced Thrombolysis
Ongoing coronary thrombus formation during acute myocardial infarction is primarily mediated by tissue factor (TF) in complex with Factor VIIa through the extrinsic coagulation pathway. The effect of adjunctive coagulation cascade inhibition at different points in the extrinsic pathway on the efficiency of tissue plasminogen activator (TPA) thrombolysis was determined.
Thirty-six dogs with electrically-induced coronary thrombus undergoing thrombolysis with tPA (1 mg/kg over 20 min) were given 1 of 4 adjunctive treatments: 9 received tick anticoagulant peptide (TAP), a selective factor Xa inhibitor, at 30 mg/kg/min for 90 min. TF-Factor VIIa complex was inhibited by recombinant tissue factor pathway inhibitor (TFPI) (100-150 mg/kg/min for 90 min) in 9 dogs, and by DEGR VIIa (1-2 mg/kg bolus) as a competitive antagonist of activated Factor VIIa in 9 dogs. Nine dogs received a saline control. Dogs were observed for 120 minutes after thrombolysis for reocclusion. The effects of these agents on the efficiency of thrombolysis are shown below in Table 18 (data as mean .+-.SD).
TABLE 18______________________________________ Saline DEGR-FVIIa TFPI TAP______________________________________Time to reflow 32 .+-. 13 20 .+-. 7* 21 .+-. 6* 18 .+-. 10*(min)Reflow duration 62 .+-. 45 70 .+-. 48 91 .+-. 35* 120(min)Cycle flow 70% 89% 56% 0%variationsReocclusion 70% 78% 67% 0%______________________________________ Value different from saline control at a level 0.05 of significance.
These data indicate that extrinsic pathway inhibition by either Factor Xa or TF-Factor VIIa blockade by DEGF VIIa or TFPI accelerated tPA-induced thrombolysis. Selective inhibition of Factor Xa more efficiently maintained arterial patency following successful reperfusion.
Example XVI
Modified Factor VIIa Inhibits Intravascular Thrombus Formation Without Affecting Systemic Coagulation
To determine whether inhibition of Factor VII binding to TF would result in antithrombotic effects, cycle flow variations (CFVs) due to recurrent thrombus formation were initiated by placing an external constrictor around endothelially-injured rabbit carotid arteries (Fotts' model). Carotid blood flow was measured continuously by a Doppler flow probe placed proximally to the constrictor. After positioning the constrictor around the artery, CFVs developed with a mean frequency of 11.+-.2 cycles/hr in 6 of 6 rabbits, whereas carotid blood flow velocity averaged 5.+-.2% of baseline values at the nadir of CFVs. After CFVs were observed for 30 min, the animals received an infusion of human recombinant active site-blocked (Phe-Phe-Arg chloromethylketone) Factor VIIa (FVIIai) (0.1 mg/kg/min for 10 min). The Factor VIIai completely abolished CFVs in 6 of 6 animals (CFV frequency=0 cycles/hr; p<0.05; carotid blood flow velocity=106.9% of the baseline values; p=NS vs. baseline). Thirty minutes following inhibition of CFVs, human recombinant FVIIa was infused at the doses of 0.1 mg/kg/min for 10 min. Infusion of the Factor VIIa restored CFVs in all animals, thus indicating that Factor VIIa binding to TF was competitive. Prothrombin times, activated partial thromboplastin times, and ex vivo platelet aggregation in response to ADP and thrombin were not different after FVIIai infusion as compared to baseline values. Thus, FVII-VIIa plays an important role in initiating thrombus formation in vivo. Administration of Factor VIIai exerts potent antithrombotic effects in this model without affecting systemic coagulation.
Example XVII
Inhibition of Microarterial Thrombosis by Topical Administration of Modified Factor VIIa
In vascular surgery, microvascular reconstructive surgery, or replantation surgery the most common cause of failure is thrombosis at the anastomotic sites. The risk of occlusive thrombus formation is highly increased when vessels have been subjected to trauma, exhibit pathological changes, or when interpositional vein grafts are used. Therefore, antithrombotic intervention in context with surgery is frequently used. The substances which are currently available and used on this indication are administered parenterally (heparin, dextran) or orally (ASA) and are all associated with hemorrhagic side-effects. Moreover, heparin and especially ASA are only partially effective in preventing (arterial) thrombus formation. Based on these drawbacks, a need exists to prevent thrombosis in vascular surgery using an agent that binds to and is effective in anastomotic regions and sites of vascular trauma by means of a substance that can be delivered locally, thereby avoiding undesirable systemic side-effects. In this Example, topical administration of active-site inactivated Factor VIIa at arterial trauma sites was used to produce an antithrombotic effect without inducing a tendency for increased bleeding or other hemostatic defect.
Methods
Swedish loop rabbits of either sex weighing .about.3.5 kg were fed a standard pellet diet and given water ad libitum. They were observed to be disease-free at the laboratory for at least one week before use.
A marginal ear vein was cannulated and anaesthesia induced with sodium pentobarbital, 18 mg/kg, and maintained by repeated injections.
Skin flaps were raised on both ears, and 3 cm long segments of the central arteries (outer diameter .about.1 mm) prepared. All branches were ligated with 10-0 sutures and cut. The operative field was superfused with isotonic saline and covered with thin plastic films. To keep blood-flow high and constant and counteract vasospasm, the animals were placed on heat pads and kept slightly hyperthermic at a body temperature of .about.39.5.degree. C. (normal body temperature .about.38.5.degree. C.) and three drops of lidocaine 10 mg/ml were applied topically to the vessels after manipulating them (after reperfusion and after testing patency at 30 minutes after reperfusion).
Vessels on both sides were simultaneously placed in double microvascular clamps (S&T 2V, S&T Marketing Ltd., Neuhausen, Switzerland), thereby isolating 7 mm artery segments between the clamps. Longitudinal arteriotomies (7 mm) were performed, whereafter the clamps were approximated, the vessels repositioned and the vascular lumina everted and flattened, exposing deep layers of the tunica media. The arteriotomies were closed with continuous 10-0 monofilament nylon sutures (Ethilon 10-0 BV-75-3, Ethicon Ltd., Edinburgh, U.K.). All surgical procedures were carried out by one surgeon using a high-quality operating microscope (Wild M-650, Leica-Heerbrugg, Heerbrugg, Switzerland).
Vessels were simultaneously reperfused by opening the vascular clamps. They were quickly covered by saline-soaked gauze pads, and inspected once a minute. The time until complete cessation of arteriotomy bleeding was recorded.
At 30 and 120 min. after reperfusion, vessel patency was assessed using a standard microsurgical empty/refill test: Vessels were gently occluded distal to the trauma area with a pair of microforceps, and emptied downstream with another pair of forceps. After release of the first pair of forceps, vessel refilling was assessed and vessels classified as patent or occluded. Occluded vessels showed no refilling, while patent vessels showed rapid or slow refilling, the latter being referred to as "reduced patency". After the final patency test, vessels were excised and opened longitudinally, whereafter the thrombotic material was removed and weighed.
Compounds
Recombinant chemically-inactivated human factor VIIa (VIIai) at a concentration of 3.1 mg/ml or vehicle were stored as 200 ml aliquots in coded vials.
Experimental protocol
Twenty rabbits were treated as follows in a blind random fashion, each rabbit serving as its own control: After performing the deep arterial trauma as described in the method section above, the exposed trauma site on one ear was superfused during 5 minutes with VIIai solution (a total of 0.5 mg) and on the other ear with vehicle. The superfused trauma fields were allowed to incubate with the solutions during an additional 5 minute period, whereafter all surplus of solution was flushed away with isotonic saline. The arteriotomies were then closed and the vessels reperfused.
Statistical methods
Patency results were compared using the sign test, and thrombus weights and arteriotomy bleeding data with the Wilcoxon test. Two-sided p values were presented.
Results
The administration of VIIai gave a distinct antithrombotic effect as measured by patency rates. In the VIIai group the vessel patency was 85% at 30 minutes and 75% at 120 after reperfusion. Corresponding values in the vehicle group were 40% and 30% respectively. The difference is statistically significant (p=0.008 and p=0.004 respectively). Median thrombus weights were 0.3 mg in the VIIai group and 0.5 mg in the vehicle group, although this difference was not significant (p=0.19). Median arteriotomy bleeding times were 1.5 minutes in the VIIai group and 2 minutes in the vehicle group. The groups are statistically indistinguishable (p=1).
This Example demonstrates that topical administration of VIIai at arterial trauma sites produces an antithrombotic effect without inducing an increased bleeding tendency. This presents a highly attractive mode of treatment for preventing thrombotic complications due to surgery, microsurgery on blood vessels, angioplasty or other trauma.
Example XVIII
Local Application of FVIIai Reduces Thrombus-Weight and Improves Patency
This Example demonstrates that local application of chemically inactivated FVIIa (FVIIai) reduces thrombus-weight and improves vascular patency.
Twenty anesthetized rabbits were used in this Example. The jugular veins were mobilized and a 10 mm segment was isolated between clamps. The thrombus was introduced by a combination of chemical (aetoxysclerol) destruction of the endothelium of the isolated segment and a semi-restricting ligature placed caudally of the segment. In a blinded randomized fashion one side was treated with 0.5 mg chemically inactivated FVIIa (FVIIai) and the other with the buffer. The test substance was injected to the isolated segment and incubated for 10 min. after the chemical destruction. 30 and 120 min. later patency was controlled with an empty/refill test. Possible thrombus was weighed after sacrifice.
______________________________________ median range of 120 thromb. thromb. p- 30 min. min. weight weights value patency value patency p-value______________________________________FVIIai 0.85 mg 0-22.3 0.035 90% 0.0070 85% 0.070 mgbuffer 9.3 mg 0-26.8 55% 50% mg______________________________________
The results showed that local application of inactivated FVIIa significantly reduced thrombus-weight and improved patency in the venous thrombosis model.
Example XIX
Application of FVIIai Reduces the Area of Risk and improves reperfusion
Methods
Experimental Preparation
Twenty-eight New Zealand white rabbits of both sexes (3.2-3.8 kg) were studied. Briefly, the animals were anesthesized with a mixture of ketamine (35 mg/kg) and xylazine (5 mg/kg) administered intramuscularly, intubated and ventilated with aconstant volume respirator (harvard Apparatus Co., Cambridge, Mass.). Polyethylene catheters were placed in the aorta through the left carotid artery and into a jugular vein for monitoring arterial pressure and administration of drugs, respectively. A thoracotomy was performed through the fifth left intercostal space and the pericardium opened. A polyethylene catheter was placed into the left atrial appendage for later injections of coloured microsperes. The large marginal branch of the circumflex coronary artery was temporarily occluded approximately 0.3 cm from its origin with a surgical suture snare. Coronary artery occlusion was maintained for 30 min, at which time the ligature was released and reperfusion allowed for additional 5.5 hours.
Systemic arterial pressure (Statham P23 DB pressure transducer) was recorded contineously during the experimant (Gould Instruments).
Experimental Protocol
At the moment of reperfusion, the animals were randomly assigned to one of the following treatment groups: A control group received a 5-ml bolus of saline into the left atrium; a group treated with human recombinant, active site-blocked factor VIIa (FVIIai, Novo Nordisk ANS, Gentofte, Denmark, 1 mg/kg bolus into the left atrium); a group treated with human recombinant activated factor VI la (FVIIai, Novo Nordisk A/S, Gentofte, Denmark, 1 mg/kg bolus into the left atrium).
Assessment of Area of Risk, Infarct Size, and No-Reflow phenomenon
To estimate the distribution of tissue perfusion at the end of the experiment ("no-reflow" phenomenon, NR) the animals received an injection of a 6% solution of thioflavin S (1 mg/kg) via the left atrial catheter. To permit the assessment of the area of risk of infarcting, the coronary artery was reoccluded immediately after injecting thioflavin, and a solution of monastral blue (E.l.DuPont; 1 mg/kg) was injected through the left atrial catheter. The heart was thereafter immediately excised and the left ventricle was dissected free from all other structures and weighed. The left ventricle was frozen at -70.degree. C. for 30 min and cut into 8 to 10 slices parallel to the atrioventricular groove. Contours of the normally perfused myocardium as well as of the area of risk, according to the distribution of monasteral blue, were traced onto a transparent plastic sheet. Myocardial slices were then observed under ultraviolet light and the normally perfused myocardium (fluorescent) was then easily differentiated and separated from the ischemic myocardium (nonfluorescent) according to the thioflavin distribution. These areas were also traced onto transparent plastic sheets. The slices were next incubated in a 2% solution of triphenyltetrazolium chloride (TTC, Sigma Chemicals) for 10 min at 37.degree. C., to visualize the area of necrosis. Again, a transparent plastic sheet was used to trace the contours of the normal myocardium (TTC-positive), and of the infarcted portion (TTC-negative).
The following variables were calculated: 1) the area of risk of infarction, as a percent of the left ventricle, assessed at the end of the reperfusion period (monastral blue distribution) (AR); 2) infarct size (IS), as a percent of the area of risk that actually evolved to necrosis by the TTC staining criterion; 3) the percentage of the area of risk that did not receive blood flow at the end of the reperfusion period (no-reflow phenomenon, NR).
Regional Myocardial Blood Flow measurements.
Regional myocardial blood flow (RMBF) was measured in all rabbits in each treatment group. Differentially coloured plastic microsperes (Blue, Red, and Yellow, triton Technology, San Diego, Calif.) were used to measure RMBF 20 min after occlusion and 10 min and 5 hrs after reperfusion.
Microsperes were 15.+-.1 .mu. in size and suspended in 10% dextran solution with 0.01% Tween 80. To ensure adequate dispersion, the microsperes were sonicated in an ultrasonic bath for 5 min immediately before use. Approximately 500,000 microsperes were injected (0.5-1.0 ml total volume) into the left atrial catheter. One minute before microspere injection, reference arterial blood flow withdrawal was begun and continued for 1 min after the injection. Tissue samples (100 to 300 mg) from the center of the ischemic areas and from nonischemic regions were taken according to TTC staining. The microsperes were then recovered from tissue by digestion in a 4M KOH solution at 72.degree. C. for 3 hrs and from reference blood samples by digestion in 16M KOH at room temperature for 3 hrs and subsequent microfiltration, according to the instructions provided by the manufacturer. The dyes were then recovered from the spheres within a known volume of a solvent (dimethylformamide) and their concentrations determined by spectrophotometry at optimal wave lenghts for each dye according to the manufacturer's instructions. The composite spectrum of each dye solution was resolved into the spectra of the single constituents by a matrix inversion technique. Blood flow to each myocardial sample was calculated by the formula: RMBF=Fr x Am/Ar, where RMBF=myocardial blood flow in ml/min, AM=absorbance in myocardial sample, and AR=absorbance in reference blood sample. Myocardial blood flow was divided by the sample wet weight and expressed as ml/min/g.
Coagulation Studies
To determine the effects of FVIIai and FVIIa administration on systemic coagulation, prothrombin time (PT) and activated partial thromboplastin time (aPTT) were measured at baseline and 30 minutes after drug administration. Blood samples (4.5 ml) were collected in 0.5 ml sodium citrate (3.8%) and centrifuged at 2000 g for 10 min at 4.degree. C. to separate the plasma. PT and aPTT were measured in duplicate within 2 hrs from blood collection.
Statistical Analyses
Results are expressed as mean .+-.SD of the mean. Analysis of variance was used for multiple comparisons among groups. Differences for individual groups were tested with Student's t-test for unpaired observations with Bonferroni's correction. For comparisons of hemodynamic variables, as well as regional myocardial blood flow among groups, a two-way analysis of variance with a design for repeated measures was used.
Results
Twenty-eight rabbits underwent the surgical procedure; two animals died during coronary occlusion for ventricular fibrillation before treatment group allocation and two additional rabbits died during reperfusion (one in the control and one in the FVIIa-treated group). These animals were excluded from subsequent statistical analysis. Therefore, eight animals in each treatment group were included in the study.
Hemodynamic Measurements
In all treatment groups, coronary occlusion induced a slight decrease in heart rate and mean arterial pressure. No differences were found among the three groups in heart rates and mean arterial pressures during the course of the experimental periods (Table I).
Assessment of area of Risk, Infarct Size, and the No-Reflow Phenomenon
Coronary occlusion produced an area of risk of infarction assessed by injection of monastral blue at the end of the experiment which was similar in the three treatment groups (31.6.+-.6.3, 28.2.+-.4.1, and 29.2.+-.5.3% of the left ventricle in control, FVIIai, and FVIIa-treated animals, respectively, p=NS).
After 30 min of coronary occlusion and 5.5 hrs of reperfusion, the amount of the area at risk that evolved toward necrosis averaged 59.8.+-.12.8% in the control group (FIG. 7). Administration of FVIIai significantly reduced infarct size to 28.1.+-.11.3% of the area at risk p<0.01 by ANOVA, FIG. 1), while FVIIa administration was associated with a significant increase in infarct size to 80.1.+-.13.1% of the area of risk (p<0.01 vs. controls and FVIIai-treated rabbits, FIG. 7).
In control rabbits, 24.4.+-.2.7% of the area of risk showed a perfusion defect, as determined by thioflavine S distribution at the end of the experiment (no-reflow phenomenon). The extent of this area of no-reflow was significantly reduced by FVIIai and significantly increased by FVIIa to 11.1.+-.6.1 and 61.9.+-.13.8% of the area of risk, respectively (p<0.01, FIG. 8).
Previous studies have established that the amount of the myocardial tissue that shows a perfusion defect during post-ischemic reperfusion is related to various parameters; the most important are the extent of the area of risk, the magnitude of infarct size, and the amount of residual collateral flow during occlusion. Controlling for these variables allows for more precise assessment of the effects of interventions on the no-reflow phenomenon. In the present study, when the no-reflow area in control rabbits was correlated to these parameters, a close relationship was observed, which fits a multiple linear regression equation: NR (% of the left ventricle [LV])=-14.62+0.75 (AR)+0.07(IS)+3.69(RMBF); r2=0.98; F test=109.3, (0.37)(0.3)(3.69), where NR is no-reflow area, AR is the area at risk, and RMBF is collateral blood flow (in ml/min/g). The number in parentheses indicate the standard errors of the coefficients. With an r2 value of 0.98, this model accounted for >95% of the variation in the no-reflow area that was observed in control animals in this study.
The equation coefficients that were obtained from the data of control rabbits in the multiple regression equation were then used to calculate the expected no-reflow areas for individual animals within each intervention group (FIG. 9). The actually observed no-reflow areas in rabbits receiving FVIIai were significantly smaller than the expected ones calculated applying the equation obtained from the multiple regression analysis. Indeed, in FVIIai-treated rabbits, for any given expected area of no-reflow, the actual observed value was smaller, such that all animals in this group distributed below the regression line obtained for control animals (FIG. 9). On the contrary, FVIIa-treated rabbits showed just the opposite, i.e., for any given expected area of no-reflow, the actual observed value was significantly bigger, as all animals distributed above the regression line of control animals (FIG. 9). Taken together, these data indicate that the reduction in the no-reflow phenomenon observed in FVIIai treated animals is not entirely accounted for by the reduction in infarct size, and suggest that activation of the extrinsic coagulation cascade during post-ischemic reperfusion contribute to the occurrence of the no-reflow phenomenon.
Regional Myocardial Blood Flow Measurements
In control animals RMBF to the non-ischemic myocardium averaged 1.20 ml/min/g of tissue throughout the study (data not shown). In the same group of animals, RMBF to the ischemic myocardium was 0.08.+-.0.02, 1.43.+-.0.28, and 0.98.+-.0.19 ml/min/g of tissue 20 min after coronary occlusion and 10 min and 5 hrs after perfusion, respectively. The various drug treatment used in the present study did not significantly change RMBF during the experimental period both in the normal and in the ischemic myocardium as compared to control animals (FIG. 10).
Coagulation Studies
To study possible systemic effects of FVIIai, which may predispose to an increased risk of bleeding, PTs and aPTTs were measured in blood samples collected at 30 minutes of CFVs, and after FVIIai and FVIIa administration. At the end of the 30 min CFVs period, PTs and aPTTs averaged 8.2.+-.0.6 seconds and 25.+-.3 seconds, respectively. A slight increase in PTs to 10.1.+-.0.6 seconds was observed after FVIIai administration (FIG. 11). This increase, however, did not reach statistical significance (p=0.09 by ANOVA and student's t-test with the Bonferroni's correction). APTT did not change significantly after FVIIai administration (FIG. 10). FVIIa administration resulted in a significant shortening in both PTs and aPTTs only with respect to the values obtained after FVIIai administration (FIG. 11).
TABLE 1______________________________________Hemodynamic variables during coronary artery occlusion-reperfusion______________________________________Heart Rate (b/min)Time afterocclusion Control SQ29548 Dazoxiben R68070 ASA______________________________________0 175 .+-. 5 170 .+-. 4 178 .+-. 6 169 .+-. 5 130 min 169 .+-. 4 165 .+-. 5 173 .+-. 5 164 .+-. 6 11 hr 169 .+-. 6 167 .+-. 4 171 .+-. 5 166 .+-. 5 12 hr 173 .+-. 5 172 .+-. 5 175 .+-. 6 170 .+-. 5 13 hr 170 .+-. 4 168 .+-. 5 177 .+-. 5 168 .+-. 6 14 hr 175 .+-. 5 168 .+-. 6 174 .+-. 5 169 .+-. 5 15 hr 175 .+-. 5 172 .+-. 2 173 .+-. 5 172 .+-. 6 16 hr 168 .+-. 5 170 .+-. 4 174 .+-. 5 168 .+-. 5 1______________________________________Mean arterial pressure (mm Hg)Time after ASA + R680occlusion Control SQ29548 Dazoxiben R68070 70______________________________________0 75 .+-. 4 78 .+-. 3 78 .+-. 4 73 .+-. 3 730 min 69 .+-. 4 65 .+-. 4 71 .+-. 3 67 .+-. 4 61 hr 69 .+-. 3 67 .+-. 4 71 .+-. 4 69 .+-. 4 62 hr 73 .+-. 4 75 .+-. 4 75 .+-. 5 72 .+-. 4 73 hr 74 .+-. 4 75 .+-. 3 77 .+-. 5 75 .+-. 3 74 hr 75 .+-. 5 78 .+-. 4 76 .+-. 4 73 .+-. 4 75 hr 73 .+-. 4 74 .+-. 5 78 .+-. 5 72 .+-. 4 76 hr 73 .+-. 5 76 .+-. 4 74 .+-. 5 73 .+-. 5 7______________________________________
TABLE II______________________________________Regional myocardial blood flow (ml/min/g of tissue) during coronaryocclusion and reperfusion Control FVIIai FVIIa______________________________________Normal myocardium20 min CAO 1.19 .+-. 0.22 1.03 .+-. 0.19 1.27 .+-. 0.2410 min REP 1.22 .+-. 0.15 1.10 .+-. 0.17 1.19 .+-. 0.202 hrs REP 1.17 .+-. 0.18 1.07 .+-. 0.16 1.22 .+-. 0.19Ischemic myocardium20 min CAO 0.09 .+-. 0.05 0.08 .+-. 0.05 0.08 .+-. 0.0410 min REP 1.53 .+-. 0.12 1.65 .+-. 0.18 1.24 .+-. 0.142 hrs REP 0.89 .+-. 0.14 1.23 .+-. 0.15 0.72 .+-. 0.13______________________________________ CAO = Coronary Artery Occlusion; REP = Reperfusion
Example XIX
Application of FVIIai Reduces Infarct size and Area at Risk of Infarction
Tissue factor exposure occurs during reperfusion of post-ischemic hearts within the coronary vasculature, leading to a decrease in coronary blood flow.
To assess whether tissue factor exposure might contribute to myocardial injury via activation of the coagulation and reduction in coronary blood flow during post-ischemic reperfusion, NZW rabbits underwent 30 min coronary occlusion followed by 5.5 hrs of reperfusion. At reperfusion, the animals randomly received: saline (n=8); human recombinant, active site-blocked factor VIIa (FVIIai, 100 .mu.g/kg/min for 10 minutes into the left atrium, n=8) or human recombinant activated factor VIIa (FVIIa, 100 .mu.g/kg/min into the left atrium, n=8). Regional myocardial blood flow (RMBF) was measured using coloured microspheres at 20 min of ischemia, and 10 min and 2 hrs following reperfusion. The area at risk of infarction (AR), infarct size (IS), and the no-reflow area (NR) were determined at the end of the experiment by monastral blue and thioflavin distribution and by TTC staining. FVIIai resulted in a significant reduction in both IS and NR with respect to controls (28.1.+-.11.3% and 11.1.+-.6.1% of AR vs. 59.8.+-.12.8% and 24.4.+-.8.2% of AR, respectively, p<0.01), while FVIIa resulted in a significant increase in both IS and NR to 80.1.+-.13.1% and 61.9.+-.13.8% of AR, respectively, p<0.01 vs. Controls. No differences in blood pressure, heart rate, AR, and RMBF at 20 min of ischemia were observed among groups. RMBF was significantly higher at 2 hrs of reperfusion in FVIIai-treated animals, while it was lower in FVIIa-treated rabbits. Thus, TF-mediated activation af the coagulation importantly contributes to the occurrence of myocardial injury during post-ischemic reperfusion.
______________________________________ FVIIai (AR) Control (AR) FVIIa (AR)______________________________________IS 28.1 .+-. 11.3 59.8 .+-. 12.8 80.1 .+-. 13.1NR 11.1 .+-. 6.1 24.4 .+-. 8.2 61.9 .+-. 13.8______________________________________ AR = The area at risk of infarction; IS = infarct size (IS); NR = the noreflow area
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.
__________________________________________________________________________# SEQUENCE LISTING- (1) GENERAL INFORMATION:- (iii) NUMBER OF SEQUENCES: 4- (2) INFORMATION FOR SEQ ID NO:1:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 2422 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: cDNA- (iii) HYPOTHETICAL: N- (iv) ANTI-SENSE: N- (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 28..1420#/codon.sub.-- start= 28RMATION: /product=- # "Factor VII"- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:- CCTCCCGACA ATACAGGGGC AGCACTGCAG AGATTTCATC ATG GTC TC - #C CAG GCC 55# Met Val Ser Gln Ala#-35- CTC AGG CTC CTC TGC CTT CTG CTT GGG CTT CA - #G GGC TGC CTG GCT GCA 103Leu Arg Leu Leu Cys Leu Leu Leu Gly Leu Gl - #n Gly Cys Leu Ala Ala20- GTC TTC GTA ACC CAG GAG GAA GCC CAC GGC GT - #C CTG CAC CGG CGC CGG 151Val Phe Val Thr Gln Glu Glu Ala His Gly Va - #l Leu His Arg Arg Arg- CGC GCC AAC GCG TTC CTG GAG GAG CTG CGG CC - #G GGC TCC CTG GAG AGG 199Arg Ala Asn Ala Phe Leu Glu Glu Leu Arg Pr - #o Gly Ser Leu Glu Arg# 15- GAG TGC AAG GAG GAG CAG TGC TCC TTC GAG GA - #G GCC CGG GAG ATC TTC 247Glu Cys Lys Glu Glu Gln Cys Ser Phe Glu Gl - #u Ala Arg Glu Ile Phe# 30- AAG GAC GCG GAG AGG ACG AAG CTG TTC TGG AT - #T TCT TAC AGT GAT GGG 295Lys Asp Ala Glu Arg Thr Lys Leu Phe Trp Il - #e Ser Tyr Ser Asp Gly# 45- GAC CAG TGT GCC TCA AGT CCA TGC CAG AAT GG - #G GGC TCC TGC AAG GAC 343Asp Gln Cys Ala Ser Ser Pro Cys Gln Asn Gl - #y Gly Ser Cys Lys Asp# 60- CAG CTC CAG TCC TAT ATC TGC TTC TGC CTC CC - #T GCC TTC GAG GGC CGG 391Gln Leu Gln Ser Tyr Ile Cys Phe Cys Leu Pr - #o Ala Phe Glu Gly Arg# 75- AAC TGT GAG ACG CAC AAG GAT GAC CAG CTG AT - #C TGT GTG AAC GAG AAC 439Asn Cys Glu Thr His Lys Asp Asp Gln Leu Il - #e Cys Val Asn Glu Asn# 95- GGC GGC TGT GAG CAG TAC TGC AGT GAC CAC AC - #G GGC ACC AAG CGC TCC 487Gly Gly Cys Glu Gln Tyr Cys Ser Asp His Th - #r Gly Thr Lys Arg Ser# 110- TGT CGG TGC CAC GAG GGG TAC TCT CTG CTG GC - #A GAC GGG GTG TCC TGC 535Cys Arg Cys His Glu Gly Tyr Ser Leu Leu Al - #a Asp Gly Val Ser Cys# 125- ACA CCC ACA GTT GAA TAT CCA TGT GGA AAA AT - #A CCT ATT CTA GAA AAA 583Thr Pro Thr Val Glu Tyr Pro Cys Gly Lys Il - #e Pro Ile Leu Glu Lys# 140- AGA AAT GCC AGC AAA CCC CAA GGC CGA ATT GT - #G GGG GGC AAG GTG TGC 631Arg Asn Ala Ser Lys Pro Gln Gly Arg Ile Va - #l Gly Gly Lys Val Cys# 155- CCC AAA GGG GAG TGT CCA TGG CAG GTC CTG TT - #G TTG GTG AAT GGA GCT 679Pro Lys Gly Glu Cys Pro Trp Gln Val Leu Le - #u Leu Val Asn Gly Ala160 1 - #65 1 - #70 1 -#75- CAG TTG TGT GGG GGG ACC CTG ATC AAC ACC AT - #C TGG GTG GTC TCC GCG 727Gln Leu Cys Gly Gly Thr Leu Ile Asn Thr Il - #e Trp Val Val Ser Ala# 190- GCC CAC TGT TTC GAC AAA ATC AAG AAC TGG AG - #G AAC CTG ATC GCG GTG 775Ala His Cys Phe Asp Lys Ile Lys Asn Trp Ar - #g Asn Leu Ile Ala Val# 205- CTG GGC GAG CAC GAC CTC AGC GAG CAC GAC GG - #G GAT GAG CAG AGC CGG 823Leu Gly Glu His Asp Leu Ser Glu His Asp Gl - #y Asp Glu Gln Ser Arg# 220- CGG GTG GCG CAG GTC ATC ATC CCC AGC ACG TA - #C GTC CCG GGC ACC ACC 871Arg Val Ala Gln Val Ile Ile Pro Ser Thr Ty - #r Val Pro Gly Thr Thr# 235- AAC CAC GAC ATC GCG CTG CTC CGC CTG CAC CA - #G CCC GTG GTC CTC ACT 919Asn His Asp Ile Ala Leu Leu Arg Leu His Gl - #n Pro Val Val Leu Thr240 2 - #45 2 - #50 2 -#55- GAC CAT GTG GTG CCC CTC TGC CTG CCC GAA CG - #G ACG TTC TCT GAG AGG 967Asp His Val Val Pro Leu Cys Leu Pro Glu Ar - #g Thr Phe Ser Glu Arg# 270- ACG CTG GCC TTC GTG CGC TTC TCA TTG GTC AG - #C GGC TGG GGC CAG CTG1015Thr Leu Ala Phe Val Arg Phe Ser Leu Val Se - #r Gly Trp Gly Gln Leu# 285- CTG GAC CGT GGC GCC ACG GCC CTG GAG CTC AT - #G GTC CTC AAC GTG CCC1063Leu Asp Arg Gly Ala Thr Ala Leu Glu Leu Me - #t Val Leu Asn Val Pro# 300- CGG CTG ATG ACC CAG GAC TGC CTG CAG CAG TC - #A CGG AAG GTG GGA GAC1111Arg Leu Met Thr Gln Asp Cys Leu Gln Gln Se - #r Arg Lys Val Gly Asp# 315- TCC CCA AAT ATC ACG GAG TAC ATG TTC TGT GC - #C GGC TAC TCG GAT GGC1159Ser Pro Asn Ile Thr Glu Tyr Met Phe Cys Al - #a Gly Tyr Ser Asp Gly320 3 - #25 3 - #30 3 -#35- AGC AAG GAC TCC TGC AAG GGG GAC AGT GGA GG - #C CCA CAT GCC ACC CAC1207Ser Lys Asp Ser Cys Lys Gly Asp Ser Gly Gl - #y Pro His Ala Thr His# 350- TAC CGG GGC ACG TGG TAC CTG ACG GGC ATC GT - #C AGC TGG GGC CAG GGC1255Tyr Arg Gly Thr Trp Tyr Leu Thr Gly Ile Va - #l Ser Trp Gly Gln Gly# 365- TGC GCA ACC GTG GGC CAC TTT GGG GTG TAC AC - #C AGG GTC TCC CAG TAC1303Cys Ala Thr Val Gly His Phe Gly Val Tyr Th - #r Arg Val Ser Gln Tyr# 380- ATC GAG TGG CTG CAA AAG CTC ATG CGC TCA GA - #G CCA CGC CCA GGA GTC1351Ile Glu Trp Leu Gln Lys Leu Met Arg Ser Gl - #u Pro Arg Pro Gly Val# 395#TGGCCTGTGG 1396C TAG C CCAGCAGCCCLeu Leu Arg Ala Pro Phe Pro400 4 - #05- AGAGAAAGCC AAGGCTGCGT CGAACTGTCC TGGCACCAAA TCCCATATAT TC - #TTCTGCAG1456- TTAATGGGGT AGAGGAGGGC ATGGGAGGGA GGGAGAGGTG GGGAGGGAGA CA - #GAGACAGA1516- AACAGAGAGA GACAGAGACA GAGAGAGACT GAGGGAGAGA CTCTGAGGAC AT - #GGAGAGAG1576- ACTCAAAGAG ACTCCAAGAT TCAAAGAGAC TAATAGAGAC ACAGAGATGG AA - #TAGAAAAG1636- ATGAGAGGCA GAGGCAGACA GGCGCTGGAC AGAGGGGCAG GGGAGTGCCA AG - #GTTGTCCT1696- GGAGGCAGAC AGCCCAGCTG AGCCTCCTTA CCTCCCTTCA GCCAAGCCCC AC - #CTGCACGT1756- GATCTGCTGG CCCTCAGGCT GCTGCTCTGC CTTCATTGCT GGAGACAGTA GA - #GGCATGAA1816- CACACATGGA TGCACACACA CACACGCCAA TGCACACACA CAGAGATATG CA - #CACACACG1876- GATGCACACA CAGATGGTCA CACAGAGATA CGCAAACACA CCGATGCACA CG - #CACATAGA1936- GATATGCACA CACAGATGCA CACACAGATA TACACATGGA TGCACGCACA TG - #CCAATGCA1996- CGCACACATC AGTGCACACG GATGCACAGA GATATGCACA CACCGATGTG CG - #CACACACA2056- GATATGCACA CACATGGATG AGCACACACA CACCAAGTGC GCACACACAC CG - #ATGTACAC2116- ACACAGATGC ACACACAGAT GCACACACAC CGATGCTGAC TCCATGTGTG CT - #GTCCTCTG2176- AAGGCGGTTG TTTAGCTCTC ACTTTTCTGG TTCTTATCCA TTATCATCTT CA - #CTTCAGAC2236- AATTCAGAAG CATCACCATG CATGGTGGCG AATGCCCCCA AACTCTCCCC CA - #AATGTATT2296- TCTCCCTTCG CTGGGTGCCG GGCTGCACAG ACTATTCCCC ACCTGCTTCC CA - #GCTTCACA2356- ATAAACGGCT GCGTCTCCTC CGCACACCTG TGGTGCCTGC CACCCAAAAA AA - #AAAAAAAA2416# 2422- (2) INFORMATION FOR SEQ ID NO:2:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 444 amino (B) TYPE: amino acid (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: protein- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:- Met Val Ser Gln Ala Leu Arg Leu Leu Trp Le - #u Leu Leu Gly Leu Gln25- Gly Cys Leu Ala Ala Val Phe Val Thr Gln Gl - #u Glu Ala His Gly Val10- Leu His Arg Arg Arg Arg Ala Asn Ala Phe Le - #u Glu Glu Leu Arg Pro# 10- Gly Ser Leu Glu Arg Glu Cys Lys Glu Glu Gl - #n Cys Ser Phe Glu Glu# 25- Ala Arg Glu Ile Phe Lys Asp Ala Glu Arg Th - #r Lys Leu Phe Trp Ile# 40- Ser Tyr Ser Asp Gly Asp Gln Cys Ala Ser Se - #r Pro Cys Gln Asn Gly# 55- Gly Ser Cys Lys Asp Gln Leu Gln Ser Tyr Il - #e Cys Phe Cys Leu Pro# 70- Ala Phe Glu Gly Arg Asn Cys Glu Thr His Ly - #s Asp Asp Gln Leu Ile# 90- Cys Val Asn Glu Asn Gly Gly Cys Glu Gln Ty - #r Cys Ser Asp His Thr# 105- Gly Thr Lys Arg Ser Cys Arg Cys His Glu Gl - #y Tyr Ser Leu Leu Ala# 120- Asp Gly Val Ser Cys Thr Pro Thr Val Glu Ty - #r Pro Cys Gly Lys Ile# 135- Pro Ile Leu Glu Lys Arg Asn Ala Ser Lys Pr - #o Gln Gly Arg Ile Val# 150- Gly Gly Lys Val Cys Pro Lys Gly Glu Cys Pr - #o Trp Gln Val Leu Leu155 1 - #60 1 - #65 1 -#70- Leu Val Asn Gly Ala Gln Leu Cys Gly Gly Th - #r Leu Ile Asn Thr Ile# 185- Trp Val Val Ser Ala Ala His Cys Phe Asp Ly - #s Ile Lys Asn Trp Arg# 200- Asn Leu Ile Ala Val Leu Gly Glu His Asp Le - #u Ser Glu His Asp Gly# 215- Asp Glu Gln Ser Arg Arg Val Ala Gln Val Il - #e Ile Pro Ser Thr Tyr# 230- Val Pro Gly Thr Thr Asn His Asp Ile Ala Le - #u Leu Arg Leu His Gln235 2 - #40 2 - #45 2 -#50- Pro Val Val Leu Thr Asp His Val Val Pro Le - #u Cys Leu Pro Glu Arg# 265- Thr Phe Ser Glu Arg Thr Leu Ala Phe Val Ar - #g Phe Ser Leu Val Ser# 280- Gly Trp Gly Gln Leu Leu Asp Arg Gly Ala Th - #r Ala Leu Glu Leu Met# 295- Val Leu Asn Val Pro Arg Leu Met Thr Gln As - #p Cys Leu Gln Gln Ser# 310- Arg Lys Val Gly Asp Ser Pro Asn Ile Thr Gl - #u Tyr Met Phe Cys Ala315 3 - #20 3 - #25 3 -#30- Gly Tyr Ser Asp Gly Ser Lys Asp Ser Cys Ly - #s Gly Asp Ser Gly Gly# 345- Pro His Ala Thr His Tyr Arg Gly Thr Trp Ty - #r Leu Thr Gly Ile Val# 360- Ser Trp Gly Gln Gly Cys Ala Thr Val Gly Hi - #s Phe Gly Val Tyr Thr# 375- Arg Val Ser Gln Tyr Ile Glu Trp Leu Gln Ly - #s Leu Met Arg Ser Glu# 390- Pro Arg Pro Gly Val Leu Leu Arg Ala Pro Ph - #e Pro395 4 - #00 4 - #05- (2) INFORMATION FOR SEQ ID NO:3:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 21 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: cDNA- (iii) HYPOTHETICAL: N- (iv) ANTI-SENSE: N- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:#21 CCCT T- (2) INFORMATION FOR SEQ ID NO:4:- (i) SEQUENCE CHARACTERISTICS:#pairs (A) LENGTH: 15 base (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: cDNA- (iii) HYPOTHETICAL: N- (iv) ANTI-SENSE: N- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:# 15__________________________________________________________________________

Number	Name	Date
4775624	Bang et al.	Oct 1988
4784950	Hagen et al.	Nov 1988
4829052	Glover et al.	May 1989
4959318	Foster et al.	Sep 1990
4994371	Davie et al.	Feb 1991
5190919	Fair et al.	Mar 1993
5278144	Wolf	Jan 1994
5288629	Berkner	Feb 1994
5326559	Miller	Jul 1994
5419760	Narcisco, Jr.	May 1995

Number	Date	Country
255771	Feb 1988	EPX
9215686	Sep 1929	WOX
8606408	Nov 1986	WOX
8990612	Oct 1989	WOX
9003390	Apr 1990	WOX
9015619	Dec 1990	WOX
9111514	Aug 1991	WOX
9427631	Dec 1994	WOX
9606637	Mar 1996	WOX

	Number	Date
Parent	660289	Jun 1996
Parent	475845	Jun 1995
Parent	327690

Modified factor VII

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Entry
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