JP5134362B2 - Modified annexin protein and method for preventing thrombosis - Google Patents

Description

FIELD OF THE INVENTION [0001] The present invention generally relates to methods and compositions for treating thrombosis. More specifically, the present invention relates to modified annexin proteins and methods of use thereof.

BACKGROUND OF THE INVENTION [0002] The thrombosis, i.e. the formation of a blood clot (thrombus) in a blood vessel, development, or presence, is the most common serious medical disorders. The most frequent example of arterial thrombosis is coronary thrombosis, which leads to blockage of the coronary arteries and often leads to myocardial infarction (heart attack). In North America, more than 1.3 million patients are hospitalized for myocardial infarction every year. In standard therapy, thrombolytic protein is administered by infusion. Thrombolytic treatment of acute myocardial infarction is estimated to save 30 lives per 1000 patients treated; nevertheless, the 30-day mortality rate of this disorder continues to be significant (Mehta et al., Lancet 356: 449-454 (2000)). The disclosures of Mehta et al., As well as the disclosures of all other patents, patent applications and publications cited herein are hereby fully incorporated by reference. Because bolus injection can be used before hospitalization with additional advantages, it would be preferable to administer antithrombotic and thrombolytic agents by this injection (see Rawles, incorporated herein). J. Am. Coll. Cardiol. 30: 1181-1186 (1997)). However, bolus injection (as opposed to more gradual intravenous infusion) significantly increases the risk of cerebral hemorrhage (Mehta et al., 2000). It would be desirable to develop agents that can prevent thrombosis and / or increase thrombolysis without increasing the risk of bleeding.

  [0003] Unstable angina, caused by occlusion of coronary arteries without proper oxygen delivery to the heart, is the most common cause of hospitalization, with 1.5 million cases per year in the United States alone. When treating patients with coronary artery occlusion with angioplasty and stent implantation, the use of antibodies against platelet gp IIb / IIIa reduces the likelihood of restenosis. However, in unstable angina without angioplasty, it has been shown that the same antibody does not benefit, and in these patients a better way to prevent coronary artery occlusion is needed.

[0004] Another important example of arterial thrombosis is cerebral thrombosis. Intravenous recombinant tissue plasminogen activator (rtPA) is the only cure for acute ischemic stroke approved by the Food and Drug Administration. The earlier the administration, the better (Ernst et al., Stroke 31: 2552-2527 (2000), incorporated herein). However, intravenous rtPA administration is associated with an increased risk of intracerebral hemorrhage. A full-blow stroke is often preceded by a transient ischemic attack (TIA), and it is estimated that approximately 300,000 people suffer from TIA each year in the United States. It would be desirable to have a safe and effective agent that can be administered as a bolus and would prevent recurrence of cerebral thrombosis for several days without increasing the risk of cerebral hemorrhage. Thrombosis also contributes to peripheral arterial occlusion in diabetics and other patients, and there is a need for effective and safe antithrombotic agents for use in such patients.

[0005] Venous thrombosis is a frequent complication of surgical procedures such as hip and knee arthroplasty. It would be desirable to prevent thrombosis without increasing bleeding to the surgical field. Similar considerations apply to venous thrombosis associated with pregnancy and parturition. Some people are prone to repeat venous thrombotic events, and now these people are treated with antithrombotic agents such as coumarin drugs. The dose of such drugs must be titrated in each patient, and the difference between an effective antithrombotic dose and a dose that increases bleeding is small. It would be desirable to have a treatment that better separates antithrombotic activity from an increased risk of bleeding. All recently introduced antithrombotic therapies are associated with increased bleeding risk, including platelet gp IIb / IIIa ligand, low molecular weight heparin, and pentasaccharide inhibitor of Factor Xa (Levine, incorporated herein by reference). Et al., Chest 119: 108S-121S (2001)). Therefore, there is a need to search for alternative strategies to prevent arterial and venous thrombosis without increasing the risk of bleeding.

[0006] In order to inhibit the expansion of arterial or venous thrombi without increasing bleeding, the potential difference between the mechanisms involved in hemostasis and the mechanisms involved in thrombosis in large vessels needs to be exploited is there. The main hemostatic mechanism involves the formation of platelet microaggregates, which aggregate the capillaries and accumulate on damaged or activated endothelial cells in small blood vessels. Inhibitors of platelet aggregation, inhibiting agent the formation or action of thromboxane _{A 2,} ligands of gp IIa / IIIb, and drugs acting on ADP receptors, which are incorporated in, for example, clopidogrel (herein, Hallopeter, Nature 409: 202-207 (2001)), which interferes with this process and thus increases the risk of bleeding (Levine et al., 2001). In contrast to microaggregate formation, occlusion with arterial or venous thrombosis requires the platelets to be continuously replenished and taken up. In large vessels, platelets must be tightly bound to each other and to the fibrin network deposited around the platelets in order to overcome shear detachment.

[0007] The formation of solid giant aggregates of platelets is facilitated by cellular and humoral amplification mechanisms, and evidence has gathered that they strengthen each other. In cellular mechanisms, the formation of relatively loose microaggregates of platelets is induced by moderate concentrations of agonists such as ADP, thromboxane A ₂ , or collagen, including the 85 kD protein Gas6 from platelet α granules. (Angelillo-Scherrer et al., Nature Medicine 7: 215-221 (2001), incorporated herein). When released Gas6 binds to receptor tyrosine kinases (Axl, Sky, Mer) expressed on the platelet surface, complete degranulation and the formation of firm, large aggregates of these cells is induced. In the humoral amplification mechanism, a prothrombinase complex is formed on the surface of activated platelets and microvesicles. This produces thrombin and fibrin. Thrombin is itself a potent platelet activator and inducer of Gas6 release (Ishimoto and Nakano, FEBS Lett. 446: 197-199 (2000), incorporated herein). Fully activated platelets bind tightly to the fibrin network deposited around the platelets. Histological observations have shown that both platelets and fibrin are necessary for the formation of stable coronary thrombus in humans (Falk et al., Intermediateship between aerosclerosis and thrombosis, incorporated herein). Vanstraete et al. (Supervised), Cardiovascular Thrombosis: Thrombology and Thromboneurology. Philadelphia: Lipincott-Raven Publishers (1998), 58. p. Another platelet adhesion molecule, amphoterin, translocates to the platelet surface during activation and binds to anionic phospholipids (Rouhainen et al . , Thromb. Hemost. 84: 1087-1094 (incorporated herein) ). 2000)). Like Gas6, amphoterin can also form crosslinks during platelet aggregation.

  [0008] The question arises as to whether it is possible to prevent thrombosis without inhibiting these amplification mechanisms, but without inhibiting the initial platelet aggregation stage, thereby increasing bleeding. The importance of cellular amplification has recently been established by studies of mice using targeted inactivation of Gas6 (Angelillo-Scherrer et al., 2001). Gas6-/-mice were found to be protected against collagen and epinephrine-induced thrombosis and embolism. However, Gas6-/-mice did not suffer from spontaneous bleeding and had normal bleeding after tail clipping. Furthermore, antibodies against Gas6 inhibited platelet aggregation in vitro, as well as thrombosis induced in vivo by collagen and epinephrine. In principle, antibodies or ligands that compete for Gas6 binding to receptor tyrosine kinases can be used to inhibit thrombosis. However, it may be preferable to inhibit this stage in view of the strength of humoral amplification. Ideally, such inhibitors will also have further suppressive activity against the cellular amplification mechanism mediated by Gas6.

[0009] A strategy for preventing both cellular and humoral amplification of platelet aggregation is provided by annexins, 10 of which are highly homologous, expressed in several human tissues It is a family of anti-thrombotic proteins (Benz and Hofmann, Biol. Chem. 378: 177-183 (1997), incorporated herein). Annexins share the property of binding to calcium and negatively charged phospholipids, both of which are necessary for clotting. Under physiological conditions, negatively charged phospholipids are primarily supplied by phosphatidylserine (PS) in activated or damaged cell membranes. In intact cells, PS is trapped in the inner leaflet of the plasma membrane bilayer and is inaccessible to the surface. When platelets are activated, the amount of PS accessible to the surface, and thus the extent of annexin binding, is greatly increased (Sun et al., Thrombosis Res. 69: 289-296, incorporated herein) . (1993)). During platelet activation, microvesicles are released from the platelet surface, greatly increasing the surface area on which anionic phospholipids with procoagulant activity are expressed (both Merten et al., Circulation 99, incorporated herein by reference). : 2577-2582 (1999); Chow et al., J. Lab. Clin. Med. 135: 66-72 (2000)). They can play an important role in platelet-mediated arterial thrombus growth.

[0010] Proteins involved in the coagulation cascade (Factors X, Xa, and Va) bind to the membrane carrying PS on the surface and bind to each other to form a stable and tightly bound prothrombinase complex. Form. Several annexins, including II, V, and VIII, bind to PS with high affinity, thereby preventing the formation of prothrombinase complexes and exerting antithrombotic activity. Annexin V binds PS with a very high affinity for negatively charged phospholipids, higher than the affinity of factors X, Xa, and Va (K _d = 1.7 nmol / l) (herein). Tiagarajan and Tait, J. Biol. Chem. 265: 17420-17423 (1990)). Tissue factor-dependent clotting on the surface of activated or damaged endothelial cells also requires surface expression of PS, and Annexin V can also inhibit this process (herein) Van Heerde et al . , Arterioscl. Thromb. 14: 824-830 (1994)), which is incorporated by reference, but annexin is less effective in this activity than in inhibiting prothrombinase production (incorporated herein). Rao et al . , Thromb.Res . 62: 517-531 (1992)).

[0011] The binding of Annexin V to activated platelets and damaged cells probably explains the selective retention of this protein in the thrombus. This has been shown in experimental animal models of venous thrombosis and arterial thrombosis (both incorporated herein by Straton et al., Circulation 92: 3113-3121 (1995); Thiagarajan and Benedict, Circulation 96 : 2339-2347 (1997)), and it has been proposed to use labeled annexins for medical imaging of vascular thrombi in humans with reduced noise and increased stability (incorporated herein). Reno and Kasina, International Patent Application PCT / US95 / 07599 (WO 95/34315) (published December 21, 1995)). The binding of strong antithrombotic agents such as Annexin V to the thrombus provides a strategy to prevent thrombus expansion or recurrence. Transient myocardial ischemia also increases annexin V binding (Dumont et al., Circulation 102: 1564-1568 (2000), incorporated herein). Annexin V imaging in humans has shown that binding of the protein increases in transplanted hearts when endocardial myocardial biopsy shows vascular rejection (Acio, incorporated herein by reference). Et al., J. Nuclear Med. 41 (5 Suppl.): 127P (2000)). This binding is probably due to damaged endothelial cells as well as PS exposed on the surface of apoptotic muscle cells in the rejected heart. Thus, administration of annexin after myocardial infarction should prevent the formation of prothrombotic complexes on both platelets and endothelial cells, thereby preventing the spread or recurrence of thrombosis. Annexin V binding is also increased in humans after cerebral hypoxia (D'Arceil et al., Stroke 2000: 2692-2700 (2000), incorporated herein), which indicates that annexin is reduced after TIA. Supports that administration may reduce the likelihood of developing a developed stroke.

[0012] Annexins are used in several in vitro thrombin-dependent assays, as well as venous thrombosis (both incorporated herein by Roemisch et al., Thrombosis Res. 61: 93-104 (1991); Van Ryn- McKenna et al., Thrombosis Hemostasis 69: 227-230 (1993)) and anti-coagulant activity in experimental animal models of arterial thrombosis (Thiagarajan and Benedict, 1997). Significantly, an antithrombotic dose of annexin has no demonstrable effect on traditional ex vivo clotting tests in treated rabbits (Thiagarajan and Benedict, 1997) and reduces the bleeding time of treated rats. It was not significantly prolonged (Van Ryn-McKennna et al., 1993). In treated rabbits, annexin did not increase bleeding to the surgical incision (Thiagarajan and Benedict, 1997). Therefore, among all agents studied so far, annexin exerts antithrombotic activity without increasing bleeding. Annexin does not inhibit platelet aggregation induced by agonists other than thrombin (van Heerde et al., 1994), and platelet aggregation is the major hemostatic mechanism. The tissue factor / VIIa complex also exerts a hemostatic effect in the damaged vessel wall and in extravascular tissue, and this system is more susceptible to inhibition by annexin V than the prothrombinase complex undergoes. Not sensitive (Rao et al., 1992). This is one argument in favor of limiting the administered annexin V to the vascular compartment as much as possible; the risk of bleeding is probably reduced.

[0013] Despite these promising results of preventing thrombosis, the main problem associated with the therapeutic use of annexins is that they have a short half-life in the circulation and are 5-15 minutes in laboratory animals. (Roemisch et al., 1991; Straton et al., 1995; Thiagarajan and Benedict, 1997); Annexin V also has a short half-life in the human circulation (Strauss et al., Incorporated herein by reference). J. Nuclear Med. 41 (5 Suppl.): 149P (2000)). Most annexins are lost in the urine, as expected for the 36 kDa protein (Thiagarajan and Benedict, 1997). Therefore, there is a need for a method that prevents annexin from being lost from the vascular compartment to the extravascular compartment and urine, thereby prolonging the antithrombotic activity after a single injection.

SUMMARY OF THE INVENTION [0014] The present invention provides compounds and methods for preventing arterial or venous thrombosis. Recombinant human annexins are modified in such a way that the half-life in the vascular compartment is increased. This can be accomplished in a variety of ways; annexin coupled to polyethylene glycol, an annexin homopolymer or heteropolymer, and a fusion protein of an annexin with another protein (eg, the Fc portion of an immunoglobulin) has three embodiments. It is. The modified annexins bind with high affinity to phosphatidylserine on the surface of activated platelets or damaged cells, thereby binding Gas6 and procoagulant proteins and forming a prothrombinase complex. To prevent. Thus, modified annexins inhibit both cellular and humoral mechanisms that amplify platelet aggregation, thereby preventing thrombosis.

  [0015] In one embodiment, the present invention provides an isolated modified annexin protein containing an annexin protein, preferably annexin V, coupled to polyethylene glycol (PEG). Preferably, at least two PEG chains, each PEG having a molecular weight of at least 5 kDa, more preferably at least 10 kDa, and most preferably at least 15 kDa, are coupled to a single annexin molecule. In another embodiment, the isolated modified annexin protein contains at least one additional protein, such as an additional annexin protein (forming a homodimer) or an annexin protein coupled to the Fc portion of an immunoglobulin. The further protein preferably has a molecular weight of at least 30 kDa. Also provided by the present invention is a pharmaceutical composition containing an antithrombotic effective amount of any of the modified annexin proteins of the present invention.

  [0016] In the method of the present invention, the modified annexin is administered to a subject at risk of thrombosis in a pharmaceutical composition having any one of the isolated modified annexin proteins of the present invention in an antithrombotic effective amount. . For example, the pharmaceutical composition can be administered after arterial thrombosis such as coronary thrombosis, cerebral thrombosis, or transient cerebral ischemic attack. It can also be administered after surgery related to venous thrombosis. It can also be administered to a subject who is susceptible to arterial or venous thrombosis, such as a subject with diabetes, pregnancy, or labor.

  [0017] The present invention also provides an isolated nucleic acid molecule encoding a homodimer of annexin, a recombinant molecule containing at least a portion of the nucleic acid molecule, and at least a portion of the nucleic acid molecule Is a recombinant cell. In the methods of the invention, recombinant cells are cultured under appropriate conditions to produce an annexin homodimer.

  [0018] The present invention also provides a method of screening for modified annexin proteins that modulate thrombosis using a thrombosis test system. The test system is contacted with the test modified annexin protein, after which thrombolytic activity is assessed and compared to the activity of the system in the absence of the test modified annexin protein. Preferably, the activated partial thromboplastin time is measured. Also provided is a method of identifying modified annexin proteins by contacting activated platelets with a test modified annexin protein and assessing platelet binding and protein S binding activity.

  [0019] The present invention also provides a method for screening in vivo for modified annexin proteins. In this method, a thrombosis animal model is contacted with a test modified annexin protein, and then the in vivo anticoagulant activity and increased bleeding of the test modified annexin protein is evaluated. Anticoagulant activity and time are compared to annexin anticoagulant activity and time, and the amount of bleeding is compared to bleeding in an animal model in the absence of the test-modified annexin protein.

  [0020] Accordingly, the present invention is a method of treating a subject at risk for thrombosis, wherein an isolated modified annexin protein comprising an annexin dimer is administered to said subject in an antithrombotic effective amount. Providing the method. The isolated modified annexin protein is administered after coronary artery thrombosis, after overt cerebral thrombosis, after transient cerebral ischemic attack, after surgery related to venous thrombosis, and when said subject is diabetic and said thrombus Administer when the disease is arterial thrombosis or during a period of time selected from the group consisting of pregnancy and delivery. Isolated modified annexin protein is administered in the range of 0.2 mg / kg to 1.0 mg / kg.

  [0021] The present invention is also a method of inhibiting leukocyte adhesion to endothelial cells, wherein an effective amount of an isolated modified annexin protein comprising an annexin dimer is administered to a patient in need of such method. Wherein said method is also provided. In some embodiments, the method further comprises reducing endothelial cell damage.

  [0022] The present invention is also a method of treating a subject at risk for thrombosis, wherein a protein having an affinity for phosphatidylserine that is at least 90% of the affinity of annexin V for phosphatidylserine is antithrombotic. The method is provided comprising administering to the subject in an effective amount, wherein the protein is a monoclonal or polyclonal antibody.

DETAILED DESCRIPTION OF THE INVENTION [0038] The present invention provides compounds and methods for preventing thrombosis in mammals without increasing bleeding. The present invention relies, in part, on the recognition that the initial mechanism of platelet aggregation is different from the mechanism that amplifies platelet aggregation that is required for the formation of arterial or venous thrombi. By inhibiting thrombus formation but not early platelet aggregation, it is also possible to prevent thrombosis without increasing bleeding.

[0039] Compounds of the invention include any product containing an annexin amino acid sequence that has been modified to increase the half-life of the product in humans or other mammals. As used herein, when referring to an amino acid sequence of a naturally occurring protein molecule, when “amino acid sequence” is listed, “amino acid sequence” and similar terms such as “polypeptide” or “protein” It is not meant to limit the sequence to the fully natural amino acid sequence associated with the listed proteins. Annexins are a family of homologous, phospholipid-binding membrane proteins, 10 of which correspond to distinct gene products expressed in mammals (Benz and Hofmann, 1997). Crystallographic analysis reveals a common tertiary structure for all family members studied so far, exemplified by Annexin V (Huber et al., EMBO Journal 9: 3867 (1990), incorporated herein). ). The core domain is a concave disk-like structure that can also be juxtaposed in close proximity to the phospholipid membrane. The protein contains four subdomains, each consisting of a 70 amino acid annexin repeat composed of five α-lesions. Annexins also have more hydrophilic tail domains that vary in length and amino acid sequence between different annexins. The sequence of the gene encoding annexin is well known (eg, Funakoshi et al., Biochemistry 26: 8087-8092 (1987) (Annexin V), incorporated herein).

  [0040] Annexin proteins include annexin family proteins such as annexin II (lipocortin 2, calpactin 1, protein I, p36, chromobindin 8), annexin III (lipocortin 3, PAP-III), annexin IV ( Lipocortin 4, endonexin I, protein II, chromobindin 4), annexin V (lipocortin 5, endonexin 2, VAC-alpha, ancholine CII, PAP-I), annexin VI (lipocortin 6, protein III, chromobindin 20, p68, p70), Annexin VII (Synnexin), Annexin VIII (VAC-beta), Annexin XI (CAP-50), and Annexin XIII (ISA) It is.

  [0041] Annexin IV shares many of the same properties as Annexin V. Like Annexin V, Annexin IV binds to acidic phospholipid membranes in the presence of calcium. Annexin IV is a close structural homologue of Annexin V. The sequence of annexin IV is known. Hamman et al., Biochem. Biophys. Res. Comm. 156: 660-667 (1988). Annexin IV belongs to the annexin family of calcium-dependent phospholipid binding proteins. The mechanism is still not clearly defined.

  [0042] Annexin IV (endonexin) is a 32 kDa calcium-dependent membrane-bound protein. The translated amino acid sequence of Annexin IV exhibits a four domain structure characteristic of this type of protein. Annexin IV has 45-59% identity with other members of the family and shares a similar size and exon-intron organization. Annexin IV isolated from human placenta has in vitro anticoagulant activity and encodes a protein that inhibits phospholipase A2 activity. Annexin IV is expressed almost exclusively in epithelial cells.

  [0043] Annexin VIII belongs to the family of CA (2 +)-dependent phospholipid binding proteins (Annexins) and has a high identity to Annexin V (56%). Haptmann et al., Eur J Biochem. 1989 Oct 20; 185 (1): 63-71. The protein was first isolated as a 2.2 kb vascular anticoagulant-beta. Annexin VIII is neither an extracellular protein nor associated with the cell surface. The protein may not play a role in blood clotting in vivo. Its physiological role remains unknown. The protein is expressed at low levels in the human placenta and exhibits expression restricted to lung, endothelium and skin, liver and kidney.

  [0044] In the present invention, annexin proteins are modified to increase half-life in humans or other mammals. In some embodiments, the annexin protein is annexin V, annexin IV or annexin VIII. One suitable modification of annexin is an increase in effective size, thereby preventing loss from the vascular compartment to the extravascular compartment and urine, thereby prolonging antithrombotic activity after a single injection. Any increase in size effective to maintain sufficient binding affinity with phosphatidylserine is within the scope of the present invention.

  [0045] In one embodiment of the invention, the modified annexin was coupled to polyethylene glycol (PEG) in such a manner as to be able to perform the function of annexin in a phosphatidylserine (PS) binding assay. Contains recombinant human annexin protein. The antithrombotic effect of an annexin-PEG conjugate administered intravenously is prolonged when compared to that of a free annexin. The recombinant annexin protein coupled to PEG can be annexin V protein or another annexin protein. In one embodiment, the annexin protein is annexin V, annexin IV or annexin VIII.

[0046] PEG consists of repeating units of ethylene oxide and terminates in linear or, in some cases, hydroxyl groups at both ends of the branched chain. The size and molecular weight of the PEG chain to be coupled can be selected depending on the number of ethylene oxide units contained. For the purposes of the present invention, any PEG size and number of PEG chains per annexin molecule that increases the half-life of the modified annexin relative to annexin while retaining the binding function of the modified molecule to PS. Can also be used. As noted above, sufficient binding is reduced from that of unmodified annexins, but still competes with the binding of factors of the Gas6 and prothrombinase complex and can therefore prevent thrombosis Includes bonds. The optimal molecular weight of PEG to be conjugated varies with the number of PEG chains. In one embodiment, two PEG molecules each having a molecular weight of at least about 15 kDa are coupled to each annexin molecule. PEG molecules can be linear or branched. Annexin calcium-dependent binding to PS is affected not only by the size of the PEG molecule to be coupled, but also by the site on the protein to which the PEG binds. Optimal selection ensures that the desired properties are retained. Knowing the three-dimensional structure of a molecule and analyzing the interaction between the phospholipid membrane and the molecule with mutational analysis and crystallographic analysis facilitates the selection of PEG attachment sites (Campos, incorporated herein). Et al., Biochemistry 37: 8004-8008 (1998)).

[0047] In the field of drug delivery, PEG derivatives are widely used covalently attached to proteins (referred to as PEGylation) to enhance solubility and reduce immunogenicity, proteolysis, and renal clearance. It has been. The excellent clinical efficacy of recombinant products coupled to PEG is well established. For example, once-weekly administered PEG-interferon-2a is significantly more effective against hepatitis C virus than three times weekly doses of unbound interferon (Heathcote et al., Incorporated herein by reference). N. Engl. J. Med. 343: 1673-1680 (2000)). Coupling to PEG is intended to increase the half-life of recombinant proteins in vivo (Knauf et al., J. Biol. Chem. 266: 2796- 2804 (1988), incorporated herein) as well as a set To prevent enzymatic degradation of the replacement protein and to reduce the immunogenicity that is sometimes observed with homologous products (Hermanson, Bioconjugate techniques. New York, Academic Press (1996), pp. 173, incorporated herein by reference). -176 reference)).

[0048] In another embodiment of the invention, the modified annexin protein is a polymer of annexin protein having an increased effective size. An increase in effective size is thought to result in increased half-life and increased antithrombotic activity in the vascular compartment. One such modified annexin is a dimer of annexin proteins. In one embodiment, the annexin dimer is an annexin V, annexin IV or annexin VIII homodimer. In another embodiment, the annexin dimer is annexin V and other annexin proteins (eg annexin IV or annexin VIII), annexin IV and another annexin protein (eg annexin V or annexin VIII) or annexin VIII and another annexin protein Heterodimers (eg, Annexin V or Annexin IV). Another such polymer is a heterotetramer of Annexin II and p11, a member of the S100 family of calcium binding proteins. When the S100 protein binds to annexin, the affinity of the annexin for Ca ²⁺ increases. Annexin homopolymers or heterotetramers can be produced by bioconjugation or recombinant methods, and the polymers can be administered alone or in PEG-conjugated form.

  [0049] In some embodiments, the modified annexin has increased affinity for PS. Human annexin V homodimer (DAV) was prepared using well-established methods of recombinant DNA technology as described in Example 4. Homodimeric annexin molecules are linked to flexible linkers through peptide bonds (FIG. 1). In some embodiments, the flexible linker contains an amino acid sequence flanked by glycine and serine residues that act as swivels at either end. The linker preferably comprises one or more such “swivel”. Preferably, the linker comprises two swivels, which are at least 2 amino acids, more particularly at least 4 amino acids, more particularly at least 6 amino acids, more particularly at least 8 amino acids, more particularly at least 10 amino acids, It is possible to be separated. Preferably, the overall length of the linker is 5-30 amino acids, 5-20 amino acids, 5-10 amino acids, 10-15 amino acids, or 10-20 amino acids. The dimer can be folded in such a way that the convex surface of the monomer, which binds to Ca 2+ and PS, is both accessible to the exposed PS. Flexible linkers are known in the art, see, for example, Arai et al., Proteins. 2004 Dec 1; 57 (4): 829-38, (GGGGGS) (n) (n = 3-4), and helical linker (EAAAAK) (n) (n = 2-5). As described in Example II, annexin V homodimers out-compete out annexin monomers upon binding to PS on the cell surface (FIG. 2).

  [0050] In another embodiment of the invention, the recombinant annexin is expressed with, or chemically coupled to, another protein, such as the Fc portion of an immunoglobulin. Such expression or coupling increases the effective size of the molecule, prevents the loss of annexins from the vascular compartment, and prolongs the anticoagulant action.

  [0051] Preferably, the modified annexin protein of the invention is an isolated modified annexin protein. The modified annexin protein can also contain annexin II, annexin IV, annexin V, or annexin VIII. In some embodiments, the protein is a modified human annexin. In some embodiments, the modified annexin contains a recombinant human annexin. In accordance with the present invention, an isolated or biologically pure protein is a protein that has been removed from its natural environment. As such, “isolated” and “biologically pure” do not necessarily reflect the degree to which the protein has been purified. The isolated modified annexin protein of the present invention can be obtained from a natural source, can be produced using recombinant DNA technology, or can be produced by chemical synthesis. As used herein, an isolated modified annexin protein can be either a full-length modified protein or a homologue of such a protein. It can also be a modified full-length protein (eg with respect to a PEGylated protein) or a modified homologue of such a protein.

  [0052] The minimum size of a protein homologue of the present invention is sufficient to be encoded by a nucleic acid molecule capable of forming a stable hybrid with the complementary sequence of the nucleic acid molecule encoding the corresponding natural protein. As such, the size of nucleic acid molecules encoding such protein homologues depends on the nucleic acid composition, as well as the percent homology between the nucleic acid molecule and the complementary sequence, and the hybridization conditions themselves (eg, temperature, salt concentration, and formamide concentration). To do. The minimum size of such a nucleic acid molecule is typically at least about 12 to about 15 nucleotides in length if the nucleic acid molecule is GC rich, and at least about 15 to about 17 bases in length if AT rich. It is. As such, the minimum size of a nucleic acid molecule used to encode a protein homologue of the present invention is about 12 to about 18 nucleotides in length. There is no limit to the maximum size of such a nucleic acid molecule, since a nucleic acid molecule can contain part of a gene, a whole gene, or many genes or parts thereof. Similarly, the minimum size of an annexin protein homologue or modified annexin protein homologue of the present invention is about 4 to about 6 amino acids in length, and the size is full length, multivalent (ie, more than 1 each with function) Depending on whether a fusion protein having a domain) protein, or a functional part of such a protein is desired. Annexin and modified annexin homologues of the invention preferably have activity corresponding to the natural subunit, such as being capable of performing the activity of an annexin protein that prevents thrombus formation.

  [0053] Annexin proteins and modified annexin homologues can be the result of natural allelic variation or natural mutation. It is also possible to produce protein homologues of the invention using techniques known in the art, including but not limited to direct modifications to the protein or, for example, random Modification of the gene encoding the protein using classical or recombinant DNA techniques to achieve mutagenesis or targeted mutagenesis is included.

[0054] Also included is an amino acid sequence, SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 12, SEQ ID NO: 15, or an allelic variant of a nucleic acid molecule that encodes a protein containing any of these sequences. At least about 75%, more preferably at least about 80%, more preferably at least about 85%, more preferably at least about 90%, more preferably at least about 95%, and most preferably at least about 98% A modified annexin protein containing an amino acid sequence that is Also included is a modified annexin protein comprising more than one of SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 12, SEQ ID NO: 15; eg, a protein comprising SEQ ID NO: 3 and SEQ ID NO: 12 and separated in a linker is there. Methods of determining percent identity between amino acid sequences and between nucleic acid sequences are known to those skilled in the art. Methods for determining percent identity between sequences include GCG® Wisconsin Package ^™ (available from Accelrys Corporation), DNAsis ^™ program (Hitachi Software, San Bruno, Calif.), Vector NTI suite (Informax, Inc.). Computer programs such as BLAST software available on the NCBI website), or North Bethesda, Maryland).

  [0055] In one embodiment, the modified annexin protein comprises at least about 5 amino acids, preferably at least about 50 amino acids, more preferably at least about 100 amino acids, more preferably at least about 200 amino acids, more preferably at least about 250 amino acids, More preferably at least about 275 amino acids, more preferably at least about 300 amino acids, and most preferably at least about 319 amino acids or the full length annexin protein, whichever amino acid sequence is included. In another embodiment, the annexin protein is a full-length protein, ie, a protein encoded by a full-length coding region, or a post-translationally modified protein thereof, eg, a mature protein from which the initiation methionine and / or signal sequence or “pro” sequence has been removed. Containing.

  [0056] Fragments of the modified annexin proteins of the present invention are preferably at least about 5 amino acids in length, more preferably at least about 10 amino acids, more preferably at least about 15 amino acids, more preferably at least about 20 amino acids, more preferably At least about 25 amino acids, more preferably at least about 30 amino acids, more preferably at least about 35 amino acids, more preferably at least about 40 amino acids, more preferably at least about 45 amino acids, more preferably at least about 50 amino acids, more preferably at least about about 55 amino acids, more preferably at least about 60 amino acids, more preferably at least about 65 amino acids, more preferably at least about 70 amino acids, more preferably at least about 75 amino acids, more preferably Containing at least about 80 amino acids, more preferably at least about 85 amino acids, more preferably at least about 90 amino acids, more preferably at least about 95 amino acids and even more preferably at least about 100 amino acids.

  [0057] In one embodiment, an isolated modified annexin protein of the invention contains a protein encoded by a nucleic acid molecule having the nucleic acid sequence, SEQ ID NO: 4. Alternatively, the modified annexin protein contains a protein encoded by a nucleic acid sequence having the nucleic acid sequence, SEQ ID NO: 1, or by an allelic variant of a nucleic acid molecule having this sequence. Alternatively, the modified annexin protein contains more than one protein sequence encoded by a nucleic acid sequence, a nucleic acid molecule having SEQ ID NO: 1, or an allelic variant of a nucleic acid molecule having this sequence.

  [0058] In one embodiment, an isolated modified annexin protein of the invention contains a protein encoded by a nucleic acid molecule having the nucleic acid sequence, SEQ ID NO: 10, or by an allelic variant of a nucleic acid molecule having this sequence. Alternatively, the modified annexin protein contains more than one protein sequence encoded by a nucleic acid molecule having the nucleic acid sequence, SEQ ID NO: 10, or by an allelic variant of a nucleic acid molecule having this sequence (eg, SEQ ID NO: 12-linker) -SEQ ID NO: 12).

  [0059] In another embodiment, an isolated modified annexin protein of the invention is a modified protein encoded by a nucleic acid sequence having the nucleic acid sequence, SEQ ID NO: 13, or by an allelic variant of a nucleic acid molecule having this sequence. Alternatively, the modified annexin protein contains more than one protein sequence encoded by a nucleic acid sequence having the nucleic acid sequence, SEQ ID NO: 13, or by an allelic variant of a nucleic acid molecule having this sequence (eg, SEQ ID NO: 15-linker) -SEQ ID NO: 15).

  [0060] In another embodiment, an isolated modified annexin protein of the invention is a nucleic acid sequence, a protein encoded by a nucleic acid molecule having SEQ ID NO: 1, and a nucleic acid sequence, a protein encoded by a nucleic acid molecule having SEQ ID NO: 10 Or modified proteins containing proteins encoded by allelic variants of these nucleic acid molecules (eg, SEQ ID NO: 3-linker-SEQ ID NO: 12 or SEQ ID NO: 12-linker-SEQ ID NO: 3).

  [0061] In another embodiment, an isolated modified annexin protein of the invention is a nucleic acid sequence, a protein encoded by a nucleic acid molecule having SEQ ID NO: 1, and a nucleic acid sequence, a protein encoded by a nucleic acid molecule having SEQ ID NO: 13 Or modified proteins containing proteins encoded by allelic variants of these nucleic acid molecules (eg, SEQ ID NO: 3-linker-SEQ ID NO: 15 or SEQ ID NO: 15-linker-SEQ ID NO: 3).

  [0062] In another embodiment, an isolated modified annexin protein of the invention is a nucleic acid sequence, a protein encoded by a nucleic acid molecule having SEQ ID NO: 10, and a nucleic acid sequence, a protein encoded by a nucleic acid molecule having SEQ ID NO: 13 Or a modified protein containing a protein encoded by an allelic variant of these nucleic acid molecules (eg, SEQ ID NO: 12-Linker-SEQ ID NO: 15 or SEQ ID NO: 15-Linker-SEQ ID NO: 12).

[0063] One embodiment of the present invention includes a non-naturally modified annexin protein encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions to an annexin gene. As used herein, stringent hybridization conditions refer to standard hybridization conditions that use nucleic acid molecules comprising oligonucleotides to identify molecules having similar nucleic acid sequences. Such standard conditions are disclosed, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Labs Press (1989), incorporated herein by reference. Stringent hybridization conditions typically allow isolation of a nucleic acid molecule having at least about 70% nucleic acid sequence identity with the nucleic acid molecule used to probe in the hybridization reaction. To do. Formulas for calculating hybridization and wash conditions suitable for achieving hybridization that allows for 30% or less nucleotide mismatches are described, for example, in Meinkoth et al . , Anal. Biochem. 138: 267-284 (1984). In some embodiments, the hybridization conditions will allow the isolation of nucleic acid molecules having at least about 80% nucleic acid sequence identity with the nucleic acid molecules used in the probe. In other embodiments, the hybridization conditions will allow isolation of nucleic acid molecules having at least about 90% nucleic acid sequence identity with the nucleic acid molecules used in the probe. In yet other embodiments, the hybridization conditions will allow isolation of nucleic acid molecules having at least about 95% nucleic acid sequence identity with the nucleic acid molecules used in the probe.

  [0064] The modified annexin protein is at least about 50 nucleotides and is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 10, SEQ ID NO: 13, or any of these nucleic acid molecules Preferably about 20% base pair mismatch, more preferably about 15% base pair mismatch, more preferably about 10% base pair mismatch. Encoded by a nucleic acid molecule that hybridizes under conditions that allow for, more preferably, conditions that allow about 5% base pair mismatch, and even more preferably, conditions that allow about 2% base pair mismatch. Contains protein.

  [0065] As used herein, an annexin gene includes, in addition to the coding region itself, all nucleic acid sequences associated with the natural annexin gene, such as a regulatory region that regulates the production of the annexin protein encoded by this gene (eg, a restriction region). Although not necessarily included, transcriptional, translational or post-translational regulatory regions). In one embodiment, the annexin gene includes the nucleic acid sequence, SEQ ID NO: 1. In another embodiment, the annexin gene includes the nucleic acid sequence, SEQ ID NO: 10. In another embodiment, the annexin gene includes the nucleic acid sequence, SEQ ID NO: 13. Since nucleic acid sequencing techniques are not completely error-free, SEQ ID NO: 1 (as well as other sequences presented herein) is, at best, an apparent nucleic acid molecule encoding an annexin protein of the invention. Note that it corresponds to a nucleic acid sequence.

  [0066] In another embodiment, the annexin gene can be an allelic variant that includes a sequence that is similar to, but not identical to, SEQ ID NO: 1. In another embodiment, the annexin gene can be an allelic variant that includes a sequence that is similar to SEQ ID NO: 10, but not identical. In another embodiment, the annexin gene can be an allelic variant that includes a sequence that is similar to SEQ ID NO: 13 but is not identical. An allelic variant of the annexin gene comprising SEQ ID NO: 1 is present in the genome at essentially the same locus (s) as the gene comprising SEQ ID NO: 1, but is a natural variation caused by, for example, mutation or recombination Therefore, it is a gene having a similar but not identical sequence. Allelic variants typically encode proteins that have similar activity to the protein encoded by the gene being compared. Allelic variants can also include modifications in the 5 'or 3' untranslated regions (eg, regulatory regulatory regions) of the gene. Allelic variants are well known to those skilled in the art and are expected to be found within a given human and / or within a population comprising two or more humans because the genome is diploid Will.

  [0067] The isolated modified annexin protein of the present invention can be obtained from natural sources, can be produced using recombinant DNA technology, or can be produced by chemical synthesis. is there. As used herein, an isolated modified annexin protein can contain either a full-length protein or a homologue of such a protein. Examples of annexins and modified annexin homologues include amino acid deletions (eg, truncated forms of the protein, such as a truncated form of the protein, eg Peptides, or introns are removed or by protein splicing reactions when two exons are joined), inserted, inverted, substituted, and / or derivatized (eg, glycosylation, phosphorylation) Acetylation, methylation, myristylation, prenylation, palmitoylation, amidation and / or addition of glycerophosphatidylinositol), annexins and modified annexin proteins. That is, when a homolog is administered to an animal as an immunogen using techniques known to those skilled in the art, the animal produces a humoral and / or cellular immune response against at least one epitope of the annexin protein. Will. Annexin and modified annexin homologues can also be selected by their ability to selectively bind immune sera. A method for measuring such activity is disclosed herein. Annexins and modified annexin homologues are also proteins capable of performing the functions of natural annexins in functional assays; Including. Methods for such assays are described in the Examples section elsewhere herein.

  [0068] The modified annexin proteins of the invention can also be identified by their ability to perform the function of an annexin protein in a functional assay. The phrase “can perform its function in a functional assay” means that the protein or modified protein has at least about 10% of the activity of the native protein in the functional assay. In other embodiments, the protein has at least about 20% of the activity of the native protein in the functional assay. In other embodiments, the protein has at least about 30% of the activity of the native protein in the functional assay. In other embodiments, the protein has at least about 40% of the activity of the native protein in the functional assay. In other embodiments, the protein has at least about 50% of the activity of the native protein in the functional assay. In other embodiments, the protein or modified protein has at least about 60% of the activity of the native protein in the functional assay. In yet other embodiments, the protein or modified protein has at least about 70% of the activity of the native protein in the functional assay. In yet other embodiments, the protein or modified protein has at least about 80% of the activity of the native protein in the functional assay. In other embodiments, the protein or modified protein has at least about 90% of the activity of the native protein in the functional assay. Examples of functional assays are described herein.

  [0069] The isolated proteins of the present invention can be produced in a variety of ways, including recovering such proteins from bacteria and producing such proteins recombinantly. . One aspect of the present invention is a method of producing an isolated modified annexin protein of the present invention using recombinant DNA technology. Such methods include the steps of (a) culturing a recombinant cell containing a nucleic acid molecule encoding a modified annexin protein of the invention to produce the protein, and (b) recovering the protein therefrom. It is. Details regarding the production of recombinant cells and their culture are presented below. The phrase “protein recovery” simply refers to recovering the entire fermentation medium containing the protein and need not necessarily include further steps of separation or purification. A variety of standard protein purification techniques can be used to purify the proteins of the invention.

  [0070] Isolated proteins of the present invention are preferably recovered in "substantially pure" form. As used herein, “substantially pure” refers to a purity that allows effective use of the protein in a functional assay.

Modified Annexin Nucleic Acid Molecules or Genes [0071] Another aspect of the invention is a heterodimer of Annexin V homodimer, Annexin IV homodimer, Annexin VIII homodimer, Annexin V and Annexin VIII An isolated nucleic acid molecule capable of hybridizing under stringent conditions with a gene encoding a modified annexin protein, such as a heterodimer of the body, annexin V and annexin IV, or a heterodimer of annexin IV and annexin VIII is there. Such nucleic acid molecules are also referred to herein as modified annexin nucleic acid molecules. Included are isolated nucleic acid molecules that hybridize with the modified annexin gene under stringent conditions. The characteristics of such genes are disclosed herein. In accordance with the present invention, an isolated nucleic acid molecule is a nucleic acid molecule that has been removed from its natural environment (ie, has been subjected to human manipulation). As such, “isolation” does not reflect the degree to which the nucleic acid molecule has been purified. An isolated nucleic acid molecule can include DNA, RNA, or derivatives of either DNA or RNA.

  [0072] As noted above, modified annexin genes include, in addition to the coding region itself, all nucleic acid sequences associated with the natural annexin gene, such as the regulatory regions that regulate the production of the annexin protein encoded by this gene (eg, Including, but not limited to, transcriptional, translational or post-translational regulatory regions). The nucleic acid molecules of the invention can also be isolated modified annexin nucleic acid molecules or homologues thereof. The nucleic acid molecules of the invention can also include one or more regulatory regions, full length or partial coding regions, or combinations thereof. The minimum size of the modified annexin nucleic acid molecule of the present invention is the minimum size capable of forming a stable hybrid with the corresponding natural gene under stringent hybridization conditions. Annexin nucleic acid molecules can also include nucleic acid molecules that encode hybrid proteins, fusion proteins, multivalent proteins or partially excised fragments.

  [0073] Isolated nucleic acid molecules of the invention can also be obtained from natural sources as part of the entire (ie, complete) gene or all genes capable of forming a stable hybrid with the gene. As used herein, the phrase “at least a portion” of an entity refers to the amount of the entity that is at least sufficient to have a functional aspect of that entity. For example, herein, at least a portion of a nucleic acid sequence is an amount of the nucleic acid sequence that can form a stable hybrid with the corresponding gene under stringent hybridization conditions.

  [0074] It is also possible to produce isolated nucleic acid molecules of the invention using recombinant DNA technology (eg, polymerase chain reaction (PCR) amplification, cloning, etc.) or chemical synthesis. Isolated modified annexin nucleic acid molecules include, but are not limited to, natural nucleic acid molecules and homologues thereof, or the nucleic acid molecule encodes an annexin protein of the invention or is naturally occurring under stringent conditions. Natural alleles in which nucleotides are inserted, deleted, substituted and / or inverted in such a way that such modifications do not substantially interfere with the ability to form stable hybrids with nucleic acid molecule isolates. Variants and modified nucleic acid molecules are included.

  [0075] Several methods known to those skilled in the art can also be used to produce modified annexin nucleic acid molecule homologues (see, eg, Sambrook et al., 1989). For example, a variety of techniques can be used to modify nucleic acid molecules, including but not limited to classical mutagenesis techniques and recombinant DNA techniques, such as site-specific mutations. Mutagenesis, chemical treatment of nucleic acid molecules to induce mutations, restriction enzyme cleavage of nucleic acid fragments, ligation of nucleic acid fragments, polymerase chain reaction (PCR) amplification and / or mutagenesis of selected regions of nucleic acid sequences, This includes the synthesis of oligonucleotide mixtures, and the linking of mixtures that “build” nucleic acid molecule mixtures, as well as combinations thereof. By screening for the function of the protein encoded by the nucleic acid (eg, the ability of the homologue to elicit an immune response against the annexin protein, and / or the ability to function in a coagulation assay or other functional assay) and / or It is also possible to select nucleic acid molecule homologues from a mixture of modified nucleic acids by hybridization with an isolated annexin-encoding nucleic acid under stringent conditions.

  [0076] An isolated modified annexin nucleic acid molecule of the present invention can also comprise a nucleic acid sequence encoding at least one modified annexin protein of the present invention, examples of such proteins are disclosed herein. The phrase “nucleic acid molecule” refers primarily to a physical nucleic acid molecule and the phrase “nucleic acid sequence” primarily refers to a sequence of nucleotides on the nucleic acid molecule, but the two phrases can be used interchangeably. In particular, with respect to nucleic acid molecules or nucleic acid sequences capable of encoding modified annexin proteins, they can be used interchangeably.

  [0077] One embodiment of the present invention is capable of hybridizing under stringent conditions to a nucleic acid strand encoding at least a portion of a modified annexin protein or homologue thereof, or to the complement of such a nucleic acid strand. A modified annexin nucleic acid molecule. The nucleic acid sequence complement of any of the nucleic acid sequences of the present invention refers to the nucleic acid sequence of the nucleic acid strand that is complementary to the sequence from which the sequence was drawn (ie, capable of forming a full duplex with such a strand). Note that the double-stranded nucleic acid molecule of the invention, whose nucleic acid sequence has been determined for one strand, ie represented by SEQ ID NO., Also includes a complementary strand having a sequence that is the complement of that SEQ ID NO. Should. As such, the nucleic acid molecules of the present invention, which can be either double-stranded or single-stranded, are subject to the defined SEQ ID NOs and / or the present specification under stringent hybridization conditions. A nucleic acid molecule that forms a stable hybrid with any of the complements of its SEQ ID NO that may or may not be shown. Methods for estimating complementary sequences are known to those skilled in the art. Corresponding region (s) of the nucleic acid sequence encoding at least a portion of the modified annexin protein and at least about 65 percent, preferably at least about 70 percent, more preferably at least about 75 percent, more preferably at least about 80 percent. More preferably at least about 85 percent, more preferably at least about 90 percent, and even more preferably at least about 95 percent homologous nucleic acid molecules comprising nucleic acid sequences having homology. A modified annexin nucleic acid molecule capable of encoding a homodimer of an annexin protein or a homologue thereof is included.

  [0078] Annexin nucleic acid molecules include SEQ ID NO: 4 and allelic variants of SEQ ID NO: 4, allelic variants of SEQ ID NO: 1 and SEQ ID NO: 1, allelic variants of SEQ ID NO: 10 and SEQ ID NO: 10, and SEQ ID NO: 13 and The allelic variant of SEQ ID NO: 13 is included.

  [0079] Knowing the nucleic acid molecule of the modified annexin protein of the present invention, one of ordinary skill in the art will make a copy of the nucleic acid molecule and, in addition, a nucleic acid molecule that includes additional portions of the annexin protein-encoding gene (eg, a translation initiation site and / or Nucleic acid molecules comprising transcriptional regulatory regions and / or translational regulatory regions), and / or annexin nucleic acid molecule homologues. Knowing part of the amino acid sequence of the annexin protein of the present invention allows one skilled in the art to clone the nucleic acid sequence encoding such an annexin protein. In addition, it is possible to obtain the desired modified annexin nucleic acid molecule in a variety of ways, including screening an appropriate expression library using an antibody that binds to the annexin protein of the present invention; Traditional libraries using the oligonucleotide probes of the present invention to screen libraries or DNA; and suitable libraries or PCR amplification of RNA or DNA (genomic libraries) using the oligonucleotide primers of the present invention. And / or cDNA libraries can be used).

  [0080] The present invention also relates to oligonucleotides capable of hybridizing under stringent conditions with other, preferably longer, complementary regions of nucleic acid molecules of the present invention that encode at least a portion of a modified annexin protein. Also includes certain nucleic acid molecules. The oligonucleotides of the present invention can be RNA, DNA, or any derivative. The minimum size of such an oligonucleotide is that required to form a stable hybrid between a given oligonucleotide and a complementary sequence on another nucleic acid molecule of the present invention. Minimum size features are disclosed herein. The size of the oligonucleotide must also be sufficient to use the oligonucleotide according to the invention. The oligonucleotides of the invention can also be used in a variety of applications, including, but not limited to, amplifying or extending nucleic acid molecules as probes for identifying additional nucleic acid molecules. Applications include as a primer or in therapeutic applications that modulate modified annexin production. Such therapeutic applications include the use of such oligonucleotides in technologies based on, for example, antisense, triplex formation, ribozymes and / or RNA agents. The invention thus includes methods for modulating the production of modified annexin proteins by use of such oligonucleotides and one or more such techniques.

Natural wild-type bacterial cells, and recombinant molecules and cells [0081] The invention also includes a modified annexin nucleic acid molecule of the invention inserted into any vector capable of delivering the nucleic acid molecule to a host cell. Also includes recombinant vectors. Such vectors contain heterologous nucleic acid sequences, ie, nucleic acid sequences that are not found naturally adjacent to the modified annexin nucleic acid molecules of the invention. Vectors can be either RNA or DNA, either prokaryotic or eukaryotic, and are typically viruses or plasmids. It is also possible to use recombinant vectors in cloning, sequencing, and / or otherwise manipulating modified annexin nucleic acid molecules of the invention. One type of recombinant vector, referred to herein as a recombinant molecule and described in more detail below, can also be used for expression of the nucleic acid molecules of the invention. Some recombinant vectors are capable of replication in transformed cells. The nucleic acid molecules contained in the recombinant vectors of the invention are disclosed herein.

  [0082] As previously disclosed, one embodiment of the present invention is by culturing cells capable of expressing a protein under conditions effective to produce the protein and recovering the protein. A method for producing the modified annexin protein of the present invention. In another embodiment, the method comprises culturing cells capable of expressing the protein under conditions effective to produce an annexin protein, recovering the protein, and coupling to an agent that increases its effective size. Producing the annexin protein by modifying the protein.

  [0083] In one embodiment, the cells to be cultured are natural bacterial cells and the modified annexins are isolated from these cells. In another embodiment, the cell to be cultured is a recombinant cell capable of expressing a modified annexin protein, the recombinant cell being produced by transforming a host cell with one or more nucleic acid molecules of the invention. ing. Transformation of the nucleic acid molecule into the cell can be accomplished by any method capable of inserting the nucleic acid molecule into the cell. Transformation techniques include, but are not limited to, transfection, electroporation, microinjection, lipofection, adsorption, and protoplast fusion. Recombinant cells can remain unicellular or can grow into tissues, organs or multicellular organisms. A transformed nucleic acid molecule of the invention can remain extrachromosomal or one or more within the chromosome of the transformed (ie, recombinant) cell in such a manner that the ability to be expressed is retained. It is also possible to be incorporated into the site. Nucleic acid molecules that transform host cells are disclosed herein.

  [0084] Suitable host cells for transformation include any cell that can be transformed and express the introduced modified annexin protein. Such cells can therefore produce the modified annexin protein of the invention after being transformed with at least one nucleic acid molecule of the invention. The host cell can be either an untransformed cell or a cell that has already been transformed with at least one nucleic acid molecule. Suitable host cells of the invention can include bacterial, fungal (including yeast), insect, animal, and plant cells. Host cells include bacterial cells, with E. coli cells being particularly preferred. Another host cell is an untransformed (wild-type) bacterial cell that produces a cognate modified annexin protein, optionally including attenuated strains with reduced pathogenicity.

[0085] Preferably, one or more recombinant molecules each comprising one or more nucleic acid molecules of the invention operably linked to an expression vector containing one or more transcriptional regulatory sequences, To produce recombinant cells. The phrase “operably linked” refers to the insertion of a nucleic acid molecule into an expression vector in such a way that the molecule can be expressed when transformed into a host cell. As used herein, an expression vector is a DNA or RNA vector capable of transforming a host cell and achieving expression of a specified nucleic acid molecule. Preferably, the expression vector is also capable of replicating in the host cell. Expression vectors can be either prokaryotic or eukaryotic and are typically viruses or plasmids. Expression vectors of the invention include any vector that functions (ie directs gene expression) in a recombinant cell of the invention, including in bacterial, fungal, insect, animal, and / or plant cells. As such, the nucleic acid molecules of the invention can be promoters, operators, repressors, enhancers, termination sequences, origins of replication, and other control sequences that are compatible with recombinant cells and regulate the expression of the nucleic acid molecules of the invention, etc. It can also be operably linked to an expression vector containing the above regulatory sequences. As used herein, transcriptional regulatory sequences include sequences that can regulate transcription initiation, elongation, and termination. Particularly important transcriptional regulatory sequences are those that regulate transcription initiation, such as promoters, enhancers, operators and repressor sequences. Suitable transcription regulatory sequences include any transcription regulatory sequence that is functional in at least one of the recombinant cells of the invention. A variety of such transcription regulatory sequences are known in the art. Transcriptional regulatory sequences include those that function in bacterial, yeast, insect and mammalian cells, such as but not limited to tac, lac, tzp, trc, oxy-pro, omp / lpp, rrnB, bacteriophage Lambda (λ) (λp _L and λp _R and fusions containing such promoters), bacteriophage T7, T7lac, bacteriophage T3, bacteriophage SP6, bacteriophage SP01, metallothionein, alpha mating factor, Pichia alcohol Oxidase, alphavirus subgenomic promoter (such as Sindbis virus subgenomic promoter), baculovirus, heliothis zea insect virus, vaccinia virus, herpes Irus, poxvirus, adenovirus, simian virus 40, retrovirus actin, retroviral long terminal repeat, rous sarcoma virus, heat shock, phosphate and nitrate transcription regulatory sequences, and prokaryotic or eukaryotic cells In which other sequences capable of regulating gene expression are included. Further suitable transcriptional regulatory sequences include tissue specific promoters and enhancers, and lymphokine inducible promoters (eg, promoters inducible by interferons or interleukins). The transcriptional regulatory sequences of the present invention can also include naturally occurring transcriptional regulatory sequences that are naturally associated with the DNA sequence encoding the annexin protein. One transcriptional regulatory sequence is a Kozak strong promoter and initiation sequence.

  [0086] The expression vectors of the invention can also contain a secretion signal (ie, a signal segment nucleic acid sequence) that allows the expressed annexin protein to be secreted from the cell that produces the protein. Suitable signal segments include either an annexin protein signal segment or a heterologous signal segment capable of directing secretion of an annexin protein of the invention, including fusion proteins. Signal segments include, but are not limited to, tissue plasminogen activator (t-PA), interferon, interleukin, growth hormone, histocompatibility, and viral envelope glycoprotein signal segments.

  [0087] The expression vector of the present invention may also contain a fusion sequence that directs the expression of the inserted nucleic acid molecule of the present invention as a fusion protein. Inclusion of a fusion sequence as part of the modified annexin nucleic acid molecule of the present invention can enhance stability during production, storage, and / or use of the protein encoded by the nucleic acid molecule. Further, the fusion segment can function as a tool that simplifies the purification of the modified annexin protein, for example, as a tool that allows purification of the fusion protein generated using affinity chromatography. One fusion segment that can be used for protein purification is the 8-amino acid peptide sequence asp-tyr-lys-asp-asp-asp-asp-lys (SEQ ID NO: 9).

  [0088] A suitable fusion segment can be any size domain having the desired function (eg, increased stability and / or purification tools). The use of one or more fusion segments is within the scope of the present invention. It is also possible to link the fusion segment to the amino terminus and / or carboxyl terminus of the annexin protein. Another type of fusion protein is a fusion protein that links a fusion segment to two or more annexin proteins or modified annexin proteins. In order to allow easy recovery of the annexin or modified annexin protein, it is also possible to construct a link between the fusion segment and the annexin protein so that it is sensitive to cleavage. Preferably, the fusion protein is produced by culturing recombinant cells transformed with a fusion nucleic acid sequence encoding a protein comprising a fusion segment attached to either the carboxyl terminus and / or the amino terminus of the annexin protein.

  [0089] The recombinant molecule of the present invention is operably linked to at least one of transcriptional regulatory sequences capable of effectively controlling the expression of a nucleic acid molecule in a cell to be transformed. A molecule capable of containing at least one of the nucleic acid molecules described above. Recombinant molecules include one or more nucleic acid molecules of the present invention, including those that encode one or more modified annexin proteins. The recombinant molecules of the invention and their production are described in the Examples section. Similarly, recombinant cells include one or more nucleic acid molecules of the present invention that encode one or more annexin proteins. The recombinant cells of the present invention include those disclosed in the Examples section.

  [0090] Using recombinant DNA technology, for example, manipulating the copy number of nucleic acid molecules in a host cell, the efficiency with which such nucleic acid molecules are transcribed, the efficiency with which the resulting transcript is translated, and the efficiency of post-translational modifications. Those skilled in the art will recognize that this can improve the expression of the transformed nucleic acid molecule. Recombinant techniques useful for increasing the expression of a nucleic acid molecule of the present invention include, but are not limited to, ligation such that the nucleic acid molecule can be functionalized into a high copy number plasmid, one or more host cells. Integration of nucleic acid molecules into chromosomes, addition of vector stability sequences to plasmids, substitution or modification of transcriptional regulatory signals (eg promoters, operators, enhancers), translational regulatory signals (eg ribosome binding sites, Shine-Dalgarno sequences) Transient cell growth from substitution or modification, modification of the nucleic acid molecules of the invention to accommodate host cell codon usage, deletion of sequences that destabilize transcription, and production of recombinant proteins during fermentation The use of regulatory signals that are separated. It is also possible to improve the activity of the expressed recombinant protein of the invention by fragmenting, modifying or derivatizing the resulting protein.

  [0091] In accordance with the present invention, recombinant cells are used under such conditions by culturing under conditions effective to produce an annexin or modified annexin protein of the present invention and recovering the protein, It is also possible to produce such proteins. Conditions effective to produce the protein include, but are not limited to, appropriate media, bioreactor, temperature, pH and oxygen conditions that allow protein production. A suitable or effective medium refers to any medium capable of producing an annexin or modified annexin protein when the cells of the invention are cultured. Such media are typically aqueous media containing assimilable carbohydrates, nitrogen and phosphate sources, and appropriate salts, minerals, metals and other nutrients such as vitamins. The medium can contain complex nutrients or can be a defined minimal medium.

  [0092] Cells of the present invention can also be cultured in conventional fermentation bioreactors, including but not limited to batch, fed-batch, cell recycling and continuous fermenters. It is. It is also possible to perform the culture in shake flasks, test tubes, microtiter plates, and petri dishes. Culturing is performed at a temperature, pH and oxygen content appropriate for the recombinant cell. Such culture conditions are within the expertise of one of ordinary skill in the art.

  [0093] Depending on the vector and host system used for production, the resulting annexin protein remains in the recombinant cell; is secreted into the fermentation medium; in the space between the two cell membranes, eg, the periplasmic space of E. coli Or can be retained on the outer surface of the cell or viral membrane. Methods for purifying such proteins are disclosed in the Examples section.

Antibodies [0094] The present invention also includes isolated anti-modified annexin antibodies and uses thereof. An anti-modified annexin antibody is an antibody that can selectively bind to a modified annexin protein. An isolated antibody is an antibody that has been removed from its natural environment. The term “isolated” does not refer to the state of purity of such antibodies. As such, isolated antibodies can include antisera containing such antibodies, or antibodies that have been purified to varying degrees. As used herein, the term “selectively binds” means that such an antibody binds preferentially to the protein from which the antibody was made (ie, the protein from unrelated components in the mixture). Refers to the ability to be distinguished. The binding affinity, generally expressed as an equilibrium association constant, typically ranges from about 10 ³ M ⁻¹ to about 10 ¹² M ⁻¹ . Binding can also be measured using a variety of methods known to those skilled in the art, including immunoblot assays, immunoprecipitation assays, radioimmunoassays, enzyme immunoassays (eg, ELISA), immunofluorescent antibody assays. And immunoelectron microscopy; see, for example, Sambrook et al., 1989.

  [0095] The antibody of the present invention can be either a polyclonal antibody or a monoclonal antibody. The antibodies of the present invention include functions such as antibody fragments and genetically engineered antibodies, including single chain antibodies capable of selectively binding to at least one epitope of the protein used to obtain the antibodies. The equivalents above are included. The antibodies of the present invention also include chimeric antibodies that can bind to more than one epitope. Antibodies are made at least in part in response to proteins encoded by the modified annexin nucleic acid molecules of the invention.

  [0096] Anti-modified annexin antibodies of the present invention include antibodies produced in animals that have received modified annexins. Anti-modified annexin antibodies of the present invention also include antibodies made in animals and then recovered from the cells against one or more modified annexin proteins of the present invention using techniques known to those skilled in the art. . Additional antibodies of the invention are produced recombinantly using the techniques disclosed herein for the modified annexin proteins of the invention. Antibodies produced against the defined proteins are substantially contaminated with antibodies to other substances that could otherwise cause interference in diagnostic assays or side effects when used in therapeutic compositions. This may be preferable.

  [0097] The anti-modified annexin antibodies of the present invention have a variety of uses that are within the scope of the present invention. Anti-modified annexin antibodies can be used as a tool to screen expression libraries and / or recover the desired protein of the invention from a mixture of proteins and other contaminants.

[0098] The anti-modified annexin antibody of the present invention can selectively bind to a modified annexin protein.
Therapy [0099] Any of the above-described modified annexin proteins are used in the methods of the invention to treat arterial or venous thrombosis caused by any medical method or condition. Generally, the therapeutic agent used in the present invention is administered to an animal in an effective amount. In general, an effective amount is effective to (1) reduce the symptoms of the disease to be treated, or (2) induce a pharmacological change corresponding to treating the disease to be treated. Amount.

  [00100] With respect to thrombosis, an effective amount is effective to exert prolonged antithrombotic activity or increase the life expectancy of the affected animal without substantially increasing the risk of bleeding. Amount included. As used herein, prolonged antithrombotic activity refers to the time of activity of the modified annexin protein relative to the time of activity of the same amount (molar concentration) as the unmodified annexin protein. Preferably, the antithrombotic activity is prolonged by at least about 2 times (factor of two), more preferably at least about 5 times (factor of five), and most preferably at least about 10 times (factor of ten). Preferably, the effective amount does not substantially increase the bleeding risk compared to the bleeding risk of the same subject not receiving the modified annexin. Preferably, the risk of bleeding is very small and, at most, lower than that provided for other antithrombotic treatments available in the prior art. The therapeutically effective amount of the therapeutic agent can be any amount or dose sufficient to cause the desired antithrombotic effect, and in part, the thrombus status, type and location, the patient's Depending on size and condition, and other factors known to those skilled in the art. It is also possible to administer the dosage as a single dose or as several doses, for example divided over the course of several weeks.

  [00101] Preferably, to prevent further thrombosis, by boluses or by intravenous infusion after thrombosis or under conditions where the subject is susceptible to or at risk of thrombosis Do the administration.

  [00102] The therapeutic agents of the invention can also be administered by any suitable means, including, for example, parenteral or topical administration, such as intravenous or subcutaneous injection, or by aerosol. Depending on the mode of administration, the therapeutic composition may be administered in a variety of unit dosage forms. Delivery methods for the therapeutic compositions of the present invention include intravenous and topical administration, eg, by injection. In a particular mode of delivery, the therapeutic composition of the present invention can be formulated in the excipient of the present invention. The therapeutic agents of the invention can be administered to any animal, preferably a mammal, and more preferably a human.

  [00103] One appropriate administration time occurs after coronary artery thrombosis, thereby preventing recurrence of thrombosis without substantially increasing the risk of bleeding. A bolus injection of the modified annexin is preferably performed shortly after thrombosis, for example, before hospitalization. The modified annexin can also be administered in combination with a thrombolytic therapeutic agent such as tissue plasminogen activator, urokinase, or bacterial enzyme.

  [00104] Methods of using the modified annexin proteins of the present invention include methods of treating cerebral thrombosis including overt cerebral thrombosis or transient cerebral ischemic attacks, which requires treating cerebral thrombosis By administering an effective amount of a modified annexin protein to a patient. A developed stroke is often preceded by a transient ischemic attack. It is also possible to administer modified annexins to diabetics and other patients who have an increased risk of thrombosis in peripheral arteries. Accordingly, the present invention is a method for reducing the risk of thrombosis in a patient at increased risk of thrombosis, wherein an effective amount of a modified annexin protein is administered to the patient in need of reducing the risk of thrombosis Providing the method. For adult patients, a modified annexin in the dosage range of about 1 to about 100 mg can be administered intravenously or as a bolus.

  [00105] The present invention is also a method for reducing the risk of venous thrombosis associated with some surgical procedures, such as hip and knee arthroplasty, and there is a need to reduce the risk of such thrombosis. The method is also provided by administering to a patient an effective amount of a modified annexin protein of the invention. Modified annexin treatment can also prevent thrombosis without increasing bleeding to the surgical field. In another aspect, the present invention provides a method for preventing thrombosis associated with pregnancy and labor without increasing bleeding, wherein a patient in need of preventing thrombosis is treated with the modified annexin protein of the present invention. The method is provided by administering an effective amount. In a further aspect, the present invention provides a method for the treatment of recurrent venous thrombosis, comprising administering an effective amount of the modified annexin protein of the present invention to a patient in need of treating recurrent venous thrombosis, Provide a method. For adult patients, a modified annexin in the dosage range of about 1 to about 100 mg can also be administered intravenously as a bolus.

  [00106] The invention is also a method of screening for a modified annexin protein that modulates thrombosis, wherein the thrombosis test system is contacted with at least one test modified annexin protein under conditions that permit thrombosis, And by comparing the antithrombotic activity in the absence of the test modified annexin protein with the antithrombotic activity in the presence of the test modified annexin protein, There is provided the method, wherein a change is an indication that the modified annexin protein modulates thrombotic activity. In one embodiment, the thrombosis test system is a system for measuring activated partial thromboplastin time. Modified annexin proteins that modulate thrombosis, as identified by this method, are also included within the scope of the present invention.

  [00107] The present invention is also a method of identifying a modified annexin protein for annexin activity, wherein the activated platelets are contacted with at least one test modified annexin protein under conditions that permit binding; and Comparing the test modified annexin binding activity and platelet protein S binding activity in the presence of the test modified annexin protein with the annexin binding activity and protein S binding activity in the presence of the unmodified annexin protein, thereby Also provided is the above method, wherein a modified annexin protein having annexin activity is identifiable. Modified annexin proteins identified by this method are also included within the scope of the present invention.

  [00108] In a further aspect, the present invention is a method of screening for a modified annexin protein that modulates thrombosis, wherein the in vivo thrombosis test system is subjected to at least one test modified annexin under conditions that permit thrombosis. The method is provided by contacting the protein and comparing the antithrombotic activity in the absence of the test modified annexin protein with the antithrombotic activity in the presence of the test modified annexin protein. A change in antithrombotic activity in the presence of the test modified annexin protein is an indicator that the modified annexin protein regulates thrombotic activity. In addition, comparing the time of antithrombotic activity in the presence of the test modified annexin protein with the time of antithrombotic activity in the presence of the unmodified annexin, the antithrombotic activity associated with the test modified annexin protein Decide on extension. The degree of bleeding in the presence of the test modified annexin protein is assessed, for example, by measuring tail bleeding time and compared to the degree of bleeding in the absence of the test modified annexin protein. In one embodiment, the in vivo thrombosis test system is a mouse model of photochemically induced thrombus in the levator muscle. Modified annexin proteins that modulate thrombosis, as identified in this method, are also included within the scope of the present invention.

  [00109] In a further embodiment, the therapeutic agents of the invention are useful for gene therapy. As used herein, the phrase “gene therapy” refers to the transfer of genetic material of interest (eg, DNA or RNA) into a host to treat or prevent an inherited or acquired disease or condition. The genetic material of interest encodes a product (eg, protein, polypeptide, peptide or functional RNA) that is desired to be produced in vivo. For example, the genetic material of interest can encode a hormone, receptor, enzyme or (poly) peptide of therapeutic value. In certain embodiments, the present invention provides a non-viral gene that can also be complexed with a nucleic acid, as described in Hughes et al., US Pat. No. 6,169,078, incorporated herein by reference. Utilizing a lipid molecular species for use in therapy, a disulfide linker is provided between the polar head group and the lipophilic tail group of the lipid.

  [00110] These therapeutic compounds of the present invention effectively complex with DNA and promote the transfer of DNA through the cell membrane into the intracellular space of cells to be transformed with heterologous DNA. In addition, these lipid molecules promote the release of heterologous DNA in the cytoplasm, thereby increasing gene transfection during gene therapy in humans or animals.

[00111] Cationic lipid-polyanionic macromolecular aggregates can also be formed by a variety of methods known in the art. Representative methods are described by Felgner et al . , Proc. Natl. Acad. Sci. USA 86: 7413-7417 (1987); Epstein et al., US Pat. No. 4,897,355; Behr et al . , Proc. Natl. Acad. Sci. USA 86: 6982-6986 (1989); Bangham et al., J. Biol. Mol. Biol. 23: 238-252 (1965); Olson et al . , Biochim. Biophys. Acta 557: 9 (1979); Szoka et al . , Proc. Natl. Acad. Sci. 75: 4194 (1978); Mayhew et al . , Biochim. Biophys. Acta 775: 169 (1984); Kim et al . , Biochim. Biophys. Acta 728: 339 (1983); and Fukunaga et al . , Endocrinol. 115: 757 (1984). Generally, lipid particles consisting of either (1) a cationic lipid or (2) a cationic lipid mixed with a colipid are prepared and then lipid particles at about room temperature (about 18-26 ° C.) It is also possible to form aggregates by adding polyanionic macromolecules to. Generally, conditions are selected that do not lead to deprotection of the protecting group. In one embodiment, the mixture is then allowed to form agglomerates over a period of about 10 minutes to about 20 hours, with about 15-60 minutes being most preferably used. Other time periods may be appropriate for specific lipid types. It is possible to form complexes over longer periods of time, but longer complex formation periods usually will not provide further enhancement of transfection efficiency.

  [00112] The compounds and methods of the present invention can also be used to deliver intracellularly desired molecules, such as polynucleotides, to target cells. The desired polynucleotide can also be composed of DNA or RNA or analogs thereof. The desired polynucleotide delivered using the methods of the invention is composed of nucleotide sequences that provide different functions or activities, such as nucleotides with regulatory functions, such as promoter sequences, or nucleotides that encode a polypeptide. Is also possible. The desired polynucleotide can also provide a nucleotide sequence that is antisense to other nucleotide sequences in the cell. For example, when a desired polynucleotide is transcribed in a cell, it can provide a polynucleotide having a sequence that is antisense to other nucleotide sequences in the cell. The antisense sequence can hybridize to the sense strand sequence in the cell. One of ordinary skill in the art can readily prepare polynucleotides that provide antisense sequences. The desired polynucleotide to be delivered to the cell can also include a nucleotide sequence that is capable of forming a triplex complex with double-stranded DNA in the cell.

  [00113] The present invention provides compounds and methods for preventing or attenuating reperfusion injury in mammals. Reperfusion injury (RI) occurs when the blood supply to an organ or tissue is interrupted and restored after an interval. Loss of phospholipid asymmetry in endothelial cells and other cells is considered a significant event in the development of RI. When PS is exposed on the surface of these cells, binding of activated monocytes is possible. This binding triggers a series of events that lead to irreversible apoptosis of endothelial and other cells, another critical event in RI. Furthermore, PS on the cell surface, and vesicles derived therefrom, are accessible to phospholipases that produce lipid mediators. These lipid mediators amplify the damage caused by the mechanisms described above and cause serious complications such as ventricular arrhythmia after acute myocardial infarction.

  [00114] The recombinant human annexin, preferably annexin V, is modified in such a way as to increase the half-life in the vascular compartment. This can be accomplished in a variety of ways; annexin coupled to polyethylene glycol, an annexin homopolymer or heteropolymer, and a fusion protein of an annexin and another protein (eg, the Fc portion of an immunoglobulin) in three embodiments. is there. Both of which are fully incorporated herein by reference, Allison, “Modified Annexins Proteins and Methods for Producing Thrombosis,” US Patent Application No. 10 / 080,370 (filed February 21, 2002) and Allison, “Modified A See Proteins and Methods for Treating Vaso-Occlusive Sickle-Cell Disease, "US patent application Ser. No. 10 / 632,694 (filed Aug. 1, 2003).

  [00115] Modified annexins bind phosphatidylserine on the surface of epithelial cells and other cells with high affinity, thereby preventing phagocytic binding and manipulation of phospholipases that release lipid mediators. Thus, modified annexins inhibit both the cellular and humoral mechanisms of reperfusion injury.

  [00116] In one embodiment, the invention comprises at least one additional protein, such as an annexin protein coupled to an additional annexin protein (forming a homodimer), polyethylene glycol, or the Fc portion of an immunoglobulin. An isolated modified annexin protein is provided. The further protein preferably has a molecular weight of at least 30 kDa. Also provided by the present invention is a pharmaceutical composition containing any of the modified annexin proteins of the present invention in an amount effective to prevent or reduce reperfusion injury.

  [00117] In some methods of the invention, a reperfusion injury in a pharmaceutical composition having any one of the modified annexin proteins of the invention in an amount effective to prevent or attenuate reperfusion injury. Administer modified annexins to subjects at risk of For example, the pharmaceutical composition may be administered before and after organ transplantation, arthroplasty, or other surgical procedures where blood supply to the organ or tissue is interrupted and restored after an interval. Is possible. It is also possible to administer the composition after coronary or cerebral thrombosis.

  [00118] Modified annexins bind to accessible PS on the cell surface (shield cells), thereby preventing irreversible steps of monocyte attachment and apoptosis. Furthermore, modified annexins inhibit the activity of phospholipases that generate lipid mediators that also contribute to RI. Modified annexins may be useful to prevent or attenuate RI in organs transplanted from cadaveric donors, in patients with coronary and cerebral thrombosis, in patients undergoing arthroplasty, and in other situations . Furthermore, modified annexins will exert prolonged antithrombotic activity without increasing bleeding. This combination of the ability to attenuate RI and antithrombotic efficacy presents a unique profile of desirable activity that is not presented to any therapeutic agent currently used or known to be in development.

  [00119] As described in Example 6, annexin homodimers are potent inhibitors of sPLA2 (Figure 4). Annexin V shields PS from degradation by sPLA2 and other phospholipases because it binds with high affinity to PS on the cell surface.

  [00120] Producing a human annexin V homodimer increases its affinity for PS, thereby improving its efficacy as a therapeutic agent; and not only increasing its size, thereby circulating The duration and duration of action are prolonged. The 36 kDa monomer was rapidly lost from the bloodstream to the kidneys. In rabbits, more than 80% of labeled annexin V injected into the circulation disappeared in 7 minutes (Thiagarajan and Benedict, Circulation 96: 2339, 1997). In cynomolgus monkeys, the half-life of injected annexin V was found to be 11-15 minutes (Romisch et al., Thrombosis Res., 61:93, 1991). In humans injected with 99MTc-labeled annexins, the half-life for the major (α) compartment was 24 minutes (Kemerink et al., J. Nucl. Med. 44: 947, 2003).

  [00121] Annexin homodimers can be produced by any suitable method. Recombinant DNA technology is used in some embodiments to avoid the need for post-translational treatment, such as linking to one available sulfhydryl group in the monomer or coupling with polyethylene glycol. Annexin homodimers are produced by recombinant DNA technology. Recombinant homodimerization was achieved by using a flexible peptide linker attached to the amino terminus of one annexin monomer and the carboxy terminus of the other monomer (Figure 1). The three-dimensional structure of annexin V and the residues that bind to Ca2 + and PS is known from X-ray crystallography and site-directed mutagenesis (Huber et al., J. Mol. Biol. 223: 683, 1992; Campos et al. 37: 8004, 1998). The Ca 2+ binding site and the PS binding site are on the convex surface of the molecule, while the amino terminus forms a loose tail on the concave surface. The annexin V homodimer shown in FIG. 1 is designed so that both convex surfaces can be folded in such a way that the PS on the cell surface is accessible. Thus, therefore, the dimer will have a higher affinity for PS than that of the monomer. This was verified experimentally as reported in Example 4. Another advantage of the annexin V homodimer is that the 36 kDa molecule (monomer) is rapidly lost from circulation to the kidney, while the 73 kDa one (dimer) exceeds the renal filtration threshold. , That will not be lost. Thus, a therapeutically useful activity will be prolonged with the dimer. This prediction was confirmed by experiments.

  [00122] In some cases, it is desirable to have a longer duration of activity in order to prevent or attenuate reinfarction and RI. Increasing the molecular weight of annexin V by homodimerizing to 76 kDa prevents loss to the kidney and prolongs survival in the circulation. Thus, such modified annexins can effectively attenuate RI even if administered several hours before the blood supply to the organ is interrupted.

  [00123] The description of the present invention is contrary to reports in the literature that suggested that Annexin V does not inhibit RI. For example, d'Amico et al. Report that in the rat heart, annexin V does not inhibit RI but lipocortin I (annexin I) inhibited (d'Amico et al., FASEB J. 14: 1867, 2000). Infusion of lipocortin I fragment into the rat ventricle has been reported to reduce infarct size and cerebral edema after cerebral ischemia (Pelton et al., J. Exp. Med. 174: 305, 1991); Did not study reperfusion. In a comprehensive review of strategies to prevent liver ischemic injury (Selzner et al., Gastroenterology 15: 917, 2003), no annexin is mentioned.

  [00124] The ability of annexin V homodimers to attenuate RI was tested in a mouse liver model (Teoh et al., Hepatology 36:94, 2002) as described in Example 7. In this model, blood flow to the left and middle lobes of the liver is interrupted for 90 minutes and then recovered. After 24 hours, the severity of liver injury is assessed by serum levels of alanine aminotransferase (ALT) and liver histology. Annexin V homodimer (DAV), molecular weight 73 kDa, and annexin V (PEG-AV) coupled to polyethylene glycol, molecular weight 57 kDa, injected 6 hours prior to clamping the hepatic artery, are both serum ALT levels (FIG. 5) and as shown in liver histology, was very effective in attenuating RI. Annexin V monomer (AV) was less protective in this model.

  [00125] Thus, experimental evidence confirms that the modified annexins of the present invention will be useful in attenuating RI in subjects. As discussed above, the types of RI that occur in different organs involve a similar pathogenesis, and thus annexin V homodimers can be used to attenuate RI in all of these.

  [00126] Because of its high affinity for PS and reduced loss from circulation, annexin V homodimers will exert prolonged antithrombotic activity. This is clinically useful in preventing reinfarction, and reinfarction is known to be an important event after coronary thrombus (Andersen et al., N. Engl. J. Med. 349: 733). 2003), and may be important for stroke. The prevention of thrombosis in patients undergoing arthroplasty is also a clinically important need. Thus, the additional activity of modified annexins as anticoagulants is valuable. In some experimental animal models, Annexin V inhibits arterial and venous thrombosis without increasing bleeding (Roemisch et al., Thromb. Res. 61:93, 1991; Van Ryn-McKenna et al., Thromb Hemost. 69: 227, 1993; Thiagarajan and Benedict, Circulation 96: 2339, 1997). Modified annexins have the ability to exert anticoagulant activity and attenuate reperfusion injury without increasing bleeding. This combination of actions may be useful in some clinical situations. None of the other therapeutic agents currently used or known to be in development share this desirable profile of activity.

  [00127] Several annexins other than annexin V bind to Ca2 + and PS. Any of these can be used to prevent or reduce reperfusion injury. It is also possible to increase the molecular weight of annexin V or another annexin by methods other than homodimerization. Such methods include the preparation of other homopolymers or heteropolymers. Alternatively, annexin can be conjugated to another protein by recombinant DNA techniques or chemical manipulation. Conjugation of annexins to polyethylene glycol or another non-peptide compound is also envisioned.

  [00128] It is expected that Annexin V homodimers will be well tolerated. Another annexin, annexin VI, is a naturally occurring homodimer of conserved annexin sequences. However, Annexin VI does not bind to PS with high affinity. PS binding proteins other than annexins can also be used in the methods of the invention. For example, monoclonal or polyclonal antibodies with high affinity for PS (Diaz et al., Bioconjugate Chem. 9: 250, 1998; Thorpe et al., US Pat. No. 6,312,694) (eg, reduce reperfusion injury) (Or to prevent) can also be used in accordance with the present invention.

  [00129] Diannexin (define-SEQ ID NO: 6) has dose-related antithrombotic activity in rats (Figure 7). Even when diannexin is administered at 5.0 mg / kg (approximately 7 times the antithrombotic dose), blood loss does not increase significantly after cutting the rat tail. In contrast, administration of 140 aXa units / kg (approximately 7 times the therapeutic dose) of fragmin (low molecular weight heparin) significantly increased blood loss in concurrent experiments (Table 4 and FIG. 10). With respect to APTT (activated prothrombin time), none of the diannexin doses used increased APTT, but neither 20aXa units / kg (Table 2) nor 140aXa units / kg (Table 5 and Figure 11) Fragmin. , APTT was increased significantly. The clearance of iodine-labeled diannexin can be expressed by a two-compartment model, a 9-14 minute alpha phase and a 6-7 hour beta phase. The latter is significantly longer than previously reported for annexin IV monomers in some species. A single bolus injection is sufficient for many clinical applications of Diannexin, so a half-life of 6.5 hours is therapeutically suitable. In the unlikely event that diannexin induces bleeding, the effect will disappear fairly quickly. Both Diannexin and Fragmin significantly increase the bleeding time of rats after tail amputation (Figure 9 and Table 4). In the case of diannexin, this may be due to inhibition of phospholipase A2 action and thromboxane production. In humans, bleeding time increases when cyclooxygenase is inhibited by drugs or as a result of gene failure. Diannexin does not significantly increase blood loss, so it is clear that Diannexin does not have a major impact on the initial hemostatic mechanism despite increased bleeding time. Diannexin administration does not affect body weight.

  [00130] The present invention also relates to therapeutic compositions comprising the modified annexin proteins of the present invention. The compositions of the present invention can also include other components such as pharmaceutically acceptable excipients, adjuvants, and / or carriers. For example, the composition of the present invention can be formulated in an excipient that the animal to be treated can tolerate. Examples of such excipients include water, saline, Ringer's solution, dextrose solution, mannitol, Hank's solution, and other aqueous physiological balanced salt solutions. It is also possible to use non-aqueous vehicles such as triglycerides. Excipients can also contain minor amounts of additives such as substances that enhance isotonicity and chemical stability. Examples of buffering agents include phosphate buffer, bicarbonate buffer, Tris buffer, histidine, citrate and glycine, or mixtures thereof, while examples of preservatives include thimerosal, m- or o-cresol, formalin and benzyl alcohol are included. A standard formulation can be either a liquid injectable substance or a solid, and the solid can be incorporated into a suitable liquid as a suspension or solution for injection. Thus, in non-liquid formulations, excipients can include dextrose, human serum albumin, preservatives, etc., and can be added to sterile water or saline prior to administration. .

  [00131] One aspect of the present invention is a sustained release formulation capable of sustained release of the composition of the present invention in an animal. As used herein, a sustained release formulation includes the composition of the present invention in a sustained release vehicle. Suitable sustained release vehicles include, but are not limited to, biocompatible polymers, other polymer matrices, capsules, microcapsules, microparticles, bolus preparations, osmotic pumps, dispersion devices, liposomes, lipospheres. ), And transdermal delivery systems. Other sustained release formulations of the invention include liquids that form solids or gels in situ when administered to animals. Preferred sustained release formulations are biodegradable (ie biodegradable).

  [00132] Generally, the therapeutic agent used in the present invention is administered to an animal in an effective amount. In general, an effective amount either (1) reduces the symptoms of the disease to be treated, or (2) induces a pharmacological change corresponding to treating the disease to be treated. Is an effective amount.

  [00133] The therapeutically effective amount of the therapeutic agent can be any amount or dose sufficient to cause the desired effect, and in part, the cancer state, type, and location, the patient Depending on the size and condition of the and other factors readily known to those skilled in the art. It is also possible to administer the dosage as a single dose or as several doses, for example divided over the course of several weeks.

[00134] The present invention also relates to therapeutic methods utilizing the therapeutic compositions of the present invention. The methods of the invention include administering a therapeutic agent to a subject in need of such administration.
[00135] The therapeutic agent of the present invention can be administered by any appropriate means, for example, parenteral administration, topical administration, oral administration, or intradermal administration, such as by local administration or injection. Or by aerosol. In one embodiment of the invention, the agent is administered by injection. Such injections can be injected locally into any affected area. Depending on the mode of administration, the therapeutic composition may be administered in a variety of unit dosage forms. Suitable methods of delivery of the therapeutic composition of the present invention include intravenous and local administration, for example by injection. For certain delivery modalities, the therapeutic composition of the invention can also be formulated in an excipient. The therapeutic reagents of the invention can be administered to any animal, preferably a mammal, and more preferably a human.

  [00136] The particular mode of administration will depend on the condition to be treated. Administration of the agents of the present invention can be via any body fluid, or any target or tissue accessible through the body fluid.

  [00137] The following examples illustrate the preparation of the modified annexin proteins of the invention, as well as in vitro and in vivo assays of the anticoagulant activity of the modified annexin proteins. It is to be understood that the present invention is not limited to the exemplary studies or specific details set forth in the examples.

Example 1
Modified Annexin Preparation [00138] Annexin can be purified from human tissue or can be produced by recombinant techniques. For example, annexin V can be purified from human placenta as described in Funakoshi et al. (1987). An example of a recombinant product is the expression of annexin II and annexin V in E. coli (Kang, HM, Trends Cardiovasc. Med. 9: 92-102 (1999); Thiagarajan and Benedict, 1997, 2000). A rapid and efficient method for the purification of recombinant annexin V based on Ca ²⁺ enhanced binding to phosphatidylserine-containing liposomes and subsequent elution with EDTA is described by Berger, FEBS Lett. 329: 25-28 (1993). It is also possible to improve this method by using phosphatidylserine coupled to a solid support.

  [00139] In a process termed PEGylation, it is also possible to couple annexin to polyethylene glycol (PEG) by any of several well-established methods (reviewed in Hermanson, 1996). . The present invention includes chemically derivatized annexin molecules having mono or poly (eg 2-4) PEG moieties. Methods for preparing PEGylated annexins generally include: (a) reacting annexin with polyethylene glycol (such as a reactive ester or aldehyde derivative of PEG) under conditions where the annexin is attached to one or more PEG groups; And (b) obtaining a single or a plurality of reaction products. In general, the optimal reaction conditions for the reaction must be determined on a case-by-case basis based on known parameters and the desired result. Further, the reaction may produce different products with different numbers of PEG chains, and further purification may be necessary to obtain the desired product.

  [00140] Conjugation of PEG to Annexin V can also be performed using a method using sulfo-NHS in addition to EDC. Using EDC (1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride) and sulfo-NHS (N-hydroxysulfosuccinamide) to form an active ester group with a carboxylate group . This increases the stability of the active intermediate, which reacts with the amine to produce a stable amide linkage. Conjugation can also be performed as described in Hermanson, 1996.

  [00141] Bioconjugate methods can also be used to produce homopolymers or heteropolymers of annexins; this method is reviewed in Hermanson, 1996. Recombinant methods can also be used to produce an annexin expressed with a fusion protein, eg, the Fc portion of an immunoglobulin or another protein. Annexin II and p11 heterotetramers have also been produced in E. coli (Kang et al., 1999). All of these methods have the potential to increase the molecular weight of annexin and increase the half-life of circulating annexin and prolong its anticoagulant effect.

[00142] A DNA construct encoding the amino acid sequence shown in Figure 1C (5'-3 'sense strand) (SEQ ID NO: 4) and shown in SEQ ID NO: 6 is used to produce an annexin V homodimer It is also possible. In this example, the annexin V gene is cloned into the expression vector pCMV FLAG2 (available from Sigma-Aldrich) at the EcoRI and BglII sites. The exact sequence before and after the annexin V sequence is unknown and is denoted by “x”. It is therefore necessary to sequence the construct prior to modification to ensure that codon alignment is appropriate. The pCMV FLAG2 vector incorporates a strong promoter and initiation sequence (Kozak) and an initiation site (ATG). Therefore, the start codon in front of each annexin V gene must be removed, and a strong stop codon for robust expression must be added at the end of the second annexin V gene. The vector is also equipped with an 8 amino acid peptide sequence (asp-tyr-lys-asp-asp-asp-asp-lys) (SEQ ID NO: 9) that can be used for protein purification. A 14 amino acid spacer with a glycine-serine swivel end allows for optimal rotation between tandem gene-encoded proteins. Addition of restriction sites, PvuII and ScaI allows removal of the linker if necessary. The addition of a protease site allows cleavage of the tandem protein after expression. PreScission ^™ protease is available from Amersham Pharmacia Bittech and can be used to cleave tandem proteins. Two annexin V homodimers were generated. In the first dimer, a “His tag” was placed at the amino terminus of the dimer to facilitate purification (FIG. 1A). The 12 amino acid linker sequence was flanked by glycine and serine residues that served as swivels. FIG. 1A shows a structural diagram. The amino acid sequence of His-tagged annexin V homodimer is provided below:

  [00143] The “swivel” amino acid of the linker is shown in bold and underlined. The His-tagged annexin V homodimer was expressed at high levels in E. coli and purified using a nickel column. The DNA in the construct was shown to have the correct sequence and the dimer had the expected molecular weight (74 kDa). MALDI-TOF mass spectrometry was accomplished using a linearly operated PerSeptive Biosystems Voyager-DE Pro workstation, a positive ion mode with a static acceleration voltage of 25 kV, and a delay time of 40 nanoseconds.

  [00144] A second human annexin V homodimer was synthesized without a His tag. A structural diagram is shown in FIG. 1B. The amino acid sequence of the (non-His tagged) annexin V homodimer is provided below:

  [00145] Again, the “swivel” amino acids of the linker are shown in bold and underlined. The dimer was expressed at high levels in E. coli and purified by ion exchange chromatography followed by heparin affinity chromatography. The ion exchange column was from Bio-Rad (Econo-pak HighQ Support) and the heparin affinity column was from Amersham Biosciences (HiTrap heparin HP). Both were used according to the manufacturer's instructions. Again, the DNA sequence of the annexin V homodimer was found to be correct. The mass spectrometer showed a 73 kDa protein as expected. The amino acid sequences of annexins and other proteins are routinely determined in this laboratory by mass spectrometry of peptide fragments. The expected sequence was obtained.

  [00146] Human annexin V has the following amino acid sequence:

  [00147] The nucleotide sequence of human annexin V inserted as shown in the DNA construct illustrated in FIG. 1C is as follows:

(Example 2)
In vitro and in vivo assays [00148] The in vitro assay determines the ability of a modified annexin protein to bind to activated platelets. Annexin V binds to platelets, and this binding is markedly increased in vitro by platelet activation with thrombin (Thiagarajan and Tait, 1990; Sun et al., 1993). Preferably, the modified annexin protein of the present invention is prepared in such a manner as to perform the function of annexin in that the annexin binds to platelets and prevents protein S from binding to platelets (Sun et al., 1993). To do. The modified annexin protein also performs the function of exhibiting the same in vitro anticoagulant activity as does the unmodified annexin protein. The method of measuring clotting time is activated partial thromboplastin time (Fritsma, Hemostasis and the thrombosis in the clinical laboratory (Corribeau, DM, and in Fritsma, Supervised, G.A., incorporated herein). JP Lipincott Co., Philadelphia (1989), pp. 92-124).

[00149] The in vivo assay measures the antithrombotic activity of annexin proteins. Annexin V has been shown to reduce laser or photochemically induced venous thrombosis in rats (Roemisch et al., 1991). Maximum anticoagulant effect was observed between 15-30 minutes after intravenous administration of Annexin V as determined functionally by thromboelastography. Preferably, the modified annexin proteins of the present invention exhibit extended activity in such models over unmodified annexins. Annexin V was also found to reduce fibrin deposits in a rabbit model of cervical venous thrombosis (Van Ryn-McKenna et al., 1993). Air jetting was used to remove the endothelium and annexin V was shown to bind to the treated vein but not to the control contralateral vein. The decrease in fibrin accumulation in the injured vein was not associated with systemic clotting. Heparin did not inhibit fibrin accumulation in the injured vein. The modified annexin proteins of the present invention preferably perform the function of annexins in this model of venous thrombosis. Thiagarajan and Benedict, 1997 used a rabbit model of arterial thrombosis. By applying the current, a partially occluded thrombus was formed in the left carotid artery. Annexin V infusion strongly inhibited thrombosis as evidenced by blood flow measurements, thrombus weight, labeled fibrin deposition and labeled platelet accumulation. Recently, a mouse model of photochemically induced thrombi in the levator ani muscle has been published (Volmar et al . Thromb. Haemost. 85: 160-164 (2001), incorporated herein). This technique can be used to induce thrombosis in either the desired artery or vein. The modified annexin proteins of the present invention preferably perform the function of annexins in such a model even when administered by bolus injection.

(Example 3)
[00150] The anticoagulant potential of human recombinant annexin V and PEGylated human recombinant annexin V was compared in vitro.

[00151] Annexin V production . The polymerase chain reaction was used to amplify cDNA from the human placenta cDNA library from the start methionine to the stop codon using specific oligonucleotide primers. The forward primer was 5′-ACCTGAGTAGTCGCCATGGCACAGTTCTC-3 ′ (SEQ ID NO: 7), and the reverse primer was 5′-CCCCGAATTCACGTTTAGCATCTTCTCCACAGAGCAG-3 ′ (SEQ ID NO: 8). The amplified 1.1 kb fragment was digested with NcoI and EcoRI and ligated into the prokaryotic expression vector pTRC 99A. The ligation product was used to transform and sequence competent E. coli strain JM105.

  [00152] Recombinant annexin V was isolated from bacterial lysates as described in Berger et al., 1993 with some modifications. An overnight culture of E. coli JM105 transformed with pTRC 99A-Annexin V was grown 50-fold in fresh Luria-Bertrani medium containing 10 mg / l ampicillin. After 2 hours, isopropyl β-D-thiogalactopyranoside was added to a final concentration of 1 mmol / l. 16 hours after induction, bacteria were pelleted at 3500 g for 15 minutes at 4 ° C. The bacterial pellet was suspended in TBS, pH 7.5, containing 1 mmol / l PMSF, 5 mmol / l EDTA, and 6 mol / l urea. The bacterial suspension was sonicated for 3 minutes on ice using an ultrasonic probe set at 6. The lysate was centrifuged at 10,000 g for 15 minutes and the supernatant was dialyzed twice against 50 volumes of TBS containing 1 mmol / l EDTA and once against 50 volumes of TBS.

  [00153] Multilayer by dissolving phosphatidylserine, lyophilized bovine brain extract, cholesterol, and dicetyl phosphate in chloroform at a molar ratio of 10: 15: 1 and drying in a conical flask with a stream of nitrogen. Liposomes were prepared. TBS (5 ml) was added to the flask and stirred vigorously for 1 minute in a vortex mixer. Liposomes were washed by centrifuging at 3500 g for 15 minutes, then incubated with the bacterial extract, and calcium chloride was added to a final concentration of 5 mmol / l. After 15 minutes incubation at 37 ° C., the liposomes were sedimented by centrifugation at 10,000 g for 10 minutes and the bound annexin V was eluted with 10 mmol / l EDTA. The eluted annexin V was concentrated by Amicon ultrafiltration and loaded onto a Sephacryl S 200 column. Annexin V was recovered in the included volume, while most liposomes were in the void volume. Fractions containing Annexin V are pooled and dialyzed in 10 mmol / l Tris and 2 mmol / l EDTA, pH 8.1, loaded onto an anion exchange column, and 0-200 mmol / l in the same buffer. Elute with a linear gradient of NaCl. The purified preparation showed a single band in SDS-PAGE under reducing conditions.

[00154] Annexin V produced as described above was PEGylated using the method of Hermanson, 1996, as described above.
[00155] Anticoagulation assay . The prolongation of clotting time (activated partial thromboplastin time) induced by annexin V and PEGylated annexin V was compared. Activated partial thromboplastin time was assayed using normal pooled plasma with citrate as described in Fritsma, 1989. Dose-response curves for prolonged clotting time were obtained using different concentrations of annexin V and PEGylated annexin V produced as described above. The results are shown in FIG. 6, which is a plot of clotting time against annexin V and PEGylated annexin V dose. As shown in the figure, the anticoagulant strength of recombinant human annexin V and PEGylated recombinant human annexin V is substantially equivalent. The small differences observed are due to changes in molecular weight after PEGylation. This experiment demonstrates the assertion made here that annexin V PEGylation can be achieved without significantly reducing the antithrombotic effect.

Example 4
[00156] The affinity of recombinant annexin V (AV) and recombinant annexin V homodimer (DAV) for PS on the cell surface was compared. Human peripheral red blood cells (RBC) were treated with Ca ²⁺ ionophore (A23187) to produce cells with PS exposed on the surface. Phospholipid transferase (Flipase), which transfers PS to the inner leaflet of the plasma membrane bilayer, was inactivated by treatment with N-ethylmaleimide (NEM) covalently bound to unbound sulfhydryl groups. As intracellular Ca ²⁺ increases, the scramblease enzyme is activated, thus increasing the amount of PS in the outer leaflet of the plasma membrane bilayer.

[00157] Washed human RBCs were resuspended in 30% hematocrit in K buffer (80 mM KCl, 7 mM NaCl, 10 mM HEPES, pH 7.4). These were incubated for 30 minutes at 37 ° C. in the presence of 10 mM NEM to inhibit flipase. NEM treated cells were washed and suspended in 16% hematocrit in the same buffer supplemented with ₂ mM CaCl ₂ . Scramblease enzyme was activated by incubation with A23187 (final concentration 4 μM) at 37 ° C. for 30 minutes. This method resulted in over 95% RBCs on the surface that could be manifested by flow cytometry.

  [00158] Recombinant AV and DAV were biotinylated using the FluReporter protein labeling kit (Molecular Probes, Eugene, OR). Biotin-AV and biotin-DAV conjugates were visualized using a final concentration of 2 μg / ml R-phycoerythrin-conjugated streptavidin (PE-SA). Flow cytometry was performed on a Becton Dickinson FACScaliber and data was analyzed with Cell Quest software (Becton Dickinson, San Jose, CA).

  [00159] Neither AV or DAV binding was detectable when normal RBC was used. However, both AV and DAV bound to at least 95% of the PS-exposed RBC. After PS exposure RBCs were mixed with various amounts of AV and DAV (a) separately or (b) in a 1: 1 molar ratio, incubated, PE-SA was added, and flow cytometry was performed. went. In such a mixture, either AV or DAV was biotinylated and the amount of each protein bound was assayed as described above. The biotin label was higher in DAV than AV, and the experiment was adjusted in this regard.

  [00160] A representative result is shown in FIG. In this experimental set, PS-exposed RBCs were (a) 0.2 μg biotinylated DAV (FIG. 2A); (b) 0.2 μg biotinylated DAV (FIG. 2B); (c) 0.2 μg biotinylated AV. And 0.2 μg non-biotinylated DAV; and (d) 0.2 μg biotinylated DAV and 0.2 μg non-biotinylated AV (FIG. 2D). Comparison of FIGS. 2B and 2D shows that 0.2 μg of non-biotinylated AV had no effect on biotinylated DAV binding. However, comparing FIG. 2A and FIG. 2C shows that the amount of biotinylated AV bound to PS exposed cells was greatly reduced when 0.2 μg of non-biotinylated DAV was present. These results indicate that DAV and AV compete for the same PS binding site on the RBC, but differ in affinity; DAV may bind to PS exposed on the cell surface with higher affinity than AV. Indicated.

(Example 5)
[00161] A cell binding assay was established using known amounts of annexin V monomer (AV) and dimer (DAV) added to mouse serum. RBCs with PC exposed as described above were incubated with serum containing dilutions of AV and DAV. After washing, labeled streptavidin was added and washed again, and RBC bound AV and DAV were assayed by flow cytometry. When RBCs without exposed PS were used, binding was not detectable. AV and DAV concentrations in mouse serum assayed by cell binding were highly correlated with those determined by an independent ELISA assay. Thus, AV and DAV in mouse plasma do not bind to other plasma proteins in a manner that impairs their ability to interact with PS exposed on the cell surface. These observations confirmed that it was justified to apply a cell binding assay to compare the survival of circulating AV and DAV.

  [00162] Mice were injected intravenously with AV and DAV, and then peripheral blood samples were collected several times. Different mice were used at each time point. A representative result is shown in FIG. Rabbits (Thiagarajan and Benedict, Circulation 96: 2339, 1977), cynomolgus monkeys (Roemisch et al., Thrombosis Res. 61:93, 1991) and humans (Kemerink et al., J. Nucl. 94, Med. 44: Med. AV has a short half-life in the circulation (7-24 minutes each), indicating that the main loss is to the kidneys. Consistent with these reports, AV was virtually undetectable in peripheral blood 20 minutes after mice were injected with AV (FIG. 3B). However, when mice were injected intravenously with DAV, a significant amount of protein was detectable in the circulation even after 120 minutes (FIG. 3E). Thus, dimerization of annexin V increases circulation persistence and thus increases the duration of the therapeutic effect.

(Example 6)
[00163] compared the inhibitory effects of human sPLA ₂ (Cayman, Ann Arbor, MI) Annexin V (AV) on the activity of and annexin V homodimer (DAV). As described above, PS exposed on NBC and RBC treated with A23187 was used as substrate. In control cells, AV and DAV were found to bind to PS-exposed RBCs as can be demonstrated by flow cytometry. Incubation of PS-exposed cells with sPLA ₂ removed PS, so the cells no longer bound to annexin. Treatment of PS-exposed cells with AV or DAV prior to incubation with PLA ₂ does not remove PS. It is also possible to expose the cells to a Ca ²⁺ chelator that dissociates the AV or DAV from the PS and subsequently binds the labeled AV, indicating PS remaining on the cell surface. In such assays, when titrated the AV and DAV, both shown to be a potent inhibitor of sPLA ₂ activity against cell surface PS.

[00164] Inhibition of phospholipase can also be demonstrated by other methods. The activity of sPLA ₂ releases lysophosphatidylcholine (LPS), which is hemolytic. It is therefore possible to compare the inhibitory effects of AV and DAV on PLA ₂ in hemolysis assays. As shown in FIG. 4, both AV and DAV inhibited the action of PLA ₂ , and DAV was somewhat more effective. After 60 minutes incubation with pPLA ₂ , hemolysis was strongly reduced in the presence compared to the absence of DAV or AV. From these results, it can be concluded that the annexin V homodimer is a potent inhibitor of secreted PLA ₂ . Thus, homodimers of annexin V should reduce the formation of thromboxane A ₂ and mediators such as lysophosphatidylcholine and lysophosphatidic acid that are thought to contribute to the development of reperfusion injury (Hashizume et al. Jpn Heart J., 38:11, 1997; Okuza et al., J. Physiol., 285: F565, 2003).

(Example 7)
[00165] A mouse liver model of warm ischemia-reperfusion injury was used to determine if the modified annexin protects against reperfusion injury (RI), to compare the activities of modified annexin and annexin V, and The duration of activity of the modified annexin was determined. The model is described in Teoh et al. (Hepatology 36:94, 2002). Female C57BL6 mice weighing 18-25 g were used. Under ketamine / xylazine anesthesia, the blood supply to the left and middle lobes of the liver was occluded for 90 minutes using an atraumatic microvascular clamp. The vascular clamp was then removed and reperfusion was established. The animals were recovered and sacrificed by exsanguination after 24 hours. Liver damage was assessed by measuring serum alanine aminotransferase (ALT) activity and histological examination. The control group was subjected to anesthesia and sham laparotomy. Groups of 4 mice were used to assay the activity of annexin V and modified annexin. Each mouse in the first group has 25 micrograms of annexin V (AV), each mouse in the second group has 25 micrograms of annexin homodimer (DAV), and a third group of mice. Mice were injected intravenously with 2.5 micrograms of annexin V (PEG-AV, 57 kDa) coupled to polyethylene glycol. Controls received saline or HEPES buffer containing annexin. In the first experimental set, annexin was administered a few minutes before clamping the hepatic artery branch. In the second experimental set, annexin and HEPES were administered 6 hours before the onset of ischemia. The results of each experiment are summarized in FIG.

  [00166] Slight protection was observed in animals that received Annexin V (AV) immediately prior to ischemia. In contrast, animals dosed with annexin dimer (DAV) or PEG-AV either immediately before or 6 hours before ischemia showed dramatic protection against RI. Histological studies confirmed that these groups had little or no hepatocyte necrosis. The results show that modified annexins (DAV and PEG-AV) are significantly more protective than ischemic against ischemia-reperfusion injury in the liver. Furthermore, modified annexins (DAV and PEG-AV) retain the ability to attenuate RI for at least 6 hours.

  [00167] In sham-operated animals, circulating ALT levels were very low. In animals that received saline immediately before ischemia or HEPES administered 6 hours before ischemia, ALT levels were very high and histology confirmed that there was severe hepatocyte necrosis. It was. HEPES administered just before ischemia was found to have protective activity against RI.

(Example 8)
[00168] Thrombosis study [00169] Six groups of 8 rats each were used. The rat for this study was a male Wistar rat weighing approximately 300 grams (Charles River Nederland, Maastricht, The Netherlands). Animals were housed in macrolon cages and optionally provided with standard rodent food pellets and acidified tap water. The experiments were in accordance with the rules and regulations promoted by the Dutch animal testing law. Rats were anesthetized with FFM (fentanyl / fluanison / midazolam) and placed on a heating pad. The femoral vein was cannulated and filled with saline. The inferior vena cava was isolated and the side branch was closed by ligation or cauterization. A loose ligation was applied around the caval vein under the left renal vein. A second loose ligature was applied 1.5 cm upstream of the first, above the branch. The test (or control) compound was administered intravenously via the femoral vein cannula and then the cannula was vigorously flushed with saline.

  [00170] Test compounds or control compounds include: phosphate buffered saline 1.0 ml / kg body weight (10 minutes); phosphate buffered saline 1.0 ml / kg body weight (12 hours); dianexin 0.04 mg / kg Diannexin 0.2 mg / kg body weight; Diannexin 1.0 mg / kg body weight (10 minutes); Diannexin 1.0 mg / kg body weight (12 hours); Fragmin 20aXa U / kg body weight. After 10 minutes (or 12 hours for group 2), recombinant human thromboplastin (0.15 ml / kg) is quickly infused into the venous cannula, the cannula is flushed with saline and exactly 10 seconds Later, the downstream ligation near the renal vein was closed. After 9 minutes, a citrated venous blood sample was obtained and placed on ice.

  [00171] After 1 minute (at 10 minutes), the upstream ligature near the bifurcation was closed and the thrombus that had formed in this segment was collected. The thrombus was easily washed in saline, dampened with moisture and weighed. Citrated plasma was prepared by centrifugation at 2000 g, 4 ° C. for 15 minutes and stored at −60 ° C. for analysis. In the two groups where thrombus induction was performed 12 hours after compound injection, different i. v. The injection method was used. Rats were s. c. Anesthesia with DDF (Dmitor / Dormicum / Fentanyl) and injection via the penile vein. The rats are then given an antidote (annexate / antisedan / naloxone) s. c. Dosage and place in cage overnight.

  [00172] The femoral vein cannula was inserted and then injected intravenously into the rat. Ten minutes after intravenous injection of the compound (12 hours after injection for the two groups), diluted thromboplastin i. v. Infusion and 10 seconds later, the inferior vena cava was ligated. Nine minutes after ligation, blood was collected and citrated plasma was prepared. Ten minutes after ligation, the thrombus-formed segment was ligated, and the thrombus was collected and weighed. aPTT (seconds) was also measured. At 0.04 mg / kg, dianexin reduced thrombus weight by approximately 40%. Thrombus formation did not differ from controls 12 hours after diannexin (1 mg / kg) injection. Body weight was not different between groups in parametric ANOVA. The thrombus wet weight (Table 1) was in the range of 0-44.5 mg.

  [00173] Table 1: Effect of treatment on wet thrombus weight (mg) in a 10 minute thrombosis study

By parametric ANOVA; F = 24.48; p <0.00001
All groups <saline control (p <0.01)
By parametric ANOVA of the three diannexin groups:
F = 4600, p <0.0001
1 mg = 0.2 mg <0.04 mg; p <0.001
[00174] Treatment had a significant effect on thrombus weight. Both Fragmin (20aXa U / kg) and Diannexin (0.04, 0.2 and 1.0 mg / kg) significantly reduced thrombus weight (p <0.0001). See Example 7 and the text table. For diannexin, the effect was dose-dependent. The APTT values are shown in Table 2 and FIG.

  [00175] Table 2: Effect of treatment on APTT (sec) in a 10 minute thrombosis study

By parametric ANOVA; F = 6.66; p = 0.0005
Fragmin group> all other groups (p <0.05)
The saline and dianexin groups are not significantly different. [00176] Fragmin significantly increased APTT compared to all other groups. APTT was slightly but significantly increased only in the Fragmin group. Diannexin group was not different from saline control group.

  [00177] In a second thrombosis study in which rats were treated 12 hours prior to inducing thrombus formation, there was no significant difference between the saline infusion control group and the dianexin treatment group (Table 3). .

  [00178] Table 3: Effect of treatment on wet thrombus weight (mg) in a 12-hour thrombosis study

^* Average time to thrombus induction: 13.6 hours No significant difference by t-test [00179] Thrombus weight in the saline group was also compared to the thrombus weight in the saline control group of the 10 minute thrombosis study (25.7 ± 12.3 mg, see Table 1). APTT values were not different (not shown).

  [00180] Intravenous infusion of diannexin (0.2 mg / kg and 1.0 mg / kg) 10 minutes before thrombus induction strongly inhibited thrombus formation in the Wesslar rat venous thrombus model.

Example 9
[00181] Bleeding study. Three groups were studied. A group of 8 rats was used as described in Example 8. Rats were anesthetized with isoflurane, intubated and supplied with oxygen and placed on a heating pad. A cannula was inserted through the femoral vein and filled with saline. Test or control compounds were infused through the cannula and then the cannula was vigorously washed with saline. Test or control compounds were phosphate buffered saline 1.0 ml / kg body weight; Diannexin 5.0 mg / kg body weight; Fragmin 140aXa Ukg body weight. Ten minutes after test compound injection, the rat tail was placed in a horizontal position and then cut by scissors at a fixed distance from the tail as defined. Subsequently, bleeding from the tail was measured by gently wiping off any blood that had oozed from the tail with a filter paper. The time when bleeding stopped was measured. Both were recorded. The experiment was terminated 30 minutes after cutting the tail. Just prior to the end of the experiment, a citrated blood sample was obtained from the cannula. Citrated plasma was prepared by centrifugation at 2000 g, 4 ° C. for 15 minutes and stored at −60 ° C. for analysis. The filter paper was extracted in 20 ml of 10 mM phosphate buffer (pH = 7.8) containing 0.05% Triton X-100 (registered trademark). Blood loss was determined by measuring the hemoglobin content of the phosphate buffer (Drabkin's potassium cyanide 1 potassium ferricyanide method). Body weight (Table 3) did not differ between groups according to parametric ANOVA. Treatment with either Diannexin (5 mg / kg) or Fragmin (140 Ukg) almost doubled the bleeding time (Figure 9, Table 3), but these effects were only marginally significant (nonparametric ANOVA). KW = 5.72, p = 0.057). Blood loss (Figure 10, Table 4) was slightly increased in the dianexin group and almost doubled in the fragmin group compared to the control group.

  [00182] Table 4. Bleeding time and blood loss in the tail bleeding study

[00183] However, these differences were not significant (nonparametric ANOVA, p = 0.490). The APTT values are shown in Table 5 and FIG.
[00184] Table 5. Effect of treatment on APTT (seconds) in the tail bleeding study

[00185] Fragmin nearly doubled the APTT, but the APTT in the dianexin group was not different from the saline control group.
[00186] Blood loss and aPTT were almost double in the Fragmin group compared to the Dianexin group in the tail bleeding study. 5.0 mg / kg i. v. The diannexin induced bleeding from severed rat tails, but had less blood loss than after infusion of 140 aXa U / kg fragmin.

(Example 10)
[00187] Clearance studies. Rats were injected with radiolabeled dianexin and blood was collected after 5 min, 10 min, 15 min, 20 min, 30 min, 45 min and 60 min, and 2 h, 3 h, 4 h, 8 h, 16 h and 24 h Samples were obtained and blood radioactivity was measured to construct a blood loss curve. The disappearance of diannexin from the blood is represented by a two-compartment model, with approximately 75-80% disappearance (t / 2, approximately 10 minutes) in the alpha phase and 15-20% disappearance in the beta period ( t / 2, about 400 minutes). Clearance can also be expressed by a two-compartment model with half lives of 9-14 minutes and 6-7 hours, respectively. Two experiments were performed using three male Wistar rats (300 grams) each. Dianexin was labeled with ¹²⁵ I by the method of Macfarlane and the labeled protein was separated by free Sephadex G-50. After injecting NaI (5 mg / kg) to prevent label uptake into the thyroid, approximately 8 × 10 6 cpm (via the femoral vein catheter (rats 1 and 2) or via the penile vein (rat 3)). 50 μl of the protein solution was diluted to 0.5 ml with physiological saline). Thereafter, at the indicated time points (see table below), blood samples (150 μl) were obtained from the tail vein and 100 μl were counted. After taking the last blood sample, Nembutal i. v. Rats were sacrificed and liver, lung, heart, spleen and kidney (pieces) collected for counting.

  [00188] Sampling scheme for clearance studies:

  [00189] From the data collected during 45 minutes and 24 hours, β-phase parameters were calculated. The alpha phase parameters were then calculated from the data between 5 and 45 minutes by the subtraction method. Blood radioactivity curves were analyzed by a two compartment model using the subtraction method. The linear correlation coefficients for the α and β phases were −0.99 and −0.99 in experiment 1 and −0.95 and −0.96 in experiment 2. The clearance parameters are shown in Table 6.

  [00190] Table 6. Diannexin clearance parameter

  [00191] FIGS. 15 and 16 show clearance curves with the alpha and beta phases superimposed. Table 7 shows cpm (after tissue digestion) collected in lung, heart, liver, spleen and kidney. Note that high counts were seen in the lung 2 hours after diannexin infusion.

[00192] Table 7. Radioactivity recovered in selected tissues at 2, 8, and 24 hours after ¹²⁵ I-Diannexin infusion

(Example 11)
[00193] A study was conducted to ascertain the onset of ischemia-reperfusion injury (IRI) and the mode of diannexin action. According to the hypothesis of the development of ischemia-reperfusion injury, during ischemia, phosphatidylserine (PD) becomes accessible on the endothelial cell (EC) luminal surface of the liver microvasculature. During the reperfusion phase, leukocytes and platelets adhere to PS on the EC surface and induce the final stage of apoptosis in the EC. Diannexin binds to PS on the EC surface and reduces leukocyte and platelet adhesion to PS. By this mechanism, Diannexin prevents irreversible damage to the EC and thereby attenuates ischemia-reperfusion injury.

  [00194] This hypothesis was tested by observing the microcirculation in mouse liver in vivo using published methods (McCuskey et al., Hepatology 40: 386, 2004). As described in Example 7, after 90 minutes of ischemia, various periods of reperfusion were performed. FIGS. 12A and 12B show that during reperfusion, many leukocytes attached to EC in both the periportal and centrilobular regions (IR). Diannexin (1 mg / kg) IV reduces such adhesion in a statistically significant manner (IR + D). FIGS. 13A and 13B show that this also applies to platelet adhesion to ECs during reperfusion. As expected, EC damage (reflected by swelling) is prominent during reperfusion and is significantly reduced by dianexin (FIGS. 14A and 14B). Thus, our hypothesis of the mode of diannexin action in attenuating ischemia-reperfusion injury is confirmed. As shown in FIGS. 15A and 15B, Diannexin does not affect the phagocytic activity of Kupffer cells at either location. Thus, Diannexin has no effect on this defense mechanism against pathogenic organisms. This finding supports other evidence that Diannexin has no side effects.

[0023] FIGS. 1A-C show structural diagrams of two modified annexin embodiments. FIG. 1A shows the structure of a human annexin V homodimer with a His tag; FIG. 1B shows the structure of a human annexin V homodimer without a His tag. FIG. 1C shows a DNA construct for making an annexin V homodimer. [0024] FIGS. 2A-D show, in each case, 0.2 μg / ml biotinylated AV (FIG. 2A); 0.2 μg / ml biotinylated DAV (FIG. 2B); 0.2 μg / ml biotinylated AV. And 0.2 μg / ml non-biotinylated DAV (FIG. 2C); and 0.2 μg / ml biotinylated DAV and 0.2 μg / ml non-biotinylated AV (FIG. 2D) followed by R-phycoerythrin- Shown are the results of flow cytometric analysis of a mixture of normal (1 × 10 7 / ml) and PS exposed (1 × 10 7 / ml) RBCs incubated with conjugated streptavidin. [0025] FIGS. 3A-E illustrate AV or DAV levels in mouse circulation at various time points after injection. 3A-B show serum samples collected 5 minutes and 20 minutes after AV was injected into mice, respectively. 3C-E show serum samples collected 5 minutes, 25 minutes, and 120 minutes after the injection of annexin V homodimer (DAV) into mice, respectively. [0026] FIG. 4 shows PLA2-induced hemolysis of RBCs exposed to PS. A mixture of normal (1 × 10 7 / ml) and PS exposed (1 × 10 7 / ml) RBCs was incubated with 100 ng / ml pancreatic PLA2 (pPLA2) or secreted PLA2 (sPLA2). Hemolysis was measured as a function of time and expressed relative to 100% hemolysis induced by osmotic shock. After labeling with biotinylated DAV and R-phycoerythrin-conjugated streptavidin, the percentage of PS exposed cells was determined by flow cytometry of the cell suspension. FIG. 4A shows hemolysis induced by 100 ng / ml pPLA2 in the absence (triangle) or presence of 2 μg / ml DAV (circle) or AV (square). FIG. 4B shows hemolysis induced by 100 ng / ml pPLA2 in the presence of various amounts of DAV (circle) or AV (square). FIG. 4C shows PS-exposed cells in cell suspension after 60 minutes incubation with 100 ng / ml pPLA2 in the presence of 2 μg / ml DAV. [0027] FIG. 5 shows a sham-operated mouse (pseudo), a mouse administered with saline, a mouse administered with HEPES buffer 6 hours before clamping the hepatic artery, and 6 hours before clamping the artery. Figure 2 shows serum alanine aminotransferase (ALT) levels in mice administered PEGylated annexin (PEG annexin) or annexin dimer, and mice administered monomeric annexin (annexin). An asterisk above PEG annexin and annexin dimer indicates p <0.001. [0028] FIG. 6 is a plot of clotting time for an in vitro clotting assay comparing the clotting strength of recombinant human annexin V and PEGylated recombinant human annexin V. [0029] FIG. 7 shows thrombus weights in 5 treatment groups of the 10 minute thrombosis study (mean ± sd; n = 8). [0030] FIG. 8 shows the APTT in the five treatment groups of the thrombosis study (mean ± sd; n = 8). [0031] FIG. 9 shows the bleeding time in the 3 groups of the tail bleeding study (mean ± sd; n = 8). [0032] FIG. 10 shows blood loss in the 3 groups of the tail bleeding study (mean ± sem; n = 8). [0033] FIG. 11 shows the APTT in the 3 groups of the tail bleeding study (mean ± sd; n = 8). [0034] FIG. 12A shows leukocyte attachment to endothelial cells during ischemia-reperfusion injury with and without diannexin for periportal sinusoids. FIG. 12B shows leukocyte attachment to endothelial cells during ischemia-reperfusion injury with and without diannexin for centrilobular sinusoids. [0035] FIG. 13A shows platelet attachment to endothelial cells during ischemia-reperfusion injury with and without diannexin for periportal sinusoids. FIG. 13B shows platelet attachment to endothelial cells during ischemia-reperfusion injury with and without dianexin for centrilobular sinusoids. [0036] FIG. 14A shows endothelial cell swelling during ischemia-reperfusion injury with and without diannexin for periportal sinusoids. FIG. 14B shows endothelial cell swelling during ischemia-reperfusion injury with and without diannexin for centrilobular sinusoids. [0037] FIG. 15A shows phagocytic activity of Kupffer cells during ischemia-reperfusion injury with and without diannexin for periportal sinusoids. FIG. 15B shows the phagocytic activity of Kupffer cells during ischemia-reperfusion injury with and without diannexin for centrilobular sinusoids.

Claims (1)

A pharmaceutical composition for preventing or attenuating reperfusion injury (RI), comprising an effective amount of annexin V dimer effective to prevent or attenuate reperfusion injury, said annexin V dimer The body has the following amino acid sequence:

Said pharmaceutical composition.

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