Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
  • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
  • C07K14/4721—Lipocortins
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
  • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the present invention relates generally to methods and compositions for treating thrombosis. More particularly, it relates to modified annexin proteins and methods for their use.
• Thrombosis the formation, development, or presence of a blood clot
• thrombus in a blood vessel
• coronary thrombosis which leads to occlusion of the coronary arteries and often to myocardial infarction (heart attack).
• myocardial infarction heart attack
• the standard therapy is administration of a thrombolytic protein by infusion.
• Intravenous recombinant tissue plasminogen activator is the only treatment for acute ischemic stroke that is approved by the Food and Drug Administration. The earlier it is administered the better (Ernst et al., Stroke 31 :2552-2557 (2000), incorporated herein by reference). However, intravenous rtPA administration is associated with increased risk of intracerebral hemorrhage. Full-blown strokes are often preceded by transient ischemic attacks (TIA), and it is estimated that about 300,000 persons suffer TIA every year in the United States.
• TIA transient ischemic attacks
• Thrombosis also contributes to peripheral arterial occlusion in diabetics and other patients, and an efficacious and safe antithrombotic agent for use in such patients is needed.
• Venous thrombosis is a frequent complication of surgical procedures such as hip and knee arthroplasties. It would be desirable to prevent thrombosis without increasing hemorrhage into the field of operation. Similar considerations apply to venous thrombosis associated with pregnancy and parturition. Some persons are prone to repeated venous thrombotic events and are currently treated by antithrombotic agents such as coumarin-type drugs. The dose of such drugs must be titrated in each patient, and the margin between effective antithrombotic doses and those increasing hemorrhage is small. Having a treatment with better separation of antithrombotic activity from increased risk of bleeding is desirable.
• Primary hemostatic mechanisms include the formation of platelet microaggregates, which plug capillaries and accumulate over damaged or activated endothelial cells in small blood vessels.
• Inhibitors of platelet aggregation including agents suppressing the formation or action of thromboxane A , ligands of gp lla / Illb, and drugs acting on ADP receptors such as clopidogrel (Hallopeter. Nature 409:202-207 (2001), incorporated herein by reference), interfere with this process and therefore increase the risk of bleeding (Levine et al., 2001).
• occlusion by an arterial or venous thrombus requires the continued recruitment and incorporation of platelets into the thrombus.
• platelets To overcome detachment by shear forces in large blood vessels, platelets must be bound tightly to one another and to the fibrin network deposited around them. [0007] Evidence has accumulated that the formation of tight macroaggregates of platelets is facilitated by a cellular and a humoral amplification mechanism, which reinforce each other. In the cellular mechanism, the formation of relatively loose microaggregates of platelets, induced by moderate concentrations of agonists such as ADP, thromboxane A 2 , or collagen, is accompanied by the release from platelet ⁇ -granules of the 85-kD protein Gas6 (Angelillo-Scherrer et al., Nature Medicine 7:215-221 (2001), incorporated herein by reference).
• a prothrombinase complex is formed on the surface of activated platelets and microvesicles. This generates thrombin and fibrin.
• Thrombin is itself a potent platelet activator and inducer of the release of Gas6 (Ishimoto and Nakano, FEBS Lett. 446:197-199 (2000), incorporated herein by reference). Fully activated platelets bind tightly to the fibrin network deposited around them.
• antibodies against Gas6 inhibited platelet aggregation in vitro as well as thrombosis induced in vivo by collagen and epinephrine.
• such antibodies, or ligands competing for Gas6 binding to receptor tyrosine kinases might be used to inhibit thrombosis.
• such an inhibitor would also have additional suppressive activity on the Gas6-mediated cellular amplification mechanism.
• annexins a family of highly homologous antithrombotic proteins of which ten are expressed in several human tissues (Benz and Hofmann, Biol. Chem. 378:177-183 (1997), incorporated herein be reference).
• Annexins share the property of binding calcium and negatively charged phospholipids, both of which are required for blood coagulation. Under physiological conditions, negatively charged phospholipid is mainly supplied by phosphatidylserine (PS) in activated or damaged cell membranes. In intact cells, PS is confined to the inner leaflet of the plasma membrane bilayer and is not accessible on the surface.
• PS phosphatidylserine
• Proteins involved in the blood coagulation cascade (factors X, Xa, and Na) bind to membranes bearing PS on their surfaces, and to one another, forming a stable, tightly bound prothrombinase complex.
• annexins including II, N, and VIII, bind PS with high affinity, thereby preventing the formation of a prothrombinase complex and exerting antithrombotic activity.
• Tissue factor-dependent blood coagulation on the surface of activated or damaged endothelial cells also requires surface expression of PS, and annexin V can inhibit this process (van Heerde et al., Arterioscl. Thromb. 14:824-830 (1994), incorporated herein by reference), although annexin is less effective in this activity than in inhibition of prothrombinase generation (Rao et al., Thromb. Res. 62:517-531 (1992), incorporated herein by reference).
• annexin V The binding of annexin V to activated platelets and to damaged cells probably explains the selective retention of the protein in thrombi.
• Transient myocardial ischemia also increases annexin N binding (Dumont et al., Circulation 102:1564-1568 (2000), incorporated herein by reference).
• Annexin V imaging in humans has shown increased binding of the protein in transplanted hearts when endomyocardial biopsy has demonstrated vascular rejection (Acio et al., J. Nuclear Med. 41 (5 Suppl.):127P (2000), incorporated herein by reference). This binding is presumably due to PS exteriorized on the surface of damaged endothelial cells, as well as of apoptotic myocytes in hearts that are being rejected.
• annexin V binding is also augmented following cerebral hypoxia in humans (D'Arceuil et al, Stroke 2000: 2692-2700 (2000), incorporated herein by reference), which supports the hypothesis that administration of annexin following TIA may decrease thq likelihood of developing a full-blown stroke.
• Annexins have shown anticoagulant activity in several in vitro thrombin-dependent assays, as well as in experimental animal models of venous thrombosis (R ⁇ misch et al., Thrombosis Res. 61 :93-104 (1991); Van Ryn-McKenna et al., Thrombosis Flemostasis 69:227-230 (1993), both incorporated herein by reference) and arterial thrombosis (Thiagarajan and Benedict, 1997).
• annexin in antithrombotic doses had no demonstrable effect on traditional ex vivo clotting tests in treated rabbits (Thiagarajan and Benedict, 1997) and did not significantly prolong bleeding times of treated rats (Van Ryn-McKenna et al., 1993). In treated rabbits annexin did not increase bleeding into a surgical incision (Thiagarajan and Benedict, 1997). Thus, uniquely among all the agents so far investigated, annexins exert antithrombotic activity without increasing hemorrhage. Annexins do not inhibit platelet aggregation triggered by agonists other than thrombin (van Heerde et al., 1994), and platelet aggregation is the primary hemostatic mechanism.
• the tissue factor / Vila complex also exerts hemostatic effects, and this system is less susceptible to inhibition by annexin V than is the prothrombinase complex (Rao et al., 1992). This is one argument for confining administered annexin V to the vascular compartment as far as possible; the risk of hemorrhage is likely to be reduced. [0013] Despite such promising results for preventing thrombosis, a major problem associated with the therapeutic use of annexins is their short half-life in the circulation, estimated in experimental animals to be 5 to 15 minutes (R ⁇ misch et al., 1991 ; Stratton et al., 1995; Thiagarajan.
• annexin V also has a short half-life in the circulation of humans (Strauss et al., J. Nuclear Med. 41 (5 Su ⁇ pl.):149P (2000), incorporated herein by reference). Most of the annexin is lost into the urine, as expected of a 36 kDa protein (Thiagarajan and Benedict, 1997). There is a need, therefore, for a method of preventing annexin loss from the vascular compartment into the extravascular compartment and urine, thereby prolonging antithrombotic activity following a single injection.
• the present invention provides compounds and methods for preventing arterial or venous thrombosis.
• Recombinant human annexins are modified in such a way that its half-life in the vascular compartment is prolonged. This can be achieved in a variety of ways; three embodiments are an annexin coupled to polyethylene glycol, a homopolymer or heteropolymer of annexin, and a fusion protein of annexin with another protein (e.g., the Fc portion of immunoglobulin).
• the modified annexin binds with high affinity to phosphatidylserine on the surface of activated platelets or injured cells, thereby preventing the binding of Gas6 as well as procoagulant proteins and the formation of a prothrombinase complex. Modified annexin therefore inhibits both the cellular and humoral mechanisms by which platelet aggregation is amplified, thereby preventing thrombosis.
• the present invention provides an isolated modified annexin protein containing an annexin protein, preferably annexin V, coupled to polyethylene glycol (PEG).
• an isolated modified annexin protein contains an annexin protein coupled to at least one additional protein, such as an additional annexin protein (forming a homodimer) or the Fc portion of immunoglobulin.
• the additional protein preferably has a molecular weight of at least 30 kDa.
• pharmaceutical compositions containing an antithrombotically effective amount of any of the modified annexin proteins of the invention.
• the modified annexin is administered to a subject at risk of thrombosis in a pharmaceutical composition having an antithrombotically effective amount of any one of the modified annexin proteins of the present invention.
• the pharmaceutical composition can be administered after an arterial thrombosis such as coronary thrombosis, cerebral thrombosis, or a transient cerebral ischemic attack. It can also be administered after a surgical operation associated with venous thrombosis. Additionally, it can be administered to subjects having conditions subject to arterial or venous thrombosis, such as diabetes, pregnancy, or parturition.
• Also provided by the present invention are an isolated nucleic acid molecule encoding a homodimer of annexin, a recombinant molecule containing at least a portion of the nucleic acid molecule, and a recombinant cell containing at least a portion of the nucleic acid molecule.
• the recombinant cell is cultured under suitable conditions in a method of the invention to produce a homodimer of annexin.
• the present invention also provides a method for screening for a modified annexin protein that modulates thrombosis using a thrombosis test system.
• the test system is contacted with a test modified annexin protein, after which the thrombolytic activity is assessed and compared with the activity of the system in the absence of the test modified annexin protein.
• the activated partial thromboplastin time is measured.
• a method for identifying a modified annexin protein by contacting activated platelets with a test modified annexin protein and assessing the platelet-binding and protein S-binding activity.
• Also provided by the present invention is a method for in vivo screening for a modified annexin protein.
• a thrombosis animal model is contacted with a test modified annexin protein, after which the in vivo anticoagulation activity and increase in hemorrhage of the test modified annexin protein is assessed.
• the anticoagulation activity and time are compared with the anticoagulation activity and time of annexin, and the amount of hemorrhage is compared with hemorrhage in the animal model in the absence of the test modified annexin protein.
• the isolated modified annexin protein is administered after coronary thrombosisk, after a overt cerebral thrombosis, after, transient cerebral ischemic attack, after a surgical operation associated with venous thrombosis, wherein said subject is diabetic and said thrombosis is arterial thrombosis, or during a condition selected from the group consisting of pregnancy and parturition.
• the isolated modified annexin protein is administered in a range from 0.2 mg/kg to 1.0 mg/kg.
• the present invention also provides a method of inhibiting the attachment of leukocytes to endothelial cells comprising administering an effective amount of an isolated modified annexin protein comprising an annexin dimer to a patient in need thereof.
• the method further comprises reducing endothelial cell damage.
• the present invention also provides a method of treating a subject at risk of thrombosis comprising administering to said subject an antithrombotically effective amount of a protein having an affinity for phosphatidylserine that is at least 90% of the affinity of annexin V for phosphatidylserine, including wherein said protein is a monoclonal or polyclonal antibody.
• FIGS. 1A-C show the structural scheme of two modified annexin embodiments.
• FIG. 1A shows the structural scheme of human annexin V homodimer with a His-tag
• FIG. IB shows the structural scheme of the human annexin V homodimer without a His-tag.
• FIG. lC shows a DNA construct for making a homodimer of annexin V.
• FIGS. 2A-D show the results of flowcytometric analysis of a mixture of normal (1 x 107/ml) and PS exposing (1 x 107/ml) RBCs incubated with 0.2 ⁇ g/ml biotinylated AV (FIG.
• FIGS. 3A-E illustrate the levels of AV or DAN in mouse circulation at various times after injection.
• FIGS. 3A-B show serum samples recovered 5 minutes and 20 minutes after injection of AV into mice, respectively.
• FIGS. 3A-E show serum samples recovered 5 minutes and 20 minutes after injection of AV into mice, respectively.
• FIG. 4 shows PLA2-induced hemolysis of PS-exposing RBC.
• pPLA2 pancreatic PLA2
• sPLA2 secretory PLA2
• FIG. 4A shows hemolysis induced by 100 ng/ml pPLA2 in absence (triangles) or presence of 2 ⁇ g/ml DAV (circles) or AV (squares).
• FIG. 4B shows hemolysis induced by 100 ng/ml pPLA2 in the presence of various amounts of DAV (circles) or AV (squares).
• FIG. 4C shows PS-exposing cells in the cell suspension after 60 minutes incubation with 100 ng/ml pPLA2 in the presence of 2 ⁇ g/ml DAN.
• FIG. 5 shows serum alanine aminotransferase (ALT) levels in mice sham operated (Sham), mice given saline, mice given HEPES buffer 6 hrs. before clamping the hepatic artery, mice given pegylated annexin (PEG Anex) or annexin dimer 6 hrs. before clamping the artery, and mice given monomeric annexin (Anex).
• PEG Anex pegylated annexin
• annexin dimer 6 hrs. mice given monomeric annexin
• FIG. 6 is a plot of clotting time of an in vitro clotting assay comparing the anticoagulant potency of recombinant human annexin V and pegylated recombinant human annexin V.
• FIG. 8 shows APTT in the five treatment groups of the thrombosis study
• FIG. 9 shows bleeding time in the three groups of the tail bleeding study
• FIG. 10 shows blood loss in the three groups of the tail bleeding study
• FIG. 12A shows attachment of leukocytes to endothelial cells during ischemia-reperfusion injury with and without diannexin for periportal sinusoids.
• FIG. 12B shows attachment of leukocytes to endothelial cells during ischemia-reperfusion injury with and without diannexin for centrilobular sinusoids.
• FIG. 13 A shows attachment of platelets to endothelial cells during ischemia-reperfusion injury with and without diannexin for periportal sinusoids.
• FIG. 12A shows attachment of leukocytes to endothelial cells during ischemia-reperfusion injury with and without diannexin for periportal sinusoids.
• FIG. 13B shows attachment of platelets to endothelial cells during ischemia-reperfusion injury with and without diannexin for centrilobular sinusoids.
• FIG. 14A shows swelling of endothelial cells during ischemia-reperfusion injury with and without diannexin for periportal sinusoids.
• FIG. 14B shows swelling of endothelial cells during ischemia-reperfusion injury with and without diannexin for centrilobular sinusoids.
• FIG. 15A shows phagocytic activity of Kupffer cells during ischemia- reperfusion injury with and without diannexin for periportal sinusoids.
• FIG. 15B shows phagocytic activity of Kupffer cells during ischemia-reperfusion injury with and without diannexin for centrilobular sinusoids.
• the present invention provides compounds and methods for preventing thrombosis in mammals without increasing hemorrhage.
• the invention relies in part on the recognition that the primary mechanisms of platelet aggregation are different from the mechanisms of amplifying platelet aggregation, which are required for the formation of an arterial or venous thrombus. By inhibiting thrombus formation but not primary platelet aggregation, thrombosis can be prevented without increasing hemorrhage.
• Compounds of the invention include any product containing annexin amino acid sequences that have been modified to increase the half-life of the product in humans or other mammals.
• amino acid sequence is recited herein to refer to an amino acid sequence of a naturally-occurring protein molecule
• amino acid sequence and like terms, such as “polypeptide” or “protein,” are not meant to limit the amino acid sequence to the complete, native amino acid sequence associated with the recited proteins.
• the annexins are a family of homologous phospholipid-binding membrane proteins, of which ten represent distinct gene products expressed in mammals (Benz and Hofmann, 1997). Crystallographic analysis has revealed a common tertiary structure for all the family members so far studied, exemplified by annexin V (Huber et al., EMBO Journal 9:3867 (1990), incorporated herein by reference).
• the core domain is a concave discoid structure that can be closely apposed to phospholipid membranes. It contains four subdomains, each consisting of a 70-amino-acid annexin repeat made up of five ⁇ -helices.
• the annexins also have a more hydrophilic tail domain that varies in length and amino acid sequence among the different annexins.
• the sequences of genes encoding annexins are well known (e.g., Funakoshi et al., Biochemistry 26:8087-8092 (1987) (annexin V), incorporated herein by reference).
• Annexin proteins include proteins of the annexin family, , such as Annexin
• Annexin IV shares many of the same properties of Annexin N.
• Annexin IV binds to acidic phospholipids 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 IN belonds to the annexin family of calcium-dependent phospholipids binding proteins. Its functions are still not clearly defined.
• Annexin IV (endonexin) is a 32kDa, calcium-dependent membrane- binding protein.
• the translated amino acid sequence of Annexin IN shows the four domain structure characteristic of proteins in this class.
• Annexin IV has 45 - 59%) identity with other members of its family and shares a similar size and exon-intron organization. Isolated from human placenta, Annexin IV encodes a protein that has in vitro anticoagulant activity and inhibits phospholipase A2 activity. Annexin IV is almost exclusively expressed in epithelial cells. [0043] Annexin VIII belonds to the family of CA (2+) dependent phospholipids binding proteins (annexins) and has high identity to Annexin V (56%>).
• annexins belonds to the family of CA (2+) dependent phospholipids binding proteins (annexins) and has high identity to Annexin V (56%>).
• Hauptmann, et al Eur J Biochem. 1989 Oct 20;185(1):63-71. It was initially isolated as a 2.2 kb vascular anticoagulant-beta.
• Annexin VIII is neither an extracellular protein nor associated with the cell surface. It may not play a role in blood coagulation in vivo. Its physiological rule remains unknown. It is expressed at low levels in human placenta and shows restricted expression in lung, endothelia and skin, liver and kidney. [0044]
• annexin proteins are modified to increase their half-life in humans or other mammals.
• the annexin protein is annexin V, annexin IV or annexin VIII.
• One suitable modification of annexin is an increase in its effective size, which prevents loss from the vascular compartment into the extravascular compartment and urine, thereby prolonging antithrombotic activity following a single injection.
• a modified annexin contains a recombinant human annexin protein coupled to polyethylene glycol (PEG) in such a way that the modified annexin is capable of performing the function of annexin in a phosphatidylserine (PS)-binding assay.
• PEG polyethylene glycol
• PS phosphatidylserine
• the antithrombotic action of the intravenously administered annexin-PEG conjugate is prolonged as compared with that of the free annexin.
• the recombinant annexin protein coupled to PEG can be annexin V protein or another annexin protein.
• the annexin protein is annexin V, annexin IV or annexin VIII.
• PEG consists of repeating units of ethylene oxide that terminate in hydroxyl groups on either end of a linear or, in some cases, branched chain. The size and molecular weight of the coupled PEG chain depend upon the number of ethylene oxide units it contains, which can be selected. For the present invention, any size of PEG and number of PEG chains per annexin molecule can be used such that the half-life of the modified annexin is increased, relative to annexin, while preserving the function of binding of the modified molecule to PS.
• sufficient binding includes binding that is diminished from that of the unmodified annexin, but still competitive with the binding of Gas6 and factors of the prothrombinase complex and therefore able to prevent thrombosis.
• the optimal molecular weight of the conjugated PEG varies with the number of PEG chains. In one embodiment, two PEG molecules of molecular weight of at least about 15 kDa each are coupled to each annexin molecule. The PEG molecules can be linear or branched. The calcium-dependent binding of annexins to PS is affected not only by the size of the coupled PEG molecules, but also the sites on the protein to which PEG is bound. Optimal selection ensures that desirable properties are retained.
• PEG attachment sites are facilitated by knowledge of the three-dimensional structure of the molecule and by mutational and crystallographic analyses of the interaction of the molecule with phospholipid membranes (Campos et al., Biochemistry 37:8004-8008 (1998), incorporated herein by reference).
• PEG derivatives have been widely used in covalent attachment (referred to as pegylation) to proteins to enhance solubility, as well as to reduce immunogenicity, proteolysis, and kidney clearance.
• pegylation covalent attachment
• PEG- interferon alpha-2a administered once weekly is significantly more effective against hepatitis C virus than three weekly doses of the free interferon (Heathcote et al., N. Engl. J. Med. 343:1673-1680 (2000), incorporated herein by reference).
• Coupling to PEG has been used to prolong the half-life of recombinant proteins in vivo (Knauf et al., J. Biol. Chem. 266:2796-2804 (1988), incorporated herein by reference), as well as to prevent the enzymatic degradation of recombinant proteins and to decrease the immunogenicity sometimes observed with homologous products (references in Hermanson, Bioconjugate techniques.
• the modified annexin protein is a polymer of annexin proteins that has an increased effective size. It is believed that the increase in effective size results in prolonged half-life in the vascular compartment and prolonged antithrombotic activity.
• One such modified annexin is a dimer of annexin proteins.
• the dimer of annexin is a homodimer of annexin V, annexin IV or annexin VIII.
• the dimer of annexin is a heterodimer of annexin V and other annexin protein (e.g., annexin IV or annexin VIII), annexin IV and another annexin protein (e.g., annexin V or annexin VIII) or annexin VIII and another annexin protein (e.g., annexin V or annexin IV).
• annexin V or annexin VIII annexin V or annexin VIII
• annexin VIII and another annexin protein e.g., annexin V or annexin IV.
• Another such polymer is the heterotetramer of annexin II with pl l, a member of the SI 00 family of calcium-binding proteins. The binding of an SI 00 protein to an annexin increases the affinity of the annexin for Ca .
• the annexin homopolymer or heterotetramer can be produced by bioconjugate methods or recombinant methods, and be administered by itself or in a PEG- conjugated form.
• the modified annexins have increased affinity for
• a homodimer of human annexin V (DAV) was prepared in using well-established methods of recombinant DNA technology.
• the annexin molecules of the homodimer are joined through peptide bonds to a flexible linker (FIG. 1).
• the flexible linker contains a sequence of amino acids flanked by a glycine and a serine residue at either end to serve as swivels.
• the linker preferably comprises one or more such "swivels.”
• the linker comprises 2 swivels which may be separated by at least 2 amino acids, more particularly by at least 4 amino acids, more particularly by at least 6 amino acids, more particularly by at least 8 amino acids, more particularly by at least 10 amino acids.
• 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 fold in such a way that the convex surfaces of the monomer, which bind Ca2+ and PS, can both gain access to externalized PS.
• a modified annexin protein of the invention is an isolated modified annexin protein.
• the modified annexin protein can contain annexin II, annexin IV, annexin V, or annexin VIII.
• the protein is modified human annexin.
• the modified annexin contains recombinant human annexin.
• an isolated or biologically pure protein is a protein that has been removed from its natural environment.
• an isolated modified annexin protein of the present invention can be obtained from its natural source, can be produced using recombinant DNA technology, or can be produced by chemical synthesis.
• an isolated modified annexin protein can be a full-length modified protein or any homologue of such a protein. It can also be (e.g., for a pegylated protein) a modified full-length protein or a modified homologue of such a protein.
• the minimal size of a protein homologue of the present invention is a size sufficient to be encoded by a nucleic acid molecule capable of forming a stable hybrid with the complementary sequence of a nucleic acid molecule encoding the corresponding natural protein.
• the size of the nucleic acid molecule encoding such a protein homologue is dependent on nucleic acid composition and percent homology between the nucleic acid molecule and complementary sequence as well as upon hybridization conditions per se (e.g., temperature, salt concentration, and formamide concentration).
• the minimal size of such nucleic acid molecules is typically at least about 12 to about 15 nucleotides in length if the nucleic acid molecules are GC-rich and at least about 15 to about 17 bases in length if they are AT-rich.
• the minimal size of a nucleic acid molecule used to encode a protein homologue of the present invention is from about 12 to about 18 nucleotides in length.
• the maximal size of such a nucleic acid molecule in that the nucleic acid molecule can include a portion of a gene, an entire gene, or multiple genes or portions thereof.
• an annexin protein homologue or a modified annexin protein homologue of the present invention is from about 4 to about 6 amino acids in length, with sizes depending on whether a full- length, multivalent (i.e., fusion protein having more than one domain, each of which has a function) protein, or functional portions of such proteins are desired.
• Annexin and modified annexin homologues of the present invention preferably have activity corresponding to the natural subunit, such as being able to perform the activity of the annexin protein in preventing thrombus formation.
• Annexin protein and modified annexin homologues can be the result of natural allelic variation or natural mutation.
• the protein homologues of the present invention can also be produced using techniques known in the art, including, but not limited to, direct modifications to the protein or modifications to the gene encoding the protein using, for example, classic or recombinant DNA techniques to effect random or targeted mutagenesis.
• modified annexin protein containing an amino acid sequence that is 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%) identical to amino acid sequence SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:12, SEQ ID NO:15, or a protein encoded by an allelic variant of a nucleic acid molecule encoding a protein containing any of these sequences.
• 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; for example, a protein comprising SEQ ID NO:3 and SEQ ID NO: 12 and separated by a linker.
• Methods to determine percent identities between amino acid sequences and between nucleic acid sequences are known to those skilled in the art. Methods to determine percent identities between sequences include computer programs such as the GCG* Wisconsin packageTM (available from Accelrys Corporation), the DNAsis 1 M program (available from Hitachi Software, San Bruno, CA), the Vector NTI Suite (available from Informax, Inc., North Bethesda, MD), or the BLAST software available on the NCBI website.
• a modified annexin protein includes an amino acid sequence of 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 is shorter.
• annexin proteins contain full-length proteins, i.e., proteins encoded by full- length coding regions, or post-translationally modified proteins thereof, such as mature proteins from which initiating methionine and/or signal sequences or "pro" sequences have been removed.
• a fragment of a modified annexin protein of the present invention preferably contains at least about 5 amino acids, 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 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 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 in length.
• an isolated modified annexin protein of the present invention contains a protein encoded by a nucleic acid molecule having the nucleic acid sequence SEQ ID NO:4.
• the modified annexin protein contains a protein encoded by a nucleic acid molecule having the nucleic acid sequence SEQ ID NO:l or by an allelic variant of a nucleic acid molecule having this sequence.
• the modified annexin protein contains more than one protein sequence encoded by a nucleic acid molecule having the nucleic acid sequence SEQ ID NO:l or by an allelic variant of a nucleic acid molecule having this sequence.
• an isolated modified annexin protein of the present 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.
• 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 (e.g., SEQ ID NO: 12-linker-SEQ ID NO: 12).
• an isolated modified annexin protein of the present invention is a modified protein encoded by a nucleic acid molecule having the nucleic acid sequence SEQ ID NO: 13 or by an allelic variant of a nucleic acid molecule having this sequence.
• the modified annexin protein contains more than one protein sequence encoded by a nucleic acid molecule having the nucleic acid sequence SEQ ID NO: 13 or by an allelic variant of a nucleic acid molecule having this sequence (e.g., SEQ ID NO: 15-linker-SEQ ID NO: 15).
• an isolated modified annexin protein of the present invention is a modified protein which contains a protein encoded by a nucleic acid molecule having the nucleic acid sequence SEQ ID NO:l and a protein encoded by a nucleic acid molecule having the nucleic acid sequence SEQ ID NO: 10, or by allelic variants of these nucleic acid molecules (e.g., SEQ ID NO: 3-linker— SEQ ID NO:12 or SEQ ID NO: 12-linker-SEQ ID NO:3).
• an isolated modified annexin protein of the present invention is a modified protein which contains a protein encoded by a nucleic acid molecule having the nucleic acid sequence SEQ ID NO:l and a protein encoded by a nucleic acid molecule having the nucleic acid sequence SEQ ID NO: 13, or by allelic variants of these nucleic acid molecules (e.g., SEQ ID NO:3-linker-SEQ ID NO: 15 or SEQ ID NO:15-linker-SEQ ID NO:3).
• an isolated modified annexin protein of the present invention is a modified protein which contains a protein encoded by a nucleic acid molecule having the nucleic acid sequence SEQ ID NO: 10 and a protein encoded by a nucleic acid molecule having the nucleic acid sequence SEQ ID NO: 13, or by allelic variants of these nucleic acid molecules (e.g., SEQ ID NO: 12-linker-SEQ ID NO: 15 or SEQ ID NO:15-linker-SEQ ID NO: 12).
• One embodiment of the present invention includes a non-native modified annexin protein encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions with an annexin gene.
• stringent hybridization conditions refer to standard hybridization conditions under which nucleic acid molecules, including oligonucleotides, are used 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 permit isolation of nucleic acid molecules having at least about 70% nucleic acid sequence identity with the nucleic acid molecule being used to probe in the hybridization reaction. Formulae to calculate the appropriate hybridization and wash conditions to achieve hybridization permitting 30% or less mismatch of nucleotides are disclosed, for example, in Meinkoth et al., Anal. Biochem.
• hybridization conditions will permit isolation of nucleic acid molecules having at least about 80% nucleic acid sequence identity with the nucleic acid molecule being used to probe. In other embodiments, hybridization conditions will permit isolation of nucleic acid molecules having at least about 90%> nucleic acid sequence identity with the nucleic acid molecule being used to probe. In still other embodiments, hybridization conditions will permit isolation of nucleic acid molecules having at least about 95%) nucleic acid sequence identity with the nucleic acid molecule being used to probe.
• a modified annexin protein includes a protein encoded by a nucleic acid molecule that is at least about 50 nucleotides and that hybridizes under conditions that preferably allow about 20%) base pair mismatch, more preferably under conditions that allow about 15%) base pair mismatch, more preferably under conditions that allow about 10% base pair mismatch, more preferably under conditions that allow about 5% base pair mismatch, and even more preferably under conditions that allow about 2% base pair mismatch with a nucleic acid molecule selected from the group consisting of SEQ ID NOT, SEQ ID NO:4, SEQ ID NO: 10, SEQ ID NO: 13, or a complement of any of these nucleic acid molecules.
• an annexin gene includes all nucleic acid sequences related to a natural annexin gene such as regulatory regions that control production of the annexin protein encoded by that gene (such as, but not limited to, transcription, translation or post- translation control regions) as well as the coding region itself.
• an annexin gene includes the nucleic acid sequence SEQ ID NOT .
• an annexin gene includes the nucleic acid sequence SEQ ID NO: 10.
• an annexin gene includes the nucleic acid sequence SEQ ID NO: 13.
• an annexin gene can be an allelic variant that includes a similar but not identical sequence to SEQ ID NO:l .
• an annexin gene can be an allelic variant that includes a similar but not identical sequence to SEQ ID NO: 10.
• an annexin gene can be an allelic variant that includes a similar but not identical sequence to SEQ ID NO: 13.
• allelic variant of an annexin gene including SEQ ID NO:l is a gene that occurs at essentially the same locus (or loci) in the genome as the gene including SEQ ID NOT, but which, due to natural variations caused by, for example, mutation or recombination, has a similar but not identical sequence.
• Allelic variants typically encode proteins having similar activity to that of the protein encoded by the gene to which they are being compared.
• Allelic variants can also comprise alterations in the 5' or 3' untranslated regions of the gene (e.g., in regulatory control regions). Allelic variants are well known to those skilled in the art and would be expected to be found within a given human since the genome is diploid and/or among a population comprising two or more humans.
• An isolated modified annexin protein of the present invention can be obtained from its 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 contain a full-length protein or any homologue of such a protein.
• annexin and modified annexin homologues include annexin and modified annexin proteins in which amino acids have been deleted (e.g., a truncated version of the protein, such as a peptide or by a protein splicing reaction when an intron has been removed or two exons are joined), inserted, inverted, substituted and/or derivatized (e.g., by glycosylation, phosphorylation, acetylation, methylation, myristylation, prenylation, palmitoylation, amidation and/or addition of glycerophosphatidyl inositol) such that the homologue includes at least one epitope capable of eliciting an immune response against an annexin protein.
• amino acids e.g., a truncated version of the protein, such as a peptide or by a protein splicing reaction when an intron has been removed or two exons are joined
• derivatized
• Annexin and modified annexin homologues can also be selected by their ability to selectively bind to immune serum. Methods to measure such activities are disclosed herein.
• Annexin and modified annexin homologues also include those proteins that are capable of performing the function of native annexin in a functional assay; that is, are capable of binding to phosphatidylserine or to activated platelets or exhibiting antithrombotic activity. Methods for such assays are described in the Examples section and elsewhere herein.
• a modified annexin protein of the present invention may be identified by its ability to perform the function of an annexin protein in a functional assay.
• the phrase "capable of performing the function of that in a functional assay” means that the protein or modified protein has at least about 10%> of the activity of the natural protein in the functional assay. In other embodiments, it has at least about 20%) of the activity of the natural protein in the functional assay. In other embodiments, it has at least about 30%) of the activity of the natural protein in the functional assay. In other embodiments, it has at least about 40% of the activity of the natural protein in the functional assay. In other embodiments, it has at least about 50%) of the activity of the natural protein in the functional assay.
• the protein or modified protein has at least about 60%) of the activity of the natural protein in the functional assay. In still other embodiments, the protein or modified protein has at least about 70%> of the activity of the natural protein in the functional assay. In yet other embodiments, the protein or modified protein has at least about 80%> of the activity of the natural protein in the functional assay. In other embodiments, the protein or modified protein has at least about 90%) of the activity of the natural protein in the functional assay. Examples of functional assays are described herein. ' [0069] An isolated protein of the present invention can be produced in a variety of ways, including recovering such a protein from a bacterium and producing such a protein recombinantly.
• One embodiment of the present invention is a method to produce an isolated modified annexin protein of the present invention using recombinant DNA technology. Such a method includes the steps of (a) culturing a recombinant cell containing a nucleic acid molecule encoding a modified annexin protein of the present invention to produce the protein and (b) recovering the protein therefrom. Details on producing recombinant cells and culturing thereof are presented below. The phrase "recovering the protein” refers simply to collecting the whole fermentation medium containing the protein and need not imply additional steps of separation or purification. Proteins of the present invention can be purified using a variety of standard protein purification techniques. [0070] Isolated proteins of the present invention are preferably retrieved in
• Another embodiment of the present invention is an isolated nucleic acid molecule capable of hybridizing under stringent conditions with a gene encoding a modified annexin protein such as a homodimer of annexin V, a homodimer of annexin IV, a homodimer of annexin VIII, a heterodimer of annexin V and annexin VIII, a heterodimer of annexin V and annexin IV or a heterodimer of annexin IV and annexin VIII.
• a modified annexin protein such as a homodimer of annexin V, a homodimer of annexin IV, a homodimer of annexin VIII, a heterodimer of annexin V and annexin VIII, a heterodimer of annexin V and annexin IV or a heterodimer of annexin IV and annexin VIII.
• nucleic acid molecule is also referred to herein as a modified annexin nucleic acid molecule. Included is an isolated nucleic acid molecule that hybridizes under stringent conditions with a modified annexin gene. The characteristics of such genes are disclosed herein.
• an isolated nucleic acid molecule is a nucleic acid molecule that has been removed from its natural milieu (i.e., that has been subject to human manipulation). As such, “isolated” does not reflect the extent 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.
• a modified annexin gene includes all nucleic acid sequences related to a natural annexin gene, such as regulatory regions that control production of an annexin protein encoded by that gene (such as, but not limited to, transcriptional, translational, or post-translational control regions) as well as the coding region itself.
• a nucleic acid molecule of the present invention can be an isolated modified annexin nucleic acid molecule or a homologue thereof.
• a nucleic acid molecule of the present invention can include one or more regulatory regions, full-length or partial coding regions, or combinations thereof.
• the minimal size of a modified annexin nucleic acid molecule of the present invention is the minimal size capable of forming a stable hybrid under stringent hybridization conditions with a corresponding natural gene.
• Annexin nucleic acid molecules can also include a nucleic acid molecule encoding a hybrid protein, a fusion protein, a multivalent protein or a truncation fragment.
• An isolated nucleic acid molecule of the present invention can be obtained from its natural source either as an entire (i.e., complete) gene or a portion thereof capable of forming a stable hybrid with that gene.
• the phrase "at least a portion of an entity refers to an amount of the entity that is at least sufficient to have the functional aspects of that entity.
• nucleic acid sequence is an amount of a nucleic acid sequence capable of forming a stable hybrid with the corresponding gene under stringent hybridization conditions.
• An isolated nucleic acid molecule of the present invention can also be produced using recombinant DNA technology (e.g., polymerase chain reaction (PCR) amplification, cloning, etc.) or chemical synthesis.
• PCR polymerase chain reaction
• Isolated modified annexin nucleic acid molecules include natural nucleic acid molecules and homologues thereof, including, but not limited to, natural allelic variants and modified nucleic acid molecules in which nucleotides have been inserted, deleted, substituted, and/or inverted in such a manner that such modifications do not substantially interfere with the ability of the nucleic acid molecule to encode an annexin protein of the present invention or to form stable hybrids under stringent conditions with natural nucleic acid molecule isolates.
• a modified annexin nucleic acid molecule homologue can be produced using a number of methods known to those skilled in the art (see, e.g., Sambrook et al., 1989).
• nucleic acid molecules can be modified using a variety of techniques including, but not limited to, classic mutagenesis techniques and recombinant DNA techniques, such as site-directed mutagenesis, chemical treatment of a nucleic acid molecule to induce mutations, restriction enzyme cleavage of a nucleic acid fragment, ligation of nucleic acid fragments, polymerase chain reaction (PCR) amplification and/or mutagenesis of selected regions of a nucleic acid sequence, synthesis of oligonucleotide mixtures, and ligation of mixture groups to "build" a mixture of nucleic acid molecules and combinations thereof.
• classic mutagenesis techniques and recombinant DNA techniques such as site-directed mutagenesis
• chemical treatment of a nucleic acid molecule to induce mutations
• restriction enzyme cleavage of a nucleic acid fragment ligation of nucleic acid fragments
• PCR polymerase chain reaction
• Nucleic acid molecule homologues can be selected from a mixture of modified nucleic acids by screening for the function of the protein encoded by the nucleic acid (e.g., the ability of a homologue to elicit an immune response against an annexin protein and/or to function in a clotting assay, or other functional assay), and/or by hybridization with isolated annexin-encoding nucleic acids under stringent conditions.
• An isolated modified annexin nucleic acid molecule of the present invention can include a nucleic acid sequence that encodes at least one modified annexin protein of the present invention, examples of such proteins being disclosed herein.
• nucleic acid molecule primarily refers to the physical nucleic acid molecule and the phrase “nucleic acid sequence” primarily refers to the sequence of nucleotides on the nucleic acid molecule, the two phrases can be used interchangeably, especially with respect to a nucleic acid molecule, or a nucleic acid sequence, being capable of encoding a modified annexin protein.
• One embodiment of the present invention is a modified annexin nucleic acid molecule that is capable of hybridizing under stringent conditions to a nucleic acid strand that encodes at least a portion of a modified annexin protein or a homologue thereof or to the complement of such a nucleic acid strand.
• a nucleic acid sequence complement of any nucleic acid sequence of the present invention refers to the nucleic acid sequence of the nucleic acid strand that is complementary to (i.e., can form a complete double helix with) the strand for which the sequence is cited. It is to be noted that a double-stranded nucleic acid molecule of the present invention for which a nucleic acid sequence has been determined for one strand, that is represented by a SEQ ID NO, also comprises a complementary strand having a sequence that is a complement of that SEQ ID NO.
• nucleic acid molecules of the present invention which can be either double-stranded or single-stranded, include those nucleic acid molecules that form stable hybrids under stringent hybridization conditions with either a given SEQ ID NO denoted herein and/or with the complement of that SEQ ID NO, which may or may not be denoted herein.
• Methods to deduce a complementary sequence are known to those skilled in the art.
• modified annexin nucleic acid molecule that includes a nucleic acid sequence having 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 homology with the corresponding region(s) of the nucleic acid sequence encoding at least a portion of a modified annexin protein.
• a modified annexin nucleic acid molecule capable of encoding a homodimer of an annexin protein or homologue thereof.
• Annexin nucleic acid molecules include SEQ ID NO:4 and an allelic variants of SEQ ID NO:4, SEQ ID NOT and an allelic variants of SEQ ID NOT, SEQ ID NO: 10 and an allelic variants of SEQ ID NO: 10; and SEQ ID NO: 13 and an allelic variants of SEQ ID NO: 13.
• nucleic acid molecule of a modified annexin protein of the present invention allows one skilled in the art to make copies of that nucleic acid molecule as well as to obtain a nucleic acid molecule including additional portions of annexin protein-encoding genes (e.g., nucleic acid molecules that include the translation start site and/or transcription and/or translation control regions), and/or annexin nucleic acid molecule homologues. Knowing a portion of an amino acid sequence of an annexin protein of the present invention allows one skilled in the art to clone nucleic acid sequences encoding such an annexin protein.
• a desired modified annexin nucleic acid molecule can be obtained in a variety of ways including screening appropriate expression libraries with antibodies that bind to annexin proteins of the present invention; traditional cloning techniques using oligonucleotide probes of the present invention to screen appropriate libraries or DNA; and PCR amplification of appropriate libraries, or RNA or DNA using oligonucleotide primers of the present invention (genomic and/or cDNA libraries can be used).
• the present invention also includes nucleic acid molecules that are oligonucleotides capable of hybridizing, under stringent conditions, with complementary regions of other, preferably longer, nucleic acid molecules of the present invention that encode at least a portion of a modified annexin protein.
• Oligonucleotides of the present invention can be RNA, DNA, or derivatives of either.
• the minimal size of such oligonucleotides is the size required to form a stable hybrid between a given oligonucleotide and the complementary sequence on another nucleic acid molecule of the present invention. Minimal size characteristics are disclosed herein.
• the size of the oligonucleotide must also be sufficient for the use of the oligonucleotide in accordance with the present invention.
• Oligonucleotides of the present invention can be used in a variety of applications including, but not limited to, as probes to identify additional nucleic acid molecules, as primers to amplify or extend nucleic acid molecules or in therapeutic applications to modulate modified annexin production.
• the present invention includes such oligonucleotides and methods to modulate the production of modified annexin proteins by use 'of one or more of such technologies.
• Natural, Wild-Type Bacterial Cells and Recombinant Molecules and Cells [0081]
• the present invention also includes a recombinant vector, which includes a modified annexin nucleic acid molecule of the present invention inserted into any vector capable of delivering the nucleic acid molecule into a host cell.
• Such a vector contains heterologous nucleic acid sequences, that is, nucleic acid sequences that are not naturally found adjacent to modified annexin nucleic acid molecules of the present invention.
• the vector can be either RNA or DNA, either prokaryotic or eukaryotic, and typically is a virus or a plasmid.
• Recombinant vectors can be used in the cloning, sequencing, and/or otherwise manipulating of modified annexin nucleic acid molecules of the present invention.
• One type of recombinant vector herein referred to as a recombinant molecule and described in more detail below, can be used in the expression of nucleic acid molecules of the present invention.
• Some recombinant vectors are capable of replicating in the transformed cell.
• Nucleic acid molecules to include in recombinant vectors of the present invention are disclosed herein.
• one embodiment of the present invention is a method to produce a modified annexin protein of the present invention by culturing a cell capable of expressing the protein under conditions effective to produce the protein, and recovering the protein.
• the method includes producing an annexin protein by culturing a cell capable of expressing the protein under conditions effective to produce the annexin protein, recovering the protein, and modifying the protein by coupling it to an agent that increases its effective size.
• the cell to culture is a natural bacterial cell, and modified annexin is isolated from these cells.
• a cell to culture is a recombinant cell that is capable of expressing the modified annexin protein, the recombinant cell being produced by transforming a host cell with one or more nucleic acid molecules of the present invention. Transformation of a nucleic acid molecule into a cell can be accomplished by any method by which a nucleic acid molecule can be inserted into the cell. Transformation techniques include, but are not limited to, transfection, electroporation, microinjection, lipofection, adsorption, and protoplast fusion. A recombinant cell may remain unicellular or may grow into a tissue, organ or a multicellular organism.
• Transformed nucleic acid molecules of the present invention can remain extrachromosomal or can integrate into one or more sites within a chromosome of the transformed (i.e., recombinant) cell in such a manner that their ability to be expressed is retained.
• Nucleic acid molecules with which to transform a host cell are disclosed herein.
• Suitable host cells to transform include any cell that can be transformed and that can express the introduced modified annexin protein. Such cells are, therefore, capable of producing modified annexin proteins of the present invention after being transformed with at least one nucleic acid molecule of the present invention.
• Host cells can be either untransformed cells or cells that are already transformed with at least one nucleic acid molecule.
• Suitable host cells of the present invention can include bacterial, fungal (including yeast), insect, animal, and plant cells.
• Host cells include bacterial cells, with E. coli cells being particularly preferred.
• Alternative host cells are untransformed (wild-type) bacterial cells producing cognate modified annexin proteins, including attenuated strains with reduced pathogenicity, as appropriate.
• a recombinant cell is preferably produced by transforming a host cell with one or more recombinant molecules, each comprising one or more nucleic acid molecules of the present invention operatively linked to an expression vector containing one or more transcription control sequences.
• an expression vector is a DNA or RNA vector that is capable of transforming a host cell and of effecting- expression of a specified nucleic acid molecule.
• the expression vector is also capable of replicating within the host cell.
• Expression vectors can be either prokaryotic or eukaryotic, and are typically viruses or plasmids.
• Expression vectors of the present invention include any vectors that function (i.e., direct gene expression) in recombinant cells of the present invention, including in bacterial, fungal, insect, animal, and/or plant cells.
• nucleic acid molecules of the present invention can be operatively linked to expression vectors containing regulatory sequences such as promoters, operators, repressors, enhancers, termination sequences, origins of replication, and other regulatory sequences that are compatible with the recombinant cell and that control the expression of nucleic acid molecules of the present invention.
• a transcription control sequence includes a sequence that is capable of controlling the initiation, elongation, and termination of transcription. Particularly important transcription control sequences are those that control transcription initiation, such as promoter, enhancer, operator and repressor sequences. Suitable transcription control sequences include any transcription control sequence that can function in at least one of the recombinant cells of the present invention.
• Transcription control sequences include those which 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 ( ⁇ ) (such as ⁇ p L and ⁇ p and fusions that include such promoters), bacteriophage T7, T71ac, bacteriophage T3, bacteriophage SP6, bacteriophage SP01, metallothionein, alpha mating factor, Pichia alcohol oxidase, alphavirus subgenomic promoters (such as Sindbis virus subgenomic promoters), baculovirus, Heliothis zea insect virus, vaccinia virus, herpesvirus, poxvirus, adenovirus, simian virus 40, retrovirus actin, retroviral long terminal repeat, Rous sarcoma
• ⁇ bacteriophage lambda
• transcription control sequences include tissue-specific promoters and enhancers as well as lymphokine- inducible promoters (e.g., promoters inducible by interferons or interleukins).
• Transcription control sequences of the present invention can also include naturally occurring transcription control sequences naturally associated with a DNA sequence encoding an annexin protein.
• One transcription control sequence is the Kozak strong promoter and initiation sequence.
• Expression vectors of the present invention may also contain secretory signals (i.e., signal segment nucleic acid sequences) to enable an expressed annexin protein to be secreted from the cell that produces the protein.
• Suitable signal segments include an annexin protein signal segment or any heterologous signal segment capable of directing the secretion of an annexin protein, including fusion proteins, of the present invention.
• Signal segments include, but are not limited to, tissue plasminogen activator (t- PA), interferon, interleukin, growth hormone, histocompatibility and viral envelope glycoprotein signal segments.
• Expression vectors of the present invention may also contain fusion sequences which lead to the expression of inserted nucleic acid molecules of the present invention as fusion proteins. Inclusion of a fusion sequence as part of a modified annexin nucleic acid molecule of the present invention can enhance the stability during production, storage and/or use of the protein encoded by the nucleic acid molecule.
• a fusion segment can function as a tool to simplify purification of a modified annexin protein, such as to enable purification of the resultant fusion protein 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).
• SEQ ID NO:9 8- amino acid peptide sequence asp-tyr-lys-asp-asp-asp-lys
• a suitable fusion segment can be a domain of any size that has the desired function (e.g., increased stability and/or purification tool). It is within the scope of the present invention to use one or more fusion segments. Fusion segments can be joined to amino and/or carboxyl termini of an annexin protein.
• fusion protein is a fusion protein wherein the fusion segment connects two or more annexin proteins or modified annexin proteins.
• Linkages between fusion segments and annexin proteins can be constructed to be susceptible to cleavage to enable straightforward recovery of the annexin or modified annexin proteins.
• Fusion proteins are preferably produced by culturing a recombinant cell transformed with a fusion nucleic acid sequence that encodes a protein including the fusion segment attached to either the carboxyl and/or amino terminal end of an annexin protein.
• a recombinant molecule of the present invention is a molecule that can include at least one of any nucleic acid molecule heretofore described operatively linked to at least one of any transcription control sequence capable of effectively regulating expression of the nucleic acid molecules in the cell to be transformed.
• a recombinant molecule includes one or more nucleic acid molecules of the present invention, including those that encode one or more modified annexin proteins. Recombinant molecules of the present invention and their production are described in the Examples section.
• a recombinant cell includes one or more nucleic acid molecules of the present invention, with those that encode one or more annexin proteins. Recombinant cells of the present invention include those disclosed in the Examples section. [0090] It may be appreciated by one skilled in the art that use of recombinant
• DNA technologies can improve expression of transformed nucleic acid molecules by manipulating, for example, the number of copies of the nucleic acid molecules within a host cell, the efficiency with which those nucleic acid molecules are transcribed, the efficiency with which the resultant transcripts are translated, and the efficiency of posttranslational modifications.
• Recombinant techniques useful for increasing the expression of nucleic acid molecules of the present invention include, but are not limited to, operatively linking nucleic acid molecules to high-copy number plasmids, integration of the nucleic acid molecules into one or more host cell chromosomes, addition of vector stability sequences to plasmids, substitutions or modifications of transcription control signals (e.g., promoters, operators, enhancers), substitutions or modifications of translational control signals (e.g., ribosome binding sites, Shine-Dalgarno sequences), modification of nucleic acid molecules of the present invention to correspond to the codon usage of the host cell, deletion of sequences that destabilize transcripts, and use of control signals that temporally separate recombinant cell growth from recombinant protein production during fermentation.
• transcription control signals e.g., promoters, operators, enhancers
• substitutions or modifications of translational control signals e.g., ribosome binding sites, Shine-Dalgarno sequences
• recombinant cells can be used to produce annexin or modified annexin proteins of the present invention by culturing such cells under conditions effective to produce such a protein, and recovering the protein.
• Effective conditions to produce a protein include, but are not limited to, appropriate media, bioreactor, temperature, pH and oxygen conditions that permit protein production.
• An appropriate, or effective, medium refers to any medium in which a cell of the present invention, when cultured, is capable of producing an annexin or modified annexin protein.
• Such a medium is typically an aqueous medium comprising assimilable carbohydrate, nitrogen and phosphate sources, as well as appropriate salts, minerals, metals and other nutrients, such as vitamins.
• the medium may comprise complex, nutrients or may be a defined minimal medium.
• Cells of the present invention can be cultured in conventional fermentation bioreactors, which include, but are not limited to, batch, fed-batch, cell recycle, and continuous fermentors. Culturing can also be conducted in shake flasks, test tubes, microtiter dishes, and petri plates. Culturing is carried out at a temperature, pH and oxygen content appropriate for the recombinant cell. Such culturing conditions are well within the expertise of one of ordinary skill in the art.
• resultant annexin proteins may either remain within the recombinant cell; be secreted into the fermentation medium; be secreted into a space between two cellular membranes, such as the periplasmic space in E. coli; or be retained on the outer surface of a cell or viral membrane. Methods to purify such proteins are disclosed in the Examples section.
• Antibodies [0094] The present invention also includes isolated anti-modified annexin antibodies and their use.
• An anti-modified annexin antibody is an antibody capable of selectively binding to a modified annexin protein.
• Isolated antibodies are antibodies that have been removed from their natural milieu. The term "isolated" does not refer to the state of purity of such antibodies.
• isolated antibodies can include anti-sera containing such antibodies, or antibodies that have been purified to varying degrees.
• selective binds to refers to the ability of such antibodies to preferentially bind to the protein against which the antibody was raised (i.e., to be able to distinguish that protein from unrelated components in a mixture). Binding affinities, commonly expressed as equilibrium association constants, typically range from about 10 3 M "1 to about 10 12 M "1 .
• Binding can be measured using a variety of methods known to those skilled in the art including immunoblot assays, immunoprecipitation assays, radioimmunoassays, enzyme immunoassays (e.g., ELISA), immunofluorescent antibody assays and immunoelectron microscopy; see, e.g., Sambrook et al, 1989.
• Antibodies of the present invention can be either polyclonal or monoclonal antibodies.
• Antibodies of the present invention include functional equivalents such as antibody fragments and genetically-engineered antibodies, including single chain antibodies, that are capable of selectively binding to at least one of the epitopes of the protein used to obtain the antibodies.
• Antibodies of the present invention also include chimeric antibodies that can bind to more than one epitope. Antibodies are raised in response to proteins that are encoded, at least in part, by a modified annexin nucleic acid molecule of the present invention.
• Anti-modified annexin antibodies of the present invention include antibodies raised in an animal administered a modified annexin.
• Anti-modified annexin antibodies of the present invention also include antibodies raised in an animal against one or more modified annexin proteins of the present invention that are then recovered from the cell using techniques known to those skilled in the art. Yet additional antibodies of the present invention are produced recombinantly using techniques as heretofore disclosed for modified annexin proteins of the present invention.
• Antibodies produced against defined proteins can be advantageous because such antibodies are not substantially contaminated with antibodies against other substances that might otherwise cause interference in a diagnostic assay or side effects if used in a therapeutic composition.
• 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 tools to screen expression libraries and/or to recover desired proteins of the present invention from a mixture of proteins and other contaminants.
• An anti-modified annexin antibody of the present invention can selectively bind to a modified annexin protein.
• any of the above-described modified annexin proteins is used in methods of the invention to treat arterial or venous thrombosis caused by any medical procedure or condition.
• the therapeutic agents used in the invention are administered to an animal in an effective amount.
• an effective amount is an amount effective either (1) to reduce the symptoms of the disease sought to be treated or (2) to induce a pharmacological change relevant to treating the disease sought to be treated.
• an effective amount includes an amount effective to exert prolonged antithrombotic activity without substantially increasing the risk of hemorrhage or to increase the life expectancy of the affected animal.
• prolonged antithrombotic activity refers to the time of activity of the modified annexin protein with respect to the time of activity of the same amount (molar) of an unmodified annexin protein.
• antithrombotic activity is prolonged by at least about a factor of two, more preferably by at least about a factor of five, and most preferably by at least about a factor of ten.
• the effective amount does not substantially increase the risk of hemorrhage compared with the hemorrhage risk of the same subject to whom the modified annexin has not been administered.
• the hemorrhage risk is very small and, at most, below that provided by alternative antithrombotic treatments available in the prior art.
• Therapeutically effective amounts of the therapeutic agents can be any amount or dose sufficient to bring about the desired antithrombotic effect and depends, in part, on the condition, type, and location of the thrombus, the size and condition of the patient, as well as other factors known to those skilled in the art.
• the dosages can be given as a single dose, or as several doses, for example, divided over the course of several weeks.
• Administration preferably occurs by bolus injection or by intravenous infusion, either after thrombosis to prevent further thrombosis or under conditions in which the subject is susceptible to or at risk of thrombosis.
• the therapeutic agents of the present invention can be administered by any suitable means, including, for example, parenteral or local administration, such as intravenous or subcutaneous injection, or by aerosol.
• a therapeutic composition can be administered in a variety of unit dosage forms depending upon the method of administration. Delivery methods for a therapeutic composition of the present invention include intravenous administration and local administration by, for example, injection. For particular modes of delivery, a therapeutic composition of the present invention can be formulated in an excipient of the present invention.
• a therapeutic agent of the present invention can be administered to any animal, preferably to mammals, and more preferably to humans.
• One suitable administration time occurs following coronary thrombosis, thereby preventing the recurrence of thrombosis without substantially increasing the risk of hemorrhage.
• Bolus injection of the modified annexin is preferably performed soon after thrombosis, e.g., before admission to hospital.
• the modified annexin can be administered in conjunction with a thrombolytic therapeutic such as tissue plasminogen activator, urokinase, or a bacterial enzyme.
• Methods of use of modified annexin proteins of the present invention include methods to treat cerebral thrombosis, including overt cerebral thrombosis or transient cerebral ischemic attacks, by administering an effective amount of modified annexin protein to a patient in need thereof.
• the modified annexin can also be administered to diabetic and other patients who are at increased risk for thrombosis in peripheral arteries. Accordingly, the present invention provides a method for reducing the risk of thrombosis in a patient having an increased risk for thrombosis including administering an effective amount of a modified annexin protein to a patient in need thereof.
• the modified annexin can be administered intravenously or as a bolus in the dosage range of about 1 to about 100 mg.
• the present invention also provides a method for decreasing the risk of venous thrombosis associated with some surgical procedures, such as hip and knee arthroplasties, by administering an effective amount of a modified annexin protein of the present invention to a patient in need thereof.
• the modified annexin treatment can prevent thrombosis without increasing hemorrhage into the operating field.
• the present invention provides a method for preventing thrombosis associated with pregnancy and parturition without increasing hemorrhage, by administering an effective amount of a modified annexin protein of the present invention to a patient in need thereof.
• the present invention provides a method for the treatment of recurrent venous thrombosis, by administering an effective amount of a modified annexin protein of the present invention to a patient in need thereof.
• the modified annexin can be administered intravenously as a bolus in the dosage range of about 1 to about 100 mg.
• the present invention also provides a method of screening for a modified annexin protein that modulates thrombosis, by contacting a thrombosis test system with at least one test modified annexin protein under conditions permissive for thrombosis, and comparing the antithrombotic activity in the presence of the test modified annexin protein with the antithrombotic activity in the absence of the test modified annexin protein, wherein a change in the antithrombotic activity in the presence of the test modified annexin protein is indicative of a modified annexin protein that modulates thrombotic activity.
• the thrombosis test system is a system for measuring activated partial thromboplastin time.
• the present invention also provides a method for identifying a modified annexin protein for annexin activity, including contacting activated platelets with at least one test modified annexin protein under conditions permissive for binding, and comparing the test modified annexin-binding activity and protein S-binding activity of the platelets in the presence of the test modified annexin protein with the annexin-binding activity and protein S-binding activity in the presence of unmodified annexin protein, whereby a modified annexin protein with annexin activity may be identified.
• modified annexin proteins identified by the method.
• the present invention provides a method of screening for a modified annexin protein that modulates thrombosis, by contacting an in vivo thrombosis test system with at least one test modified annexin protein under conditions permissive for thrombosis, and comparing the antithrombotic activity in the presence of the test modified annexin protein with the antithrombotic activity in the absence of the test modified annexin protein. A change in the antithrombotic activity in the presence of the test modified annexin protein is indicative of a modified annexin protein that modulates thrombotic activity.
• the time over which antithrombotic activity is sustained in the presence of the test modified annexin protein is compared with a time of antithrombotic activity in the presence of unmodified annexin to determine the prolongation of antithrombotic activity associated with the test modified annexin protein.
• the extent of hemorrhage in the presence of the test modified annexin protein is assessed, e.g., by measuring tail bleeding time, and compared with the extent of hemorrhage in the absence of the test modified annexin protein.
• the in vivo thrombosis test system is a mouse model of photochemically-induced thrombus in cremaster muscles.
• the therapeutic agents of the present invention are useful for gene therapy.
• gene therapy refers to the transfer of genetic material (e.g., DNA or RNA) of interest into a host to treat or prevent a genetic or acquired disease or condition.
• the genetic material of interest encodes a product (e.g., a protein polypeptide, peptide or functional RNA) whose production in vivo is desired.
• the genetic material of interest can encode a hormone, receptor, enzyme or (poly)peptide of therapeutic value.
• the subject invention utilizes a class of lipid molecules for use in non-viral gene therapy which can complex with nucleic acids as described in Hughes et al., U.S. Patent No. 6,169,078, incorporated herein by reference, in which a disulfide linker is provided between a polar head group and a lipophilic tail group of a lipid.
• These therapeutic compounds of the present invention effectively complex with DNA and facilitate the transfer of DNA through a cell membrane into the intracellular space of a cell to be transformed with heterologous DNA.
• these lipid molecules facilitate the release of heterologous DNA in the cell cytoplasm thereby increasing gene transfection during gene therapy in a human or animal.
• Cationic lipid-polyanionic macromolecule aggregates may be formed by a variety of methods known in the art. Representative methods are disclosed by Feigner et al., Proc. Natl. Acad. Sci. USA 86: 7413-7417 (1987); Eppstein et al., U.S. Patent No. 4,897,355; Behr et al., Proc. Natl. Acad. Sci. USA 86:6982-6986 (1989) ; Bangham et al., J. Mol. Biol. 23:238-252 (1965); Olson et al, Biochim. Biophys. Acta 557:9 (1979); Szoka, et al., Proc. Natl.
• aggregates may be formed by preparing lipid particles consisting of either (1) a cationic lipid or (2) a cationic lipid mixed with a colipid, followed by adding a polyanionic macromolecule to the lipid particles at about room temperature (about 18 to 26 °C). In general, conditions are chosen that are not conducive to deprotection of protected groups.
• the mixture is then allowed to form an aggregate over a period of about 10 minutes to about 20 hours, with about 15 to 60 minutes most conveniently used. Other time periods may be appropriate for specific lipid types.
• the complexes may be formed over a longer period, but additional enhancement of transfection efficiency will not usually be gained by a longer period of complexing.
• the compounds and methods of the subject invention can be used to intracellularly deliver a desired molecule, such as, for example, a polynucleotide, to a target cell.
• the desired polynucleotide can be composed of DNA or RNA or analogs thereof.
• the desired polynucleotides delivered using the present invention can be composed of nucleotide sequences that provide different functions or activities, such as nucleotides that have a regulatory function, e.g., promoter sequences, or that encode a polypeptide.
• the desired polynucleotide can also provide nucleotide sequences that are antisense to other nucleotide sequences in the cell.
• the desired polynucleotide when transcribed in the cell can provide a polynucleotide that has a sequence that is antisense to other nucleotide sequences in the cell.
• the antisense sequences can hybridize to the sense strand sequences in the cell.
• Polynucleotides that provide antisense sequences can be readily prepared by the ordinarily skilled artisan.
• the desired polynucleotide delivered into the cell can also comprise a nucleotide sequence that is capable of forming a triplex complex with double-stranded DNA in the cell.
• the present invention provides compounds and methods for preventing or attenuating reperfusion injury in mammals.
• Reperfusion injury (Rl) occurs when the blood supply to an organ or tissue is cut off and after an interval restored. The loss of phospholipids asymmetry in endothelial cells and other cells is considered a significant event in the pathogenesis of Rl.
• the PS exposed on the surfaces of these cells allows the binding of activated monocytes.
• a recombinant human annexin preferably annexin V, is modified in such a way that its half-life in the vascular compartment is prolonged.
• annexin coupled to polyethylene glycol, a homopolymer or heteropolymer of annexin, and a fusion protein of annexin with another protein (e.g., the Fc portion of immunoglobulin).
• another protein e.g., the Fc portion of immunoglobulin.
• the modified annexin binds with high affinity to phosphatidylserine on the surface of epithelial and other cells, thereby preventing the binding of phagocytes and the operation of phospholipases, which release lipid mediators.
• the modified annexin therefore inhibits both cellular and humoral mechanisms of reperfusion injury.
• the present invention provides an isolated modified annexin protein containing an annexin protein coupled to at least one additional protein, such as an additional annexin protein (forming a homodimer), polyethylene glycol, or the Fc portion of immunoglobulin.
• the additional protein preferably has a molecular weight of at least 30 kDa.
• compositions containing an amount of any of the modified annexin proteins of the invention that is effective for preventing or reducing reperfusion injury.
• the modified annexin is administered to a subject at risk of reperfusion injury in a pharmaceutical composition having an amount of any one of the modified annexin proteins of the present invention effective for preventing or attenuating reperfusion injury.
• the pharmaceutical composition may be administered before and after organ transplantation, arthroplasty or other surgical procedure in which the blood supply to organ or tissue is cut off and after an interval restored. It can also be administered after a coronary or cerebral thrombosis.
• the modified annexin binds PS accessible on cell surfaces (shielding the cells), thereby preventing the attachment of monocytes and the irreversible stage of apoptosis.
• the modified annexin inhibits the activity of phospholipases that generate lipid mediators that also contribute to Rl.
• the modified annexin will be useful to prevent or attenuate Rl in organs transplanted from cadaver donors, in patients with coronary and cerebral thrombosis, in patients undergoing arthroplasties, and in other situations.
• the modified annexin will exert prolonged antithrombotic activity without increasing hemorrhage.
• annexin homodimer is a potent inhibitor of sPLA2 (FIG. 4). Because annexin N binds to PS on cell surfaces with high affinity, it shields PS from degradation by sPLA2 and other phospholipases. [00120] Producing a homodimer of human annexin V both increased its affinity for
• the annexin homodimer may be produced by any convenient method.
• the annexin homodimer is produced by recombinant D ⁇ A technology as this avoids the necessity for post-translation procedures such as linkage to the one available sulfhydryl group in the monomer or coupling with polyethylene glycol.
• annexin V Recombinant homodimerization was achieved by the use of a flexible peptide linker attached to the amino terminus of one annexin monomer and the carboxy terminus of the other (FIG. 1).
• the three-dimensional structure of annexin V, and the residues binding Ca2+ and PS, are known from X-ray crystallography and site-specific mutagenesis (Huber et al., J. Mol. Biol. 223: 683, 1992; Campos et al., 37: 8004, 1998).
• the Ca2+- and PS- binding sites are on the convex surface of the molecule while the amino terminus forms a loose tail on the concave surface.
• annexin N Increasing the molecular weight of annexin N by homodimerization to 76 kDa prevents renal loss and extends survival in the circulation. Accordingly, such modified annexins may effectively attenuate Rl, even when administered several hours before the blood supply to an organ is cut off.
• annexin V does not inhibit RL
• lipocortin I annexin I
• ALT alanine aminotransferase
• DAN annexin V homodimer
• PEG-AV polyethylene glycol
• AV annexin V monomer
• the modified annexins of the present invention will be useful to attenuate Rl in subjects.
• the annexin N homodimer may be used to attenuate Rl in all of them.
• the annexin V homodimer will exert prolonged antithrombotic activity. This is clinically useful to prevent reinfarction, which is known to be an important event following coronary thrombosis (Andersen et al., ⁇ . Engl. J. Med. 349: 733, 2003), and is likely to be important in stroke.
• annexin V inhibits arterial and venous thrombosis without increasing hemorrhage (R ⁇ misch et al., Thromb. Res. 61 : 93, 1991; Van Ryn-McKenna et al., Thromb. Hemost. 69: 227, 1993; Thiagarajan and Benedict, Circulation 96: 2339, 1997).
• a modified annexin has the capacity to exert anticoagulant activity without increasing hemorrhage and to attenuate reperfusion injury.
• annexin V bind Ca2+ and PS. Any of these might be used to prevent or diminish reperfusion injury.
• the molecular weight of annexin V, or another annexin may be increased by procedures other than homodimerization. Such procedures include the preparation of other homopolymers or heteropolymers.
• an annexin might be conjugated to another protein by recombinant DNA technology or chemical manipulation. Conjugation of an annexin to polyethylene glycol or another nonpeptide compound are also envisaged.
• annexin V homodimer will be well-tolerated.
• annexin VI is a naturally existing homodimer of the conserved annexin sequence. However, annexin VI does not bind PS with high affinity
• a PS-binding protein other than an annexin may also be used in the methods of the invention.
• a monoclonal or polyclonal antibody with a high affinity for PS (Diaz et al., Bioconjugate Chem. 9:250, 1998; Thorpe et al., U. S. Patent No. 6,312,694) may be used according to the present invention (e.g., for decreasing or preventing reperfusion injury).
• Diannexin (PLEASE DEFINE-IS THIS SEQ ID NO: 6) has dose-related antithrombotic activity in the rat (fig. 7). Even when Diannexin is administered at 5.0 mg/kg (approximately 7x the antithrombotic dose) it does not significantly increase blood loss after transecting rat tails. In contrast, Fragmin (low molecular weight heparin) administered at 140 aXa units/kg (approx. 7x therapeutic dose) significantly increased blood loss in experiments conducted simultaneously (table 4 and fig. 10).
• Diannexin induces hemorrhage its effects will disappear fairly soon. Both Diannexin and Fragmin significantly increase the bleeding time in the rat following tail transection (fig. 9 and table 4). In the case of Diannexin this may be due to inhibition of phospholipase A2 action and thromboxane generation. In humans bleeding times are increased when cyclooxygenase is irhbited by a drug or as a result of a genetic deficiency. Because Diannexin does not significantly increase blood loss, despite increasing the bleeding time, it is clear that Diannexin has ' no major effect on early hemostatic mechanisms. Diannexin administration has no effect on body weight.
• compositions of the present invention can also include other components such as a pharmaceutically acceptable excipient, an adjuvant, and/or a carrier.
• compositions of the present invention can be formulated in an excipient that the animal to be treated can tolerate.
• excipients include water, saline, Ringer's solution, dextrose solution, mannitol, Hank's solution, and other aqueous physiologically balanced salt solutions.
• Nonaqueous vehicles, such as triglycerides may also be used.
• Excipients can also contain minor amounts of additives, such as substances that enhance isotonicity and chemical stability.
• buffers include phosphate buffer, bicarbonate buffer, Tris buffer, histidine, citrate, and glycine, or mixtures thereof
• preservatives include thimerosal, m- or o-cresol, formalin and benzyl alcohol.
• Standard formulations can either be liquid injectables or solids which can be taken up in a suitable liquid as a suspension or solution for injection.
• the excipient can comprise dextrose, human serum albumin, preservatives, etc., to which sterile water or saline can be added prior to administration.
• One embodiment of the present invention is a controlled release formulation that is capable of slowly releasing a composition of the present invention into an animal.
• a controlled release formulation comprises a composition of the present invention in a controlled release vehicle.
• Suitable controlled release vehicles include, but are not limited to, biocompatible polymers, other polymeric matrices, capsules, microcapsules, microparticles, bolus preparations, osmotic pumps, diffusion devices, liposomes, lipospheres, and transdermal delivery systems.
• Other controlled release formulations of the present invention include liquids that, upon administration to an animal, form a solid or a gel in sil .
• Preferred controlled release formulations are biodegradable (i.e., bioerodible).
• the therapeutic agents used in the invention are administered to an animal in an effective amount.
• an effective amount is an amount effective to either (1) reduce the symptoms of the disease sought to be treated or (2) induce a pharmacological change relevant to treating the disease sought to be treated.
• Therapeutically effective amounts of the therapeutic agents can be any amount or doses sufficient to bring about the desired effect and depend, in part, on the condition, type and location of the cancer, the size and condition of the patient, as well as other factors readily known to those skilled in the art.
• the dosages can be given as a single dose, or as several doses, for example, divided over the course of several weeks.
• the present invention is also directed toward methods of treatment utilizing the therapeutic compositions of the present invention. The method comprises administering the therapeutic agent to a subject in need of such administration.
• the therapeutic agents of the instant invention can be administered by any suitable means, including, for example, parenteral, topical, oral or local administration, such as intradermally, by injection, or by aerosol.
• the agent is administered by injection.
• Such injection can be locally administered to any affected area.
• a therapeutic composition can be administered in a variety of unit dosage forms depending upon the method of administration. Suitable delivery methods for a therapeutic composition of the present invention include intravenous administration and local administration by, for example, injection. For particular modes of delivery, a therapeutic composition of the present invention can be formulated in an excipient.
• a therapeutic reagent of the present invention can be administered to any animal, preferably to mammals, and more preferably to humans.
• Annexins can be purified from human tissues or produced by recombinant technology.
• annexin V can be purified from human placentas as described by Funakoshi et al. (1987).
• recombinant products are the expression of annexin II and annexin V in Escherichia coli (Kang, H.-M., Trends Cardiovasc. Med. 9:92-102 (1999); Thiagarajan and Benedict, 1997, 2000).
• Annexins can be coupled to polyethylene glycol (PEG) by any of several well-established procedures (reviewed by Hermanson, 1996) in a process referred to as pegylation.
• the present invention includes chemically-derivatized annexin molecules having mono- or poly-(e.g., 2-4) PEG moieties.
• Methods for preparing a pegylated annexin generally include the steps of (a) reacting the annexin with polyethylene glycol (such as a reactive ester or aldehyde derivative of PEG) under conditions whereby the annexin becomes attached to one or more PEG groups and (b) obtaining the reaction product or products.
• polyethylene glycol such as a reactive ester or aldehyde derivative of PEG
• the optimal reaction conditions for the reactions must be determined case by case based on known parameters and the desired result.
• the reaction may produce different products having a different number of PEG chains, and further purification may be needed to obtain the desired product.
• Conjugation of PEG to annexin V can be performed using the EDC plus sulfo-NHS procedure.
• EDC l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride
• sulfo- NHS N-hydroxysulfosuccinamide
• This increases the stability of the active intermediate, which reacts with an amine to give a stable amide linkage.
• the conjugation can be carried out as described in Hermanson, 1996.
• Bioconjugate methods can be used to produce homopolymers or heteropolymers of annexin; methods are reviewed by Hermanson, 1996.
• Recombinant methods can also be used to produce fusion proteins, e.g., annexin expressed with the Fc portion of immunoglobulin or another protein.
• the heterotetramer of annexin II with PI 1 has also been produced in E. coli (Kang et al., 1999). All of these procedures increase the molecular weight of annexin and have the potential to increase the half-life of annexin in the circulation and prolong its anticoagulant effect.
• a homodimer of annexin V can be produced using a DNA construct shown schematically in FIG. 1C (5'-3' sense strand) (SEQ ID NO:4) and coding for an amino acid sequence represented by SEQ ID NO:6.
• the annexin V gene is cloned into the expression vector pCMV FLAG 2 (available from Sigma-Aldrich) at EcoRI and Bglll sites.
• the exact sequences prior to and after the annexin V sequence are unknown and denoted as "x". It is therefore necessary to sequence the construct prior to modification to assure proper codon alignment.
• the pCMV FLAG 2 vector comes with a strong promotor and initiation sequence (Kozak) and start site (ATG) built in. The start codon before each annexin V gene must therefore be removed, and a strong stop for tight expression should be added at the terminus of the second annexin V gene.
• the vector also comes with an 8-amino acid peptide sequence that can be used for protein purification (asp-tyr-lys-asp-asp-asp-asp-lys) (SEQ ID NO:9).
• a 14-amino acid spacer with glycine- serine swivel ends allows optimal rotation between between tandem gene-encoded proteins.
• Addition of restriction sites PvuII and Seal allow removal of the linker if necessary.
• Addition of a protease site allows cleavage of tandem proteins following expression.
• PreScissionTM protease is available from Amersham Pharmacia Biotech and can be used to cleave tandem proteins. Two annexin V homodimers were generated.
• FIG. 1A The linker sequence of 12 amino acids was flanked by a glycine and a serine residue at either end to serve as swivels. The structural scheme is shown in FIG. 1A.
• the amino acid sequence of the His-tagged annexin V homodimer is provided below: MHHHHHHQAQVLRGTVTDFPGFDERADAETLRKAMKGLGTDEESILTLLTSRSNAQRQEI SAAFKTLFGRDLLDDLKSELTGKFEKLIVALMKPSRLYDAYELKHALKGAGTNEKVLTEI IASRTPEELRAIKQVYEEEYGSSLEDDVVGDTSGYYQRMLVVLLQANRDPDAGIDEAQVE QDAQALFQAGELK GTDEEKFITIFGTRSVSHLRKVFDKYMTISGFQIEETIDRETSGNL EQLLLAVVKSIRSIPAYLAETLYYAMKGAGTDDHTLIRVMVSRSEIDLFNIRKEFRKNFA TSLYSMIKGDTSGDYKKALLLLCGEDDGSLEVLFQGPSGKLAQVLRGTVTDFPGFDERAD AETLRKA KGLGTDEESILTLLTSRSNAQRQEISAAFKTLFGRDLLDDLKSEL
• His-tagged annexin V homodimer was expressed at a high level in Escherichia coli and purified using a nickel column.
• the DNA in the construct was shown to have the correct sequence and the dimer had the predicted molecular weight (74kDa).
• MALDI-TOF mass spectrometry was accomplished using a PerSeptive Biosystems Voyager-DE Pro workstation operating in linear, positive ion mode with a static accelerating voltage of 25kV and a delay time of 40 nsec.
• a second human annexin V homodimer was synthesized without the His tag. The structural scheme is shown in FIG. IB.
• This dimer was expressed at a high level 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 manufacturers' instructions.
• the DNA sequence of the annexin V homodimer was found to be correct.
• Mass spectrometry showed a protein of 73kDa, as expected. The amino acid sequence of annexin and other proteins is routinely determined in this laboratory by mass spectrometry of peptide fragments. Expected sequences were obtained.
• Human Annexin V has the following amino acid sequence: AQVLRGTVTDFPGFDERADAETLRKAMKGLGTDEESILTLLTSRSNAQRQEISAA FKTLFGRDLLDDLKSELTGKFEKLIVALMKPSRLYDAYELKHALKGAGTNEKVL TEIIASRTPEELRAIKQVYEEEYGSSLEDDVVGDTSGYYQRMLVVLLQANRDPDA GIDEAQVEQDAQALFQAGELKWGTDEEKFITIFGTRSVSHLRKVFDKYMTISGFQI EETIDRETSGNLEQLLLAVVKSIRSIPAYLAETLYYAMKGAGTDDHTLIRVMVSRS EIDLFNIRKEFRKNFATSLYSMIKGDTSGDYKKALLLLCGEDD (SEQ ID NO:3) [00147] The nucleotide sequence of human annexin V, inserted as indicated in the
• DNA construct illustrated in FIG. 1C is as follows: GCACAGGTTCTCAGAGGCACTGTGACTGACTTCCCTGGATTTGATGAGCGGGC TGATGCAGAAACTCTTCGGAAGGCTATGAAAGGCTTGGGCACAGATGAGGAG AGCATCCTGACTCTGTTGACATCCCGAAGTAATGCTCAGCGCCAGGAAATCTC TGCAGCTTTTAAGACTCTGTTTGGCAGGGATCTTCTGGATGACCTGAAATCAG AACTAACTGGAAAATTTGAAAAATTAATTGTGGCTCTGATGAAACCCTCTCGG CTTTATGATGCTTATGAACTGAAACATGCCTTGAAGGGAGCTGGAACAAATG AAAAAGTACTGACAGAAATTATTGCTTCAAGGACACCTGAAGAACTGAGAGC CATCAAACAAGTTTATGAAGAAGAATATGGCTCAAGCCTGGAAGATGACGTG GTGGGGGACACTTCAGGGTACTACCAGCGGATGTTGGTGGTTCCTTCAGGC TAACAGAGACCCTGATGAA
• Annexin V binds to platelets, and this binding is markedly increased in vitro by activation of the platelets with thrombin (Thiagarajan and Tait, 1990; Sun et al., 1993).
• the modified annexin proteins of the present invention are prepared in such a way that perform the function of annexin in that they bind to platelets and prevent protein S from binding to platelets (Sun et al., 1993).
• the modified annexin proteins also perform the function of exhibiting the same anticoagulant activity in vitro that unmodified annexin proteins exhibit.
• a method for measuring the clotting time is the activated partial thromboplastin time (Fritsma, in Hemostasis and thrombosis in the clinical laboratory (Corriveau, D.M. and Fritsma, G.A., eds) J.P. Lipincott Co., Philadelphia (1989), pp. 92- 124, incorporated herein by reference).
• In vivo assays determine the antithrombotic activity of annexin proteins.
• Annexin V has been shown to decrease venous thrombosis induced by a laser or photochemically in rats (R ⁇ misch et al., 1991). The maximal anticoagulant effect was observed between 15 and 30 minutes after intravenous administration of annexin V, as determined functionally by thromboelastography.
• the modified annexin proteins of the present invention preferably show more prolonged activity in such a model than unmodified annexin.
• Annexin V was also found to decrease fibrin accretion in a rabbit model of jugular vein thrombosis (Van Ryn-McKenna et al., 1993).
• annexin V was shown to bind to the treated vein but not to the control contralateral vein. Decreased fibrin accumulation in the injured vein was not associated with systemic anticoagulation. Heparin did not inhibit fibrin accumulation in the injured vein.
• the modified annexin proteins of the present invention preferably perform the function of annexin in this model of venous thrombosis.
• a rabbit model of arterial thrombosis was used by Thiagarajan and Benedict, 1997.
• a partially occlusive thrombus was formed in the left carotid artery by application of an electric current.
• Annexin N infusion strongly inhibited thrombosis as manifested by measurements of blood flow, thrombus weight, labeled fibrin deposition and labeled platelet accumulation.
• thrombosis can be induced in any desired artery or vein.
• the modified annexin proteins of the present invention preferably perform the function of annexin in such models, even when administered by bolus injection.
• Example 3 The anticoagulant ability of human recombinant annexin V and pegylated human recombinant annexin V were compared in vitro.
• Annexin V production The polymerase chain reaction was used to amplify the cD ⁇ A from the initiator methionine to the stop codon with specific oligonucleotide primers from a human placental cD ⁇ A library.
• the forward primer was 5'-ACCTGAGTAGTCGCCATGGCACAGGTTCTC-3' (SEQ ID NO: 7) and the reverse primer was 5'-CCCGAATTCACGTTAGTCATCTTCTCCACAGAGCAG-3' (SEQ ID NO: 8).
• the amplified 1.1 -kb fragment was digested with Nco I and Eco Rl and ligated into the prokaryotic expression vector pTRC 99A.
• the ligation product was used to transform competent Escherichia coli strain JM 105 and sequenced.
• Recombinant annexin V was isolated from the bacterial lysates as described by Berger et al., 1993, with some modification. An overnight culture of E. coli JM 105 transformed with pTRC 99A-annexin V was expanded 50-fold in fresh Luria- Bertrani medium containing 100 mg/L ampicillin.
• isopropyl ⁇ -D- thiogalactopyranoside was added to a final concentration of 1 mmol/L.
• the bacteria were pelleted at 3500g 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 with an ultrasonic probe at a setting of 6 on ice for 3 minutes.
• the lysate was centrifuged at 10,000g for 15 minutes, and the supernatant was dialyzed twice against 50 vol TBS containing 1 mmol/L EDTA and once against 50 vol TBS.
• Multilamellar liposomes were prepared by dissolving phosphatidylserine, lyophilized bovine brain extract, cholesterol, and dicetylphosphate in chloroform in a molar ration of 10:15:1 and dried in a stream of nitrogen in a conical flask. TBS (5 mL) was added to the flask and agitated vigorously in a vortex mixer for 1 minute. The liposomes were washed by centrifugation at 3500g for 15 minutes, then incubated with the bacterial extract, and calcium chloride was added to a final concentration of 5 mmol/L.
• the liposomes were sedimented by centrifugation at 10,000g- for 10 minutes, and the bound annexin V was eluted with 10 mmol/L EDTA.
• the eluted annexin V was concentrated by Aniicon ultrafiltration and loaded onto a Sephacryl S 200 column. The annexin V was recovered in the included volume, whereas most of the liposomes were in the void volume.
• the anticoagulant potency of the recombinant human annexin V and the pegylated recombinant human annexin V are substantially equivalent.
• the small difference observed is attributable to the change in molecular weight after pegylation.
• This experiment validates the assertion made herein that pegylation of annexin V can be achieved without significantly reducing its antithrombotic effects.
• Example 4 [00156] The affinities of recombinant annexin N (AV) and recombinant annexin V homodimer (DAV) for PS on the surface of cells were compared.
• AV and DAV were biotinylated using the FluReporter protein-labeling kit (Molecular Probes, Eugene OR). Biotin-AV and biotin-DAV conjugates were visualized with R-phycoerythrin-conjugated streptavidin (PE-SA) at a final concentration of 2 ⁇ g/ml. Flow cytometry was performed on a Becton Dickinson FACScaliber and data were analyzed with Cell Quest software (Becton Dickinson, San Jose CA). [00159] No binding of AV or DAV was detectable when normal RBCs were used.
• PE-SA R-phycoerythrin-conjugated streptavidin
• both AV and DAV were bound to at least 95% of RBCs exposing PS.
• RBCs exposing PS were incubated with various amounts of AV and DAV, either (a) separately or (b) mixed in a 1 : 1 molar ratio, before addition of PE-SA and flow cytometry.
• either AV or DAV was biotinylated and the amount of each protein bound was assayed as described above.
• the experiments were controlled for higher biotin labeling in DAV than AV. [00160] Representative results are shown in FIG. 2. In this set of experiments,
• RBCs exposing PS were incubated with (a) 0.2 ⁇ g of biotinylated DAV (FIG. 2A); (b) 0.2 ⁇ g of biotinylated DAV (FIG. 2B); (c) 0.2 ⁇ g of biotinylated AV and 0.2 ⁇ g nonbiotinylated DAV; and (d) 0.2 ⁇ g of biotinylated DAV and 0.2 ⁇ g nonbiotinylated AV (FIG. 2D). Comparing FIG. 2B and FIG. 2D shows that the presence of 0.2 ⁇ g of nonbiotinylated AV had no effect on the binding of biotinylated DAV. However, comparing FIG. 2A and FIG.
• Example 5 A cell-binding assay was established using known amounts of annexin N monomer (AV) and dimer (DAV) added to mouse serum. RBCs with externalized PS, as described above, were incubated with serum containing dilutions of AV and DAV. After washing, addition of labeled streptavidin and washing again, AV and DAV bound to the RBCs were assayed by flow cytometry. No binding was detectable when RBCs without externalized PS were used. Concentrations of AV and DAV in mouse serum, assayed by cell binding, were highly correlated with those determined by independent ELISA assays.
• AV annexin N monomer
• DAV dimer
• Example 7 A mouse liver model of warm ischemia-reperfusion injury was used to ascertain whether modified annexins protect against reperfusion injury (Rl), compare the activity of annexin V with modified annexins, and determine the duration of activity of modified annexins. The model has been described by Teoh et al. (Hepatology 36:94, 2002).
• mice Female C57BL6 mice weighing 18 to 25 g were used. Under ketamine/xylazine anesthesia, the blood supply to the left lateral and median lobes of the liver was occluded with an atraumatic microvascular clamp for 90 minutes. Reperfusion was then established by removal of the vascular clamp. The animals were allowed to recover, and 24 hours later they were killed by exsanguination. Liver damage was assessed by measurement of serum alanine aminotransferase (ALT) activity and histological examination. A control group was subjected to anesthesia and sham laparotomy. To assay the activity of annexin V and modified annexins, groups of 4 mice were used.
• ALT serum alanine aminotransferase
• mice in the first group were injected intravenously with 25 micrograms of annexin V (AV), each of the second group received 25 micrograms of annexin homodimer (DAV), and each of the third group received 2.5 micrograms of annexin V coupled to polyethylene glycol (PEG-AV, 57 kDa).
• Controls received saline or the HEPES buffer in which the annexins were stored.
• the annexins were administered minutes before clamping branches of the hepatic artery.
• annexins and HEPES were administered 6 hours before initiating ischemia. Representative experimental results are summarized in FIG. 5.
• HEPES administered just before ischemia was found to have protective activity against RL
• Example 8 [00168] Thrombosis study [00169] Six groups of eight rats each were used. The rats for this study were male
• Wistar rats Wistar rats, weighing about 300 grams (Charles River ⁇ ederland, Maastricht, the Netherlands). Animals were housed in macrolon cages, and given standard rodent food pellets and acidified tap water ad lib. Experiments conformed to the rules and regulations set forward by the Netherlands Law on Animal Experiments. Rats were anaesthetized with FFM (Fentanyl / Fluanison / Midazolam), and placed on a heating pad. A cannula was inserted into the femoral vein and filled with saline. The vena cava inferior was isolated, and side branches were closed by ligation or cauterization. A loose ligature was applied around the caval vein below the left renal vein.
• FFM Fluanison / Midazolam
• Test or control compounds include phosphate-buffered saline 1.0 ml/kg bodyweight (10 min); Phosphate-buffered saline 1.0 ml/kg bodyweight (12 hrs); Diannexin 0.04 mg/kg body weight; Diannexin 0.2 mg/kg body weight; Diannexin 1.0 mg/kg body weight (10 min); Diannexin 1.0 mg/kg body weight (12 hrs); Fragmin 20 aXa U/kg body weight.
• recombinant human thromboplastin (0.15mL/kg) was rapidly injected into the venous cannula, the cannula was flushed with saline, and exactly ten seconds later the downstream ligature near the renal vein was closed. After nine minutes, a citrated venous blood sample was obtained and put on ice. [00171] One minute later (at ten minutes) the upstream ligature near the bifurcation was closed and the thrombus that had formed in the segment was recovered. The thrombus was briefly washed in saline, blotted, and its wet weight was determined.
• Citrated plasma was prepared by centrifugation for 15 min at 2000g at 4°C, and stored at -60°C for analysis. In the two groups in which thrombus induction took place at 12 hrs after compound injection, a different i.v. injection procedure was used. Rats were anaesthetized with s.c. DDF (Domitor/Dormicum/Fentanyl) and injected via the vein of the penis. Rats were then s.c. given an antidote (Anexate / Antisedan / Naloxon) and kept overnight in their cage.
• DDF Domitor/Dormicum/Fentanyl
• rats were intravenously injected with At 10 minutes after the intravenous injection of compound (in two groups: at 12 hrs after injection), diluted thromboplastin was injected i.v., and ten seconds later the vena cava inferior ligated. At nine minutes after ligation, blood was collected and citrated plasma was prepared. At ten minutes after ligation, the thrombosed segment was ligated, and the thrombus was recovered and weighed. aPTT (sec) was also measured. At 0.04 mg/kg , Diannexin reduced thrombus weight by about 40%).
• Thrombus wet weights ranged from 0 to 44.5 mg. [00173] Table 1 : Effect of Treatment on Thrombus Wet Weight (mg) in the 10- min Thrombosis study.
• Fragmin increased the APPT significantly, compared to all other groups.
• Example 9 [00181] Bleeding study Three groups were studied. Groups of eight rats, as described in Example 8, were used. Rats were anaesthetized with isoflurane, intubated and ventilated, and placed on a heating pad. A cannula was inserted into the femoral vein, and filled with saline. Test or control compounds were i.v. injected via the cannula, and the cannula was then flushed with saline. Test or control compound were phosphate- buffered saline 1.0 ml/kg bodyweight; Diannexin 5.0 mg/kg body weight; Fragmin 140 aXa Ukg body weight.
• Diannexin group did not differ from the saline control group.
• Blood loss and the aPTT were approximately twice as large in the Fragmin group as in the Diannexin group in the tail bleeding study.
• Diannexin induced bleeding from a transected rat tail, though less blood was lost than after injection of 140 aXa U/kg of Fragmin.
• Example 10 Clearance study Rats were injected with radiolabeled Diannexin, blood samples were obtained at 5, 10, 15, 20, 30, 45, and 60 min and 2, 3, 4, 8, 16 and 24 hrs, and blood radioactivity was determined to construct a blood disappearance curve. Disappearance of Diannexin from blood could be described by a two-compartment model, with about 75-80% disappearing in the ⁇ -phase (t/2 about 10 min), and 15-20%o in the ⁇ - phase (t/2 about 400 min). Clearance could be described by a two-compartment model, with half-lives of 9-14 min and 6-7 hrs, respectively. Two experiments were performed, each with three male Wistar rats (300 gram).
• Diannexin was labelled with 123 I by the method of Macfarlane, and the labeled protein was separated from free Sephadex G-50.
• Nai 5 mg/kg
• about 8 x 106 cpm 50 ⁇ L of protein solution diluted to 0.5 mL with saline
• blood samples 150 ⁇ L were obtained from a tail vein and 100 ⁇ L was counted.
• rats were sacrificed by Nembutal i.v., and (pieces of) liver, lung, heart, spleen and kidneys were collected for counting.
• the ⁇ -phase parameters were then calculated from the data between 5 and 45 min by the subtraction method.
• the blood radioactivity curves were analysed by a two-compartment model, using the subtraction method.
• the linear correlation coefficients for the ⁇ - and the ⁇ -phase 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 parameters.
• Figures 15 and 16 show the clearance curves with the alpha- and beta- phases superimposed.
• Table 7 are shown the cpm recovered in lung, heart, liver spleen and kidneys (after digestion of the tissues). Of note is the high number of counts in the lung at 2 hrs after Diannexin injection.
• Table 7. Radioactivity Recovered in Selected Tissues at 2, 8 and 24 hours 1 S after injection of " I-Diannexin.
• phosphatidylserine becomes accessible on the luminal surface of endothetial cells (EC) in the hepatic micravasculature.
• EC endothetial cells
• Diannexin binds to PS on the surface of EC and decreases the attachment of leukocytes and platelets to them. By this mechanism Diannexin prevents irreversible damage to EC and thereby attenuates ischemia-reperfusion injury.
• Diannexin As predicted, EC damage (reflected by swelling) is prominent during reperfusion and is significantly decreased by Diannexin (Fig 14A and 14B). Our hypothesis of the mode of action of Diannexin in attenuating ischemia-reperfusion injury is therefore confirmed. As shown in figs. 15A and 15B, Diannexin does not influence the phagocytic activity of Kupffer cells in either location. Hence, Diannexin has no effect on this defense mechanism against pathogenic organisms. This finding supports other evidence that Diannexin does not have adverse effects.

Landscapes

• Health & Medical Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Organic Chemistry (AREA)
• General Health & Medical Sciences (AREA)
• Medicinal Chemistry (AREA)
• Engineering & Computer Science (AREA)
• Hematology (AREA)
• Biochemistry (AREA)
• Genetics & Genomics (AREA)
• Gastroenterology & Hepatology (AREA)
• Molecular Biology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Zoology (AREA)
• Toxicology (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Diabetes (AREA)
• Biophysics (AREA)
• Chemical Kinetics & Catalysis (AREA)
• General Chemical & Material Sciences (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Pharmacology & Pharmacy (AREA)
• Animal Behavior & Ethology (AREA)
• Public Health (AREA)
• Veterinary Medicine (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
• Peptides Or Proteins (AREA)
• Investigating Or Analysing Biological Materials (AREA)

Priority Applications (4)

Applications Claiming Priority (4)

Publications (2)

Family

ID=34976262

Family Applications (1)

Country Status (5)

Cited By (5)

Families Citing this family (2)

Family Cites Families (8)

• 2005
  • 2005-03-10 CA CA002559167A patent/CA2559167A1/en not_active Abandoned
  • 2005-03-10 AU AU2005221195A patent/AU2005221195B2/en not_active Ceased
  • 2005-03-10 WO PCT/US2005/008193 patent/WO2005086955A2/en active Application Filing
  • 2005-03-10 JP JP2007503052A patent/JP5134362B2/ja not_active Expired - Fee Related
  • 2005-03-10 EP EP05725390A patent/EP1734983A4/en not_active Withdrawn
• 2012
  • 2012-07-20 JP JP2012161761A patent/JP2012254992A/ja not_active Withdrawn

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events