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WO1995030438A2 - Methodes et compositions pour augmenter l'activite fibrinolytique endogene - Google Patents

Methodes et compositions pour augmenter l'activite fibrinolytique endogene Download PDF

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Publication number
WO1995030438A2
WO1995030438A2 PCT/CA1995/000279 CA9500279W WO9530438A2 WO 1995030438 A2 WO1995030438 A2 WO 1995030438A2 CA 9500279 W CA9500279 W CA 9500279W WO 9530438 A2 WO9530438 A2 WO 9530438A2
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WIPO (PCT)
Prior art keywords
vitronectin
pai
binding
vimentin
antibody
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PCT/CA1995/000279
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English (en)
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WO1995030438A3 (fr
Inventor
Thomas J. Podor
Jeffry I. Weitz
Jack Hirsh
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Hamilton Civic Hospitals Research Development, Inc.
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Priority to AU24039/95A priority Critical patent/AU2403995A/en
Publication of WO1995030438A2 publication Critical patent/WO1995030438A2/fr
Publication of WO1995030438A3 publication Critical patent/WO1995030438A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/38—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against protease inhibitors of peptide structure
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/39—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/55—Protease inhibitors
    • A61K38/57—Protease inhibitors from animals; from humans

Definitions

  • the present invention relates generally to methods and compositions for the treatment of cardiovascular disease. More particularly, the present invention relates to novel methods and compositions for enhancing fibrinolytic activity by inhibiting the accumulation of type 1 plasminogen activator inhibitor (PAI-1) at sites of vascular injury and subsequent thrombus or clot formation.
  • PAI-1 type 1 plasminogen activator inhibitor
  • Thrombus formation is a pathological manifestation of clotting in blood vessels.
  • the clotting cascade is a complex biological process which results in the formation of a clot or thrombus composed of platelets and fibrin.
  • thrombus formation that occurs on atherosclerotic plagues can accelerate plaque growth, leading to partial or total occlusion of an affected blood vessel.
  • Clots can also form in the venous system when a vein is damaged by trauma or surgery.
  • pathological clotting is a primary cause of cardiovascular diseases, including unstable angina, acute yocardial infarction (heart attack) , cerebral vascular accidents (stroke), pulmonary embolism, deep vein thrombosis, arterial thrombosis, and the like.
  • cardiovascular diseases including unstable angina, acute yocardial infarction (heart attack) , cerebral vascular accidents (stroke), pulmonary embolism, deep vein thrombosis, arterial thrombosis, and the like.
  • stroke cerebral vascular accidents
  • pulmonary embolism pulmonary embolism
  • deep vein thrombosis deep vein thrombosis
  • arterial thrombosis and the like.
  • Atherosclerosis is characterized by a gradual deposit of substances, such as clotting proteins, cellular debris, cholesterol, fats, calcium and the like, and cells, such as smooth muscle cells and mononuclear cells, into the walls of
  • Thrombus or clot within the vasculature is normally broken down by the fibrinolytic system.
  • the fibrinolytic system relies on the release of plasminogen activators, most notably tissue plasminogen activator (tPA) from cells lining the blood cells.
  • tPA tissue plasminogen activator
  • tPA converts plasminogen into a clot- digesting enzyme referred to as plasmin. Plasmin slowly degrades the clot or thrombus, thereby restoring vessel patency and blood flow.
  • the extent of fibrinolytic activity is itself regulated by the presence of endogenous plasminogen activator inhibitors.
  • PAI-1 is the major inhibitor of tPA.
  • Urokinase (uPA) is the other naturally occurring plasminogen activator.
  • PAI-1 accumulates at sites of vascular injury where fibrin deposits. Because PAI-1 locally inhibits tPA and uPA, the generation of plasmin is reduced. As a result, fibrinolysis of clots that form in damaged blood vessels can be significantly diminished.
  • Thrombus formation and growth can be inhibited by either of two basic approaches.
  • the first is to prevent clotting with anticoagulants and other thrombus-inhibiting drugs.
  • Many anticoagulants such as heparin, can be employed, but such drugs are not fully effective, have serious side effects, and require careful monitoring of the patient.
  • Newer thrombus-inhibiting drugs, such as hirulog, may be safer but have not yet proven to be more effective.
  • plasminogen activators such as tPA, streptokinase, and urokinase. While the use of such plasminogen activators has proven to be of great value, these drugs are not suitable for all patients or for all thrombus-related conditions. Moreover, in some instances, plasminogen activators can have serious side effects.
  • a heparin-binding form of vitronectin that forms non-covalently associated vitronectin multimers is described in Hess et al. "Multimeric Vitronectin: Structure and Function,," in Biology of Vitronectives and their Receptors, Preissner et al., eds., Elsevier Science Publishers (1993), pages 21-29.
  • U.S. Patent No. 5,321,127 describes a platelet glycoprotein lb receptor fragment having antiplatelet and an ithro botic activity useful for blocking platelet adhesion.
  • Grabarek et al. (1981) describes troponin fragments which formed complexes with ATP- ase inhibitory subunit.
  • a synthetic vitronectin fragment which modulates the activity of PAI-1 to reduce excessive fibrinolysis is described in EP 589181 Shohet et al. (1994)
  • THROM. HAEMOST. 71:124-128 and Paoni et al. (1993) THROM. HAEMOST. 70:307-312 described PAI-1 resistant forms of tPA. Madison et al. (1990) PROC. NATL. ACAD. SCI USA 87:3530-3533 and (1989) NATURE 339:721 describe the regions of tPA which interact with PAI-1.
  • the present invention comprises methods and compositions for inhibiting the accumulation of type 1 plasminogen activator inhibitor (PAI-1) in a ternary complex composed of a vitronectin component, a PAI-1 component, and a fibrillar protein component selected from the group consisting of intracellular vimentin and fibrin present as the major component in clot.
  • PAI-1 type 1 plasminogen activator inhibitor
  • fibrillar protein component selected from the group consisting of intracellular vimentin and fibrin present as the major component in clot.
  • at least one substance that inhibits binding between vitronectin and either PAI-1 or the fibrillar protein component, or which inhibit binding of vitronectin subunits to each other to reduce formation of a more functionally active multimeric conformation of vitronectin is exposed to an aqueous environment which includes the ternary complex and/or each individual component of the complex.
  • the aqueous environment may be a blood vessel, typically the inner wall of an arterial or venous lumen, where fibrin dissolution is enhanced by reducing the accumulation of PAI-1 in a region of pre-existing clot.
  • Compositions that enhance fibrinolysis according to the present invention will also be useful in damaged tissues which are susceptible to persistent extravascular fibrin deposition at an injury site which can lead to pathological fibrosis in blood vessels (e.g., after angioplasty) and in other tissues, including heart, lung, liver, brain, and the like.
  • compositions that enhance fibrinolysis according to the present invention will further be useful as prophylactic agents which may prevent or reduce the initial incorporation of PAI-I and vitronectin in a newly forming clots.
  • the compositions may be introduced to an aqueous environment, typically the blood plasma or interstitial fluid prior to clotting, and interfere with vitronectin and PAI-1 incorporation into clots after injury to a blood vessel or tissue, or may interfere with incorporation of vitronectin and PAI-1 into newly forming fibrin during the process of fibrin accretion onto a pre-existing clot.
  • the present invention further comprises pharmaceutical compositions comprising or consisting essentially of a substance which inhibits binding between the vitronectin and PAI-1, or vitronectin and the fibrillar protein component, or vitronectin monomeric subunits to each other which results in formation of the more functionally active multimeric conform of vitronectin, wherein the substance is present in a pharmaceutically acceptable carrier.
  • suitable substances which bind to PAI-1 and inhibit complex formation are selected from the group consisting of vitronectin fragments, vitronectin analogs, antibody and antibody fragments that bind to a vitronectin-binding site on PAI-1, and antibody and antibody fragments that alter the conformation of PAI-1, thus reducing or eliminating its ability to form the ternary complex.
  • Suitable substances which bind to vitronectin to inhibit self-association and multimerization are selected from the group consisting of vitronectin fragments, vitronectin analogs, antibody and antibody fragments that bind to vitronectin multimerization sites, and vitronectin fragments, vitronectin analogs, antibody and antibody fragments that bind to vitronectin and alter the conformation of vitronectin in order to inhibit self-association.
  • Suitable substances which bind to vitronectin to inhibit complex formation are selected from the group consisting of PAI-1 fragments, PAI-1 analogs, antibody and antibody fragments that bind to a PAI-1-binding site on vitronectin, vimentin fragments, vimentin analogs, antibodies and antibody fragments that bind to a vimentin-binding site on vitronectin, and antibody and antibody fragments that alter the conformation of vitronectin in order to inhibit formation of the complex.
  • Suitable substances which bind to vimentin 1 to inhibit complex formation are selected from the group consisting of vitronectin fragments, vitronectin analogs, antibody and antibody fragments that bind to a vitronectin- binding site on vimentin, and antibody and antibody fragments that alter the conformation of vimentin in order to inhibit complex formation.
  • Suitable substances which bind to fibrin to inhibit complex formation are selected from the group consisting of vitronectin fragments, vitronectin analogs, antibody and antibody fragments that bind to the vitronectin- binding site on fibrin, and antibody and antibody fragments that alter the conformation of fibrin in order to inhibit complex formation.
  • the present invention still further provides an inhibitor of PAI-1 comprising a hybrid molecule having a first moiety which binds to PAI-1 and inhibits binding of PAI-1 to vitronectin and a second moiety which binds to a reactive center on PAI-1 and prevents it from interacting with plasminogen activators.
  • the first and second moieties are spaced-apart by distance sufficient to permit simultaneous binding of both moieties to their respective binding sites on PAI-1, usually being in the range from 5 A to 50 A.
  • the first moiety may be selected from the group consisting of vitronectin fragments, vitronectin analogs, antibody and antibody fragments that bind to a vitronectin-binding site on PAI-1, and antibody and antibody fragments that bind and alter the conformation of PAI-1.
  • the second moiety may be selected from the group consisting of a plasminogen activator fragment, a plasminogen activator analog, and an antibody or antibody fragment which binds to the PAI-1 reactive center, and the like.
  • the present invention still further provides an inhibitor of PAI-1 comprising a hybrid molecule having a first moiety which binds to vitronectin but does not interfere with the binding of PAI-1 to vitronectin and a second moiety which binds to the PAI-1 to prevent it from binding to plasminogen activators.
  • the first and second moieties are spaced-apart by distance sufficient to permit simultaneous binding of both moieties to their respective binding sites on vitronectin and PAI-1, usually in the range from 5 A to 50 A.
  • the first moiety may be selected from a group consisting of vimentin fragments, vimentin analogs, antibody or antibody fragments that bind to the vimentin binding site on vitronectin, and fibrin fragments, fibrin analogs and antibody or antibody fragments that bind to the fibrin binding site on vitronectin.
  • the second moiety may be selected from the group consisting of a plasminogen activator fragment, a plasminogen activator analog, and a antibody or antibody fragment which binds to the PAI-1 reactive center, or binds to moieties on PAI-1 that inhibit interactions with plasminogen activators, and the like.
  • the present invention still further comprises methods for screening test compounds to determine whether a test compound can inhibit the accumulation of PAI-1 in the ternary complex described above.
  • the screening method comprises exposing the test compound to an aqueous environment including the ternary complex or each component which comprise the ternary complex. Test compounds having the desired inhibition activity are identified by their ability to
  • test system will comprise vimentin or fibrin immobilized on a solid phase within the aqueous environment, where the immobilized vimentin or fibrin is exposed to vitronectin, PAI-1, and the test compound within the aqueous environment.
  • the vitronectin and PAI -1 may be present in the aqueous environment as a preformed conjugate.
  • Detection is usually accomplished by measuring a detectable label bound to PAI-l or vitronectin where incorporation of the label into the ternary complex and/or failure to decrease the inhibition activity of PAI-l within the ternary complex is an indication that the test compound has not inhibited accumulation of the PAI-l.
  • the methods and compositions of the present invention will rely on the use of novel substances having the activities and binding affinities described above.
  • the present invention specifically excludes the use of known thrombolytic agents which might have an effect on formation of maintenance of the ternary complex, specifically excluding glycosaminoglycans, such as heparin, heparin fragments, and der atan sulfate.
  • Fig. 1A-1C illustrate the mechanism of endothelial cell injury which results in formation of a ternary vimentin- vitronectin-PAI-1 complex and which is mediated or controlled by the methods and compositions of the present invention.
  • Vimentin an insoluble cytoskeleton protein, is exposed when the cell is damaged as illustrated in Fig. IA. Vitronectin (Vn) and/or complexes of Vn and PAI-l enter into the damaged cell, as illustrated Fig. IB, and bind to the vimentin, as illustrated in Fig. 1C.
  • Fig. ID illustrates the mechanism of ternary complex formation in the region of fibrin in clot.
  • Fig. 2 is a schematic illustration of the ternary vimentin-vitronectin-PAI-1 complex whose formation is mediated by the methods and compositions of the present invention.
  • Fig. 3 is a schematic illustration of the structure of vitronectin showing the relative positions of the cell adhesion (RGD) domain, the heparin-binding domain which contains the plasmin/plasminogen-binding domain, and three
  • Fig. 4 is a schematic illustration of the structure of latent and active PAI-l.
  • the reactive center is masked in latent PAI-l and vitronectin cannot bind to this conformation.
  • the reactive center is exposed in active PAI-l which binds to vitronectin.
  • the binding of vitronectin to PAI-l stabilizes PAI-l in its active conformation and enhances its specificity toward proteinases, particularly thrombin.
  • Fig. 5 is a schematic illustration showing the relative positions of the heparin-binding domain, the vitronectin-binding domain, and the reactive center on PAI-l.
  • Fig. 6A is a schematic illustration of a bivalent molecule comprising a first moiety Ml that binds to a vitronectin-binding domain on PAI-l and a second moiety, M2 that binds to the reactive center on PAI-l.
  • the moieties are held together by a linking region L.
  • Fig. 6B is a schematic illustration showing the relative positions of a bivalent molecule comprising a first moiety Ml that binds to a vimentin- or fibrin-binding domain on vitronectin and a second moiety, M2 that binds to the reactive center of PAI-l.
  • the moieties are linked together by a linking region L.
  • Fig. 7 is a graph illustrating the binding of biotinylated PAI-l to immobilized vimentin in the presence of varying concentrations of vitronectin (Vn) .
  • Fig. 8 is a graph illustrating the inhibition of tPA activity by PAI-l alone, vitronectin alone, and an equimolar combination of vitronectin and PAI-l, all in the presence of immobilized vimentin.
  • Fig. 9 is a graph illustrating vimentin-binding inhibition of biotinylated vitronectin in the presence of polyclonal anti-vimentin antibody.
  • Fig. 10 is a graph illustrating inhibition of biotinylated vitronectin binding to vimentin in the presence of polyclonal anti-vitronectin antibody.
  • Fig. 11 is a figure illustrating the displacement of biotinylated vitronectin complexed to vimentin in the presence of F(ab) 2 fragments of polyclonal anti-vitronectin antibody.
  • Fig. 12 is a diagram showing insertion of 1.1 kb 3 1 - vimentin cDNA into the EcoRI restriction site of the multiple cloning region of pMAL-C2 vector for recombinant expression of the carboxy-terminus of human vimentin.
  • Fig. 13 is a diagram showing insertion of 500 bp
  • FIG. 14 is a representation of human vimentin protein (465 amino acids) , showing the regions encoded by the different constructs.
  • IA, IB, 2A and 2B denote four ⁇ -helical domains in the rod (310 aa) , and flanking nonhelical head (102 aa) and tail (54 aa) regions.
  • TH indicates the thrombin cleavage site at aa 78-79.
  • Fig. 15 shows the amino-terminal vimentin (VIM) sequence which binds vitronectin.
  • VIM amino-terminal vimentin
  • Purified vimentin 133 mer was subjected to thrombin cleavage and analyzed by SDS-PAGE, followed by ligand blotting with biotinylated Vn and epitope mapping with various antibodies directed against specific sequences in the amino-terminus of human vimentin.
  • Vn binding activity was detected in the 1 through 78 amino acid sequence of the amino-terminus and later defined to a sequence between residues 51 and 78.
  • Figs. 16A and 16B show the binding of 125 I-urea denatured (Fig.
  • Fig. 17 is a Scatchard analysis of multimeric 125 I-labelled vitronectin binding to a recombinant amino- terminal vimentin fragment.
  • Microtiter place wells were coated with purified VIM 133 and incubated with various doses of iodinated multimeric Vn for 1 hr and the dissociation constant was calculated using the Table Curve software program (Jandel Scientific) .
  • Affinity (calculated) of multimeric Vn for the vimentin coated wells was approximately 0.5 nM and of monomeric Vn for vimentin coated wells was undetectable.
  • Fig. 18 is an analysis of plasma proteins pre- and post-clotting.
  • Fig. 19 is an analysis of the incorporation of vitronectin into fibrin clots forming in solution.
  • Various concentrations of purified human Vn mixture of monomeric and multimeric forms) were incubated with purified human fibrinogen in the presence of thrombin and calcium. After 1 hr at 37°C the clot was pelleted by centrifugation and the concentration of Vn antigen in the pre- and post-clotted supernatants determined by ELISA. The specifically bound Vn was calculated by subtracting the mOD/min reading of the post- clot supernatants from the mOD/min reading of the pore-clot supernatants.
  • Fig. 20 is an analysis of the selective depletion of monomeric vitronectin during fibrin clot formation. Aliquots of the supernatants from the samples in Figure 19 were subjected to native polyacrylamide electrophoresis (PAGE) transferred to nitrocellulose and the Vn detected by immunoblotting. Note that in the supernatants prior to the addition of thrombin, the Vn was almost evenly distributed as monomeric and multimeric forms. However, after clotting, the remaining supernatants were selectively depleted of the monomeric conformations of Vn.
  • PAGE polyacrylamide electrophoresis
  • Fig. 21 is an analysis of the diffusion of 125 I-vitronectin from purified clots in the presence or absence of 2M NaCl.
  • Purified hanging fibrin clots were formed in the presence 125 I-Vn or 125 I-labelled thrombin or PPACK ARG 93-thrombin and the rate of diffusion of the radiolabelled proteins was determined. Note that the normal thrombin and PPACK-treated thrombin both show a marked increase in the rate of diffusion in the presence of buffer containing 2.0 M NaCl. In contrast, only a modest amount of the 125 I-Vn was displaced by 2.0 M NaCl (compare closed triangles to open triangles).
  • Fig. 22A is an analysis of the binding of vitronectin to purified fibrin clots.
  • Microtiter well plates were coated with purified human fibrinogen treated with thrombin and calcium to form clots and allowed to air dry at 4°C overnight.
  • the clots were then treated with various concentrations of purified biotinylated Vn in the presence or absence of 20-fold excess unlabelled human Vn.
  • the amount of biotinylated Vn bound was determined by incubating wells with streptavidin-conjugated alkaline phosphatase and color substrates as above.
  • the open circles represent the net specific binding of Vn with a half-maximal binding concentration of Vn binding to fibrin calculated to be approximately 20 nM.
  • Fig. 22B is a graph showing the time course of binding 125 I-Vn (denatured) to fibrin in the presence or absence of excess cold.
  • Fig. 22C is a graph showing the time course of binding 125 I-Vn (native) in the presence or absence of excess cold.
  • Fig. 23 is an analysis of vitronectin mAB-mediated displacement of vitronectin from fibrin. Fibrin-coated microtiter wells were incubated with saturating concentrations of biotinylated Vn in the presence or absence of various purified monoclonal antibodies directed against different epitopes on Vn.
  • mAB 1244 is directed against an epitope in the amino-terminal half of the protein which does not interfere with the binding of Vn to PAI-l.
  • Monoclonal antibody mAB 153 is directed against binding site in the somatomedian B domain in the amino-terminus (residues 1-40) of Vn.
  • Monoclonal antibody mAB 8E6 is directed against an epitope in the amino-terminal half (residues 1-256) of vitronectin and inhibits PAI-l binding.
  • NM-IgG is preim une, normal mouse IgG.
  • Both mABs 153 and 8E6 are directed against anionic and cationic regions respectively, on vitronectin which are believed to be involved in vitronectin multimerization.
  • Fig. 24 is an analysis of the displacement of vitronectin bound to fibrin by purified vimentin peptides.
  • the saturating concentrations of biotinylated-Vn were incubated in the presence (+PAI-1) or the absence (-PAI-1) of PAI-l and then added to fibrin-coated microtiter wells. After 1 hr the wells were washed and then incubated for 1 hr with various concentrations of the vimentin 133 mer (VM 133) , synthetic vimentin 28 (VM 28) , or with the synthetic scrambled peptide (SCR) .
  • the bo ⁇ nd biotinylated-Vn was then determined using streptavidin alkaline phosphatase and color substrate. Note that the VIM 28 mer peptide, and to a lesser extent the VIM 133 mer, were very effective in displacing Vn bound to fibrin and particularly Vn bound to PAI-l.
  • Fig. 25A is an analysis of the displacement of BAI-1 associated w.
  • Purified fibrin-coated microtiter wells were coated with saturating concentrations of purified Vn and PAI-l pre ⁇ formed complexes, or equi olar purified PAI-l for 1 hr at 37°C. After washing, the plates were then incubated for 1 hr in buffer containing various concentrations of the VIM 133 mer peptide. The residual functionally active PAI-l was directly detected using 125 I-MAI 12 IgG.
  • This monoclonal antibody is selective for active PAI-l and is directed against an epitope within the carboxy-terminus of PAI-l. Note that only in the presence of Vn, there is a vimentin 133 mer dose-dependent decrease in the amount of active PAI-l present on the fibrin clot.
  • Fig. 25B illustrates dose-dependent enhancement of fibrinolysis by displacement of the vitronectin/PAI-1 complexes with the vimentin 133 mer peptide.
  • Purified 125 ⁇ - labelled fibrin coated wells were incubated with saturating concentrations of purified Vn and PAI-l, or Vn alone for 1 h at 37°C. After washing, the wells were incubated with various concentrations of VIM 133 mer for 1 h.
  • Fig. 26A is an analysis of the displacement of fibrin-bound vitronectin by recombinant vimentin 28-mer peptide in the presence or absence of human plasma.
  • Purified fibrin-coated microtiter wells were pre-treated with saturating concentrations of biotinylated-Vn. After washing, the clots were incubated with various concentrations of the synthetic VIM 28-mer peptide in the presence or absence of 20% citrated human plasma and the bound biotinylated-Vn was detected with streptavidin-conjugated alkaline phosphatase as above. Note that the presence of 20% serum alone displaced approximately 25% and the addition of the vimentin 28 mer decreased the bound Vn by a further 25%.
  • Fig. 26B illustrates the doe-dependent prothrombolytic effects of the vimentin 133 mer peptide (VM- 133) on whole platelet-rich plasma clot lysis in 96-well microtiter plates.
  • Fig. 27 is a schematic representation of proposed interactions between different conformations of Vn with PAI-l, fibrin, and vimentin.
  • Fig. 28 is a graph illustrating the inhibition of PAI-l activity in the presence of plasminogen, tPA, or plasmin chromogenic substrate and a synthetic 14-mer peptide comprising amino acids 296-308 of the tPA light chain (NH-Ile Phe Ala Lys His Arg Arg Ser Pro Gly Gly Arg Phe Leu-COOH [SEQ ID No. :2] .
  • Fig. 29A is a graph illustrating inhibition of PAI-l binding to Vn in the presence of mABs 153 and 8E6. Microtiter plate wells were coated overnight with urea-treated Vn (1 ⁇ g/ml, 0.05 ml).
  • PAI-l bound is expressed as a percentage of the PAI-l bound to Vn in the absence of the competing mABs.
  • Fig. 29B is a graph illustrating the reduction of PAI-l activity in the presence of mABs 153 and 8E6.
  • Microtiter plate wells were coated overnight with urea-treated Vn (1 ⁇ g/ml, 0.05 ml). After washing, the wells were blocked (3% BSA in PBS, pH 7.4) and incubated with increasing concentrations of PAI-l in the presence of 5 ⁇ g/ml of mAB 153 (closed circles) , mAB 8E6 (open triangles) or buffer alone (open circles) .
  • Fig. 30 is a graph showing the effects of anti-Vn mABs on PAI-l binding to native Vn.
  • Microtiter plate wells were coated overnight with native Vn (2 ⁇ g/ml, 0.05 ml). After washing, the wells were blocked (3%, BSA in PBS, pH 7.4) and incubated with PAI-l (2 ⁇ g/ml) in the presence of increasing concentrations of either mABs 153 (open circles) or 8E6 (closed circles) .
  • Bound PAI-l was detected with biotin- conjugated, affinity-purified rabbit anti-human PAI-l IgG and 125 I-streptavidin. The amount of PAI-l bound is expressed as a percentage of the PAI-l bound to Vn in the absence of the competing mABs.
  • Fig. 31A and 3IB showing the availability of different PAI-l binding sites on Vn.
  • microtiter wells were coated overnight with either mAB 153 (open symbols) or 8E6 (closed symbols) .
  • the wells were blocked and then incubated for 1 hr with excess (2.0 ⁇ g/ml) urea-treated Vn (triangles) and native Vn (circles) .
  • excess Vn was detected using biotin-conjugated, affinity-purified rabbit anti-human Vn IgG and streptavidin-conjugated alkaline- phosphatase/pNPP substrate.
  • Vn The specific binding of Vn was determined by subtracting the change in absorbance at 405 nm in control wells coated with BSA alone.
  • Fig. 3IB microtiter wells were coated overnight with either mAB 153 (open symbols) or 8E6 (closed symbols) . After blocking, the wells were incubated for 1 hr with urea-treated (circles) and native Vn (triangles) . After washing, the wells were incubated with increasing concentrations of PAI-l for 45 min and the bound PAI-l detected with biotin-conjugated, affinity- purified rabbit anti-human PAI-l IgG and 125 I-streptavidin.
  • Fig. 32 is a graph showing the availability of PAI-l binding sites following binding of urea-treated Vn with immobilized mABs 153 and 8E6.
  • Microtiter wells were coated overnight with either mAB 153 (open circles) or mAB 8E6 (closed circles). After blocking (3% BSA in PBS, pH 7.4) and incubated with urea-treated Vn (2 ⁇ g/ml) and after washing the unbound vitronectin the wells were then incubated with PAI-l (2 ⁇ g/nl) in the presence of increasing concentrations of the opposite mAB.
  • the amount of PAI-l bound is expressed as a percentage of the PAI-l bound to immobilized mAB 153 or 8E6 in the absence of the second competing mAB.
  • Figs. 33A and 33B are graphs showing the stoichiometry of PAI-l binding to urea-treated and native Vn.
  • Fig. 33A microtiter wells were coated overnight with affinity-purified, rabbit polyclonal anti-human Vn IgG (2 ⁇ g/ml) . After blocking, the wells were incubated for
  • the wells were incubated with increasing concentrations of either urea-treated (open circles) or native (closed circles) Vn for 45 min at 37°C. After washing the wells were incubated for 1 hr with biotin-conjugated, affinity-purified rabbit anti-human PAI-l IgG and 125 ⁇ - streptavidin conjugated to alkaline phosphatase as described above.
  • the present invention provides methods and compositions for inhibiting the accumulation of type 1 plasminogen activator inhibitor (PAI-l) in a ternary complex which is present in preexisting or newly forming clot in a blood vessel or which is formed as a result of cell damage, particularly to cells which line the blood vessels, e.g., as the result of angioplasty.
  • PAI-l type 1 plasminogen activator inhibitor
  • PAI-l is a potent inhibitor of plasminogen activators, such as tissue plasminogen activators (tPA) and urokinase, which are responsible for the conversion of plasminogen to plasmin, where plasmin in turn is responsible for the degradation of fibrin in a thrombus or clot.
  • tPA tissue plasminogen activators
  • urokinase urokinase
  • Fibrin is the major component of clot or thrombus, and the present invention relies on inhibition of PAI-l accumulation at sites of preexisting clot and/or vascular injury to restore the activity of endogenous and (if present) exogenous plasminogen activators, thus increasing the degradation (fibrinolysis) and/or reducing the formation of thrombus or clot.
  • the methods and compositions of the present invention are useful in in vitro systems.
  • the present invention provides in vitro assays and test systems for determining the ability of a test compound to inhibit the formation of the ternary complex and/or binding between particular components of the ternary complex.
  • Screening assays are run by exposing a test compound to an aqueous environment including at least two components of the ternary complex and preferably the entire complex.
  • the complex will usually be immobilized on a solid phase, usually a plastic surface such as a microtiter well.
  • isolated tissue or cells from blood vessels may be introduced to and/or cultured on the solid phase, and formation of the ternary complex induced.
  • test may then be run by measuring the ability of the test compound to inhibit formation of the complex in the aqueous cellular environment.
  • assays and test systems are useful for identifying test substances which are suitable for further testing as drugs for treating patients suffering from thrombus-related conditions.
  • a damaged cell (Fig. IA) exposes intracellular components to plasma proteins and other substances present in the blood.
  • vimentin an insoluble intermediate filament cytoskeleton component
  • plasma constituents such as complement, fibrinogen, or immunoglobulins.
  • exposed intracellular vimentin binds vitronectin
  • PAI-l acts to prevent or inhibit clot breakdown, and the clot can cause narrowing of blood vessel lumen.
  • the binding of the vimentin, vitronectin, and PAI-l results in a ternary complex where the vitronectin binds as an intermediate to both the vimentin and the PAI-l, as illustrated in Fig. 2.
  • the binding sites of the vimentin and the PAI-l are distinct, as illustrated.
  • a ternary complex comprising PAI-l and vitronectin also forms by binding to fibrin located in preexisting clot in blood vessels and elsewhere.
  • PAI-l bound to vitronectin and fibrin within preexisting clot has a substantially greater activity than unbound PAI-l, as discussed in detail in the Experimental section hereinafter.
  • endogenous and administered fibrinolytic agents such as tPA, urokinase, and the like.
  • Vitronectin is a 78 kD adhesive glycoprotein which is produced in the liver and released into the blood circulation. Vitronectin is also known as complement "S- protein". Vitronectin has at least four distinct binding domains of interest, including a heparin-binding domain whi'ch contains the plasmin/plasminogen-binding domain, and three PAI-1-binding domains. The structure of vitronectin is shown in Fig. 3, including three amino acid sequences (including amino acids 1-40, 115-121, and 348-379) which are presently believed to contribute to PAI-1-binding, and is described in Seiffert et al. (1994), supra.; Preissner et al. (1990), supra .
  • vitronectin binding between vitronectin and PAI-l can be inhibited by antibodies, such as mAB 153 and mAB 8E6 directed against the amino-terminal and carboxy-terminal binding region, respectively, as demonstrated in the Experimental section hereinafter.
  • antibodies such as mAB 153 and mAB 8E6 directed against the amino-terminal and carboxy-terminal binding region, respectively, as demonstrated in the Experimental section hereinafter.
  • Other characteristics of vitronectin are well described in the literature. Vitronectin is present in plasma at concentrations of 200-400 ⁇ g/ml and as both a monomeric (>95%) and in various multimer ( ⁇ 5%) forms from dimers, trimers up to 18 mer of molecular weights up approximately 1200 kDa.
  • PAI-l circulates with vitronectin multimers (dimer-tetrameres) . It is believed that the multimeric form is that form of vitronectin associated with matices and solid surfaces and is believed to be the most functionally active form with regards to binding to antithrombin Ill-thrombin complexes, complement, cell surfaces and PAI-l. Multimerization is believed to be mediated by electrostatic interactions between adjacent anionic amino-termini and cationic carboxy-terminal heparin binding domain of overlapping monomeric subunits and these multimers can be further stabilized by disulfide exchange. See, e .g.
  • IFs vascular endothelial cells platelets, and smooth muscle cells.
  • a primary therapeutic use of the methods and compositions of the present invention is in the inhibition of PAI-l at vascular sites where vimentin will be the principal exposed IF.
  • Pathological fibrin accretion can occur in other tissues where other IF's, such as desmins, keratins, and the like, may be exposed by cellular damage and which may provide a site for formation of the ternary complex.
  • the remaining disclosure will be directed specifically at vascular therapy and reference will be made to vimentin.
  • the invention also includes treatment of other tissue types, such as heart, lung, liver, brain, skin, and the like, where other IF's may be responsible for binding of the ternary complex the ternary complex.
  • PAI-l is an approximately 50 kd (379 amino acid) serine protease inhibitor of the serpin gene family.
  • PAI-l is produced by a variety of cells, particularly endothelial and smooth muscle cells, and is a fast-acting inhibitor of plasminogen activators, including tissue plasminogen activator (tPA) and urokinase (uPA) .
  • tPA tissue plasminogen activator
  • uPA urokinase
  • PAI-l has a binding domain for heparin and vitronectin comprising particular residues among amino acids 55-123 which are exposed on the surface of the folded molecule, and a reactive center which inhibits plasminogen activator activity, as illustrated in Fig. 5.
  • fibrin refers to polymerized fibrin monomer which accumulates in blood clot as a result of the endogenous clotting cascade. Briefly, fibrinogen hydrolyzes in the presence of thrombin into fibrin and fibrinopeptide fragments. Initially, fibrin forms soft clots which can be readily dispersed. Over time, thrombin activates fibrin-stabilizing factor and the fibrin is cross-linked, resulting in hardened clot, often referred to as plaque.
  • Methods and compositions of the present invention will act by reducing the presence and/or activity of PAI-l in clot (as well as at sites of vascular injury) , which in turn will increase the fibrinolytic activity of endogenous and administered fibrinolytic agents, thus reducing the presence and accumulation of fibrin in both soft and hardened clots.
  • the present invention relies on methods and compositions which inhibit the accumulation of PAI-l, typically at vascular injury sites where intracellular vimentin has been exposed as a result of the rupture of endothelial cells, platelets, and/or smooth muscle cells.
  • Such inhibition is achieved by exposing a vascular lumen or other aqueous cellular environment to a substance which blocks or competes with binding between at least two of the three components of the ternary complex described above.
  • binding between PAI-l and vitronectin can be blocked, inhibited, or displaced by introducing a substance to the vascular or other aqueous cellular environment which binds to PAI-l or vitronectin in a manner which blocks or sterically inhibits binding between the two components.
  • the substance may bind to PAI-l, being selected from the group consisting of vitronectin fragments, vitronectin analogs, antibody and antibody fragments that bind to vitronectin-binding site on PAI-l, and antibody and antibody fragments that alter the conformation of PAI-l.
  • the substance may bind to vitronectin, being selected from the group consisting of PAI-l fragments, PAI-l analogs, antibody and antibody fragments that bind to a PAI-1-binding site on vitronectin, and antibody and antibody fragments that alter the conformation of vitronectin.
  • a binding affinity of at least 10 5 M -1 will be sufficient to prevent initial binding, while a higher affinity of at least 10 7 M -1 will be sufficient to disrupt established equilibrium binding in a formed ternary complex.
  • Such substances will preferably have binding affinities of at least 10 7 M "1 , more preferably being at least 10 8 M "1 , and still more preferably being at least 10 9 M "1 , with higher affinity substances being capable of both blocking initial binding and disrupting established equilibrium binding in formed ternary complexes.
  • Substances useful in the present invention may also be selected to block initial binding between the binary complex of PAI-l and vitronectin to the intracellular vimentin or to disrupt established binding between the binary complex and the vimentin in a formed ternary complex.
  • the substance may bind to the vimentin-binding site on vitronectin, being selected from the group consisting of vimentin fragments, vimentin analogs, antibody and antibody fragments that bind to a vimentin-binding site on vitronectin, and antibody and antibody fragments that alter the conformation of vitronectin in such a way that binding to vimentin is inhibited or disrupted.
  • the substance may bind to the vitronectin-binding site on vimentin, being selected from the group consisting of vitronectin fragments, vitronectin analogs, antibody and antibody fragments that bind to a vitronectin-binding site on vimentin, and antibody and antibody fragments that alter the conformation of vimentin in such a way that blocks or disrupts binding to vitronectin.
  • the substance may bind to the fibrin binding site on vitronectin, being selected from a group consisting of fibrin fragments, fibrin analogs, antibodies or antibody fragments which bind to the fibrin binding site on vitronectin which may alter its conformation in such a way to block or disrupt the binding interaction of vitronectin with vimentin.
  • the binding affinities of such vitronectin-binding and vimentin-binding substances will generally be at least 10 5 M "1 , preferably being at least 10 7 M "1 , more preferably being at least 10 8 M "1 , and most preferably being at least 10 9 M _1 .
  • binding substances having lower affinities generally from 10 5 M _1 to 10 7 M -1 will be suitable for blocking initial binding between vimentin and the binary complex of vitronectin and PAI-l.
  • Disrupting established equilibrium binding between the binary complex and vimentin in formed ternary complexes will generally require higher affinities, usually greater than 10 7 M -1 .
  • Substances having higher binding affinities usually above 10 7 M "1 are preferable since they will generally be able to both block initial binding between the binary complex and vimentin as well as to disrupt equilibrium binding between the binary complex and vimentin in the formed complexes.
  • Substances useful in the present invention may further be selected to block initial binding between the binary complex PAI-l and vitronectin to fibrin present in existing clot, thrombus, plaque, and the like, or to disrupt established binding between the binary complex and fibrin in a formed ternary complex.
  • a substance may bind to the fibrin-binding site on vitronectin, being selected from the group consisting of vimentin fragments, vimentin analogs, fibrin fragments, fibrin analogs, antibody and antibody fragments that bind to a fibrin-binding site on vitronectin, and antibody and antibody fragments that alter the conformation of vitronectin in such a way that binding to fibrin is inhibited or disrupted.
  • the substance may bind to the vimentin binding site on vitronectin, being selected from a group consisting of vimentin fragments, vimentin analogs, antibodies or antibody fragments which bind to the vimentin binding site on vitronectin, and other substances which may alter the conformation of vitronectin and block or disrupt its binding to fibrin.
  • the substance may bind to the vitronectin-binding site on fibrin, being selected from the group consisting of vitronectin fragments, vitronectin analogs, antibody and antibody fragments that bind to a vitronectin-binding site on fibrin, and antibody and antibody fragments that alter the conformation of fibrin in such a way that blocks or disrupts binding of the binary complex of vitronectin and PAI-l.
  • the binding affinities of such vitronectin-binding and fibrin-binding substances will generally be at least 10 5 M -1 , preferably being at least 10 7 M "1 , more preferably being at least 10 8 M _1 , and most preferably being at least 10 9 M "1 .
  • binding substances having lower affinities generally from 10 5 M “1 to 10 7 M -1 will be suitable for blocking initial binding between fibrin and the binary complex of vitronectin and PAI-l. Disrupting established equilibrium binding between the binary complex and fibrin in a formed ternary complex will generally require binding substances having affinities, usually greater that 10 7 M -1 .
  • Substances having higher binding affinities are preferably since they will generally be able to both block initial binding between the binary complex and fibrin as well as to disrupt equilibrium binding between the binary complex and fibrin in formed complexes.
  • Fragments and analogs of vitronectin, PAI-l, fibrin and vimentin will be selected to have a suitable binding affinity, as described above, so that they can block or displace binding between native components of the ternary complex. It will of course be necessary that the fragments and analogs not be so closely related to the native molecules that the fragments and analogs will actively participate in the fibrinolytic system.
  • the fragments and analogs of the present invention should display binding specificity to the target component which is generally comparable to or greater than that of the native component, but which lack at least some of the other native activity or activities which are responsible for the inhibition of plasminogen activators.
  • suitable vitronectin, PAI-l, fibrin and vimentin fragments may be polypeptides which comprise or consist essentially of the target-binding region or domain of the native molecule (i.e., the region or domain which binds to the target molecule) , but which lack some or all of the other regions of the molecule.
  • Exemplary vitronectin fragments binding to PAI-l may comprise portions or all of fragments including amino acids 1-40 and 348-379 which are brought together by folding of the molecule, as illustrated in Fig. 4. Fragments comprising amino acids 115-121 of the putative binding region (Fig. 3) presently does not appear to play a significant role in PAI-l binding and are less likely suitable for use in the present invention. These fragments may be joined by the natural molecular linking region or by any other linking sequence which maintains the binding fragments in proper orientation for binding to the vitronectin-binding region of PAI-l with the requisite affinity.
  • Exemplary vitronectin fragments binding to vimentin may comprise amino acids 40-348 (except RGD) and 40-379 to the C-terminus.
  • Exemplary PAI-l fragments binding to vitronectin may comprise portions or all of fragments including amino acids 55-123, as illustrated in Fig. 5.
  • Exemplary vimentin fragments binding to vitronectin may comprise all or portions of the 78 amino acid amino-terminal thrombin cleavage fragment of vimentin. In all cases, it will be appreciated that the fragments should include a sufficient number of amino acids so that the three-dimensional structure and binding properties of the intact molecule are retained sufficiently to preserve the desired binding characteristics.
  • testing could be performed by immobilizing or solubilizing any one or two of the components and screening for the ability of test compounds to inhibit formation of the ternary complex in the presence of the remaining components of the complex. Specific testing techniques are described in detail in the Experimental section hereinafter.
  • An exemplary vimentin fragment which has been found to disrupt the binding of the binary complex of vitronectin and PAI-l to fibrin in clots comprises amino acid residues 51 through 78 of vimentin, according to the numbering of SEQ ID No.:l.
  • the vimentin-binding site on vitronectin is the same as, sufficiently close to, or of greater affinity than the fibrin-binding site to block binding of vitronectin to PAI-l.
  • Such peptidic analogs can be prepared by conventional solid phase synthesis or recombinant techniques, as are well described in the patent and scientific literature.
  • Solid-phase synthesis techniques are based on the sequential addition of amino acid residues to a growing chain on a solid- phase substrate, as first described by Merrifield (1963) J. AM. CHEM. SOC. 85:2149-2156.
  • Commercial systems for automated solid-phase synthesis are now widely available from suppliers, such as Applied BioSystems, Inc., Foster City, California.
  • Recombinant polypeptide production techniques are widely described in the technical and scientific literature. See, for example, MOLECULAR CLONING: A LABORATORY MANUAL, Sambrook et al., Eds., Cold Spring Harbor Press, Cold Spring Harbor, New York (1989) Vol. 1-3.
  • Analogs of the binding sites of vitronectin, PAI-l, fibrin, and vimentin may also be prepared as small molecule mimetics.
  • Small molecule mimetics are non-peptidic molecules, usually having molecular weight below 2 kD, more usually below 1 kd, and frequently in the range from 300 D to 1 kD, with structures which may be derived using techniques well known to those working in the area of drug design. Such techniques include, but are not limited to, self-consistent field (SCF) analysis, configuration interaction (CI) analysis, and normal mode dynamics computer programs, all of which are now readily available.
  • SCF self-consistent field
  • CI configuration interaction
  • normal mode dynamics computer programs all of which are now readily available.
  • Antibodies and antibody fragments which bind to the specified binding sites (epitopes) on vitronectin, PAI-l, fibrin and vimentin, as described above, may be prepared by conventional techniques, typically using each of these molecules as an immunogen. Specific techniques for preparing polyclonal and monoclonal antibodies are well described in the scientific and patent literature. See, for example, ANTIBODIES: A LABORATORY MANUAL, Harlow and Lane, Eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, (1988) . Once an antibody is prepared, its sequence may be determined and peptidic analogs and mimetic compounds prepared based on such a sequence using the techniques described above.
  • preparation of antibodies to vitronectin, PAI-l vimentin or fibrin may allow for isolation of specific or randomly selected cDNA sequences from the total DNA of the immunized animals spleen cells. Once the sequence is determined to an antibody which interacts with any of these components of the ternary complexes, recombinant antibody fragments or chimerics may be synthesized and "humanized" as described in the scientific patent literature.
  • Compositions according to the present invention will also include multivalent, usually bivalent, PAI-l inhibitors comprising a hybrid molecule having a first moiety which binds to PAI-l and inhibits binding of PAI-l to vitronectin and a second moiety which binds to the reactive center on PAI-l (Fig. 6A) and inhibits plasminogen activator inhibition activity.
  • the first moiety may comprise any of the PAI-1-' binding substances described above, or substances (such as VIM 133 described in the Experimental section) which bind the Vn moiety of the Vn-PAI-1 complex (Fig. 6B) , while the second moiety will be selected to bind to, block or sterically hinder the reactive center on PAI-l.
  • Suitable second moieties include the sequences that include the active sites on tPA, urokinase, thrombin, or portions thereof. Also useful would be oligopeptides, as described in JP 5032695, and EP 320 840, which block binding between PAI-l and tPA.
  • a bivalent molecule BV comprises a first moiety Ml and a second moiety M2 joined by a linking region L.
  • the linking region or group is selected to provide the necessary covalent bridge between the first binding moiety and the second binding moiety.
  • the linking region will be derived from a bifunctional compound having a reactive group at one end which is capable of binding to the first binding moiety and second reactive group at the second end which is capable of binding to the second binding moiety.
  • the linking region may be synthesized together with either the first binding moiety, the second binding moiety, or both, and will include only a single reactive functionality for covalent binding therebetween.
  • linking region is not critical, but it should provide sufficient spacing and flexibility between the two binding moieties so that the first binding moiety Ml may bind to the vitronectin-binding site on PAI-l or to the vimentin binding site on vitronectin (Fig. 6B) and the second binding moiety M2 may bind to the reactive center, as illustrated in Figs. 6A and 6B.
  • the length of the linking region will usually be between 5 A and 50 A, preferably being between 5 A and 15 A.
  • the linking region should be resistant to degradation when administered to a patient as part of a therapy as according to the present invention and further should not contribute to nonspecific adhesion of the hybrid molecule, i.e., adhesion or binding to other than the vitronectin binding site and reactive center on PAI-l. It will be appreciated that the hybrid molecules should also display no or minimum immunogenicity so that the drugs they comprise may be administered to a patient over an extended ' period of time without initiating an undesirable immune reaction.
  • compositions according to the present invention comprise a substance which binds to PAI-l, vitronectin, fibrin, or vimentin, with a specificity and affinity as described above, present in a pharmaceutical carrier.
  • Such compositions will be useful to treat or prevent thrombus-related cardiovascular conditions, as described above, and may be administered by themselves or in combination with anticoagulants and/or exogenous fibrinolytic agents, such as tPA, streptokinase, urokinase, and the like.
  • the pharmaceutical composition will find particular use with concurrent intravascular interventions which may cause injury to arterial endothelial and/or smooth muscle cells, such as angioplasty, atherectomy, laser ablation, ultrasound ablation, rotational ablation, and the like.
  • Suitable pharmaceutical compositions will contain a therapeutic amount of the binding substance present in a pharmaceutically acceptable carrier.
  • a therapeutic amount it is meant that sufficient substance will be present in order to inhibit formation of the ternary complex or promote degradation of formed ternary complexes, generally as described above.
  • substances will be present in a pharmaceutical composition in a concentration of from about 0.01 ⁇ g per dose to 10 mg per dose, usually being in the range from 1 ⁇ g per dose to 1 mg per dose.
  • Daily dosages may vary widely, depending on the activity of the particular substance, usually being between about 1 ⁇ g per kg of body weight to about 5 mg per kg of body weight per day usually being from about 10 ⁇ g per kg of body to about 1 mg per kg of body weight per day.
  • the pharmaceutically acceptable carrier can be any compatible, non-toxic substance suitable to deliver the ternary complex- inhibiting or displacing substance to the patient.
  • Sterile water, alcohol, fats, waxes and inert solids may be used as the carrier.
  • Pharmaceutically acceptable adjuvants, buffering agents, dispersing agents and the like may also be incorporated into the pharmaceutical compositions.
  • Such compositions will be suitable for oral, nasal, transdermal, pulmonary, and/or parenteral administration, preferable being suitable for parenteral administration, i.e., subcutaneous, intravascular, and intravenous administration.
  • parenteral administration i.e., subcutaneous, intravascular, and intravenous administration.
  • Triton X-100, caprylic acid, normal rabbit immunoglobulin (IgG) and isotype- matched, non-specific mouse IgG were obtained from Sigma Chemical Co. (St. Louis, MO) .
  • the monoclonal antibody to the heparin binding domain in vitronectin (clone 8E6) was obtained from Boehringer Mannheim (Montreal, Que.).
  • the monoclonal antibody to the somatomedian B domain of vitronectin (clone 153) was kindly provided by Dr. D. Seiffert. Vimentin isolated from bovine lens tissue was obtained from Boehringer Mannheim (Montreal, Que.).
  • Vitronectin was isolated by heparin-affinity chromatography from human serum as described by Yatohgo, T., et al. (1988) CELL STRUCT. FUNCTION 13:281-292 and by affinity chromotography using affinity purified sheep anti-human vitronectin IgG coupled to an Affigel resin.
  • Human PAI-l purified from the conditioned media of human HT 1080 fibrosarcoma cells and Spectrolyse PI chromogenic substrate was obtained from Biopool Inc. (Burlington, Ont.).
  • IgG fractions of monoclonal and polyclonal antibodies were prepared by caprylic acid precipitation (Hum, B.A., et al. :1980) METHODS IN ENZYMOLOGY 70:104-142).
  • Antisera to human PAI-l, human V ⁇ and bovine vimentin were raised in rabbits and sheep using Freund's Complete Adjuvant for the initial immunization (75 ⁇ g total) and incomplete Freund's adjuvant for subsequent booster injections.
  • the IgG fractions were further purified by affinity chromatography on immobilized PAI-l, Vn or vimentin, respectively.
  • PAI-l and Vn (1 mg) were coupled to a 1 ml bed-volume of cyanogen bromide- activated Sepharose 4B (Pharmacia, Sweden) as described by the manufacturer. Vimentin was coupled to 0.5 ml bed-volume of Affi-Gel 15 (Bio Rad Laboratories, Mississauga, Ont.) according to manufacturer's instructions.
  • Affinity purification of specific IgG fractions was performed by loading respective IgG fractions diluted in PBS, pH 7.4 onto equilibrated ligand affinity columns and washing out the nonspecifically bound IgG with 10 bed-volumes each of PBS, pH 7.4 with 0.5 M sodium chloride (NaCl), PBS, pH 7.4, 0.15 M NaCl and then unbuffered saline (0.15 M NaCl).
  • the specific IgG fractions were eluted with 5 bed-volumes of 0.1 M glycine buffer containing 0.15 M NaCl, pH 2.3 and the fractions (1 ml) collected into 0.2 ml of 0.5 M glycine, 0.75 M NaCl, pH 8.9.
  • the antibodies to PAI-l, Vn and vimentin were shown to be monospecific by immunoblot analysis of extracts from human and bovine endothelial cells or rat liver extracts.
  • Biotinylation of purified proteins Purified proteins (IgG, PAI-l, Vn, vimentin and BSA) were biotinylated with amino-hexanoyl-biotin-N-hydroxysuccinimide ester (AHNS; Boehringer Mannheim, Montreal, Que.). Briefly, 100 ⁇ g of protein was dialyzed against 0.1 M bicarbonate buffer, pH 8.4 and then incubated with 10 ⁇ g AHNS in dimethylformamide (25 mg/ l) for 4 h at room temperature. Samples of biotinylated PAI-l were dialyzed against 4 M guanidine hydrochloride as previously described (Hekman, CM., et al. (1988) ARCH.
  • AHNS amino-hexanoyl-biotin-N-hydroxysuccinimide ester
  • substrate-coated e.g., vitronectin or tPA
  • Bound PAI-l or Vn was detected directly with streptavidin-conjugated alkaline phosphatase or indirectly with rabbit anti-PAI-1 or anti-Vn IgG and goat anti-rabbit IgG-conjugated alkaline phosphatase followed by pNPP substrate and quantitation of absorbance at 405 n using a microtiter plate reader (EL 340, Bio-tek Instruments Inc., Highland Park, VT) . Biotinylation did not significantly interfere with the binding of PAI-l to immobilized Vn or to human recombinant tPA (Eli Lilly Co., Indianapolis, IN) . Microtiter plates having 96 wells were coated with vimentin or vitronectin by incubation over night at 4°C with 50 ⁇ l of a coating solution. Non-specific binding sites were blocked with bovine serum albumin (BSA) .
  • BSA bovine serum albumin
  • vitronectin to mediate binding between PAI-l and immobilized vimentin was demonstrated by incubating microtiter plates having immobilized vimentin (varying coating concentrations) with 20 nM of biotinylated PAI-l in the presence of increasing concentrations of vitronectin.
  • PAI-l concentrations were determined by the addition of streptavidin-conjugated alkaline phosphatase and pNPP substrate and measuring absorbency at 405 nm. Background binding of PAI-l to BSA-coated wells was subtracted. The results are set forth in Fig.
  • the wells were then incubated for 1 hour at 37°C with varying concentrations of affinity-purified anti- vimentin IgG or pre-immune rabbit IgG. After washing, the wells were incubated with biotinylated vitronectin (2 ⁇ g/ml) for 2 hours, and the amount of biotinylated vitronectin determined by measuring the change in absorbency at 405 nM after the addition of streptavidin-conjugated alkaline phosphatase and pNPP substrate. Background binding of vitronectin to BSA-coated wells was subtracted from the results. Referring to Fig.
  • Binding is expressed as the percentage of vitronectin binding to vimentin (mOD/min) in the presence of the vimentin-specific antibodies compared with binding in the presence of pre-immune rabbit IgG.
  • anti-vitronectin antibody to compete with binding of vitronectin to vimentin was confirmed as follows. Microtiter wells were coated with vimentin (lOOnM coating concentration) as described above. Varying concentrations of affinity-purified anti-vitronectin antibody or pre-immune rabbit IgG were pre-incubated for one hour with vitronectin (2 ⁇ g/ml) prior to addition of the vitronectin and antibody to the vimentin-coated wells for 2 hours at 37°C. After washing, the amount of biotinylated vitronectin was determined by measuring the change in absorbency at 405 nM after the addition of streptavidin-conjugated alkaline phosphatase and pNPP substrate.
  • FIG. 10 Background binding of the vitronectin to the BSA-coated wells was subtracted. Referring to Fig. 10, the decrease of relative binding in the presence of increasing antibody concentrations is illustrated. The binding is expressed as percentage of binding (mOD/min) in the presence of the vitronectin-specific antibody compared with binding in the presence of the pre-immune rabbit IgG.
  • the ability of anti-vitronectin antibody fragments to displace vitronectin from preformed vitronectin-vimentin complexes was confirmed as follows. Microtiter wells were coated with vimentin (lOOnM coating concentration) as described above. The wells were then incubated for 2 hours at 37°C with biotinylated vitronectin (2 ⁇ g/ml) .
  • the amount of bound biotinylated vitronectin was determined by measuring the change in absorbency at 405 nM after the addition of streptavidin-conjugated alkaline phosphatase and pNPP substrate. Background binding of biotinylated vitronectin to BSA-coated wells was subtracted. Referring to Fig. 11, it can be seen that the relative binding of vitronectin to vimentin decreases with increasing concentrations of the anti-vitronectin antibody fragment. Relative binding is expressed as percentage of vitronectin binding (mOD/min) in the presence of the anti-vitronectin antibody fragments compared with binding in the presence of the pre-immune rabbit antibody fragments.
  • Vn binds vimentin in the absence of PAI-l, but in the presence of functionally active PAI-l, there is a 5- to 10-fold increase in the Vn binding to vimentin.
  • Vimentin binds with high-affinity (Ki ⁇ 0.5 nM) to the more functionally active multimeric Vn, particularly multimeric Vn-PAI-1 complexes. In contrast, the binding of monomeric Vn to vimentin is extremely low or near absent.
  • soluble low molecular weight vimentin-derived peptides have been synthesized and used to competitively disrupt the binding of Vn-PAI-1 complexes to fibrin clots in vitro (see below) .
  • soluble vimentin peptides selectively bind the multimeric Vn-bound form of PAI-l, and can block or displace Vn-PAI-1 complexes from fibrin.
  • mAB V.9 monoclonal antibody directed against the carboxy-terminal rod domain
  • polyclonal antibodies directed against the amino-terminal head domain obtained from Dr.
  • bovine vimentin was cleaved using various purified proteinases, including thrombin and plasmin, and the resulting fragments analyzed by SDS-PAGE, Western and ligand blotting. These studies confirmed the fragment of bovine vimentin released from damaged BAEs contains the amino-terminus of vimentin.
  • RNA isolated umbilical vein endothelial cells was screened with specific oligonucleotide primers to develop variable length vimentin fragments using reverse transcription-polymerase chain reaction (RT-PCR) .
  • RT-PCR reverse transcription-polymerase chain reaction
  • the resulting cDNA fragments were cloned into pBLUESCRIPT and used to transform DH5 ⁇ E. coli ' cells. Colonies were screened by restriction enzyme digestion and positive clones were then sequenced as follows.
  • a 1.1 Kb fragment which represents the 3' end of the coding region was subcloned into pMAL-c2 to produce a carboxy terminus fusion protein with maltose binding protein (MBP, M, 40 kDa) (Fig. 12) .
  • MBP maltose binding protein
  • Fig. 12 Transformed E. coli were induced with IPTG, and screened using Western blots with a polyclonal anti- vimentin IgG and the monoclonal antibody (Mab V.9) directed against the carboxy-terminal "tail" domain of vimentin.
  • Purified MBP-vimentin protein was isolated using amylose resin. This MBP-vimentin fragment was detected with Mab V.9 IgG.
  • methylmercury hydroxide was used to denature the RNA, prior to RT-PCR using specific primers.
  • a 460 bp fragment was obtained, which was cloned into pBLUESCRIPT, and sequenced.
  • the plasmid DNA was then digested with EcoRIIXhoI , and subcloned into a modified pFL-1 vector which contained a heart muscle kinase recognition site (HMK) for endogenous 32 p- labelling of the expressed amino-terminus fragment.
  • HMK heart muscle kinase recognition site
  • a 1.1 Kb fragment (pMAL-c2-3'-vim) which represents approximately 80% of the vimentin coding sequence from the carboxy-terminal "tail” and central ⁇ -helical "rod” domain which does not bind biotinylated-Vn.
  • a 0.5 Kb fragment (pET-21(b)-5'-vim) which codes for the entire amino-terminal "head” domain including a 100 bp region which overlaps with the "rod” domain in pMAL-c2-3'-vim.
  • the 21 kDa amino-terminal fusion protein binds to biotinylated-Vn and competes for its binding to native VIM.
  • VIM 133 was used to produce an affinity purified sheep anti- vimentin IgG. Additional cleavage and epitope mapping studies with VIM 133 peptide narrowed the Vn binding site to a sequence between the (amino-terminal) major protein kinase C and A phosphorylation sites (residues 18-50) and a (carboxy- terminal) thrombin cleavage site at residue 78-79 (Fig. 15) . A 28 amino acid vimentin peptide (N-blocked) comprising residues 51-78 and a control scramble peptide which contains the same amino acids in random order were synthesized (95% purity) .
  • The- sequence of the entire 133 residue human vimentin peptide which encompasses the specific 28 amino acid (Residues 51-78) peptide is shown in SEQ ID No.:l.
  • the sequence begins at the N-terminal methionine of the signal peptide cleavage site and includes residues 1 though 133 (VIM 133) .
  • VIM 133 C. Scatchard Analysis of 125 I-Labelled Vitronectin Binding to Vimentin Fragment (VIM 133)
  • VIM 133 the dose-dependent coating efficiency and coating concentration of purified VIM 133 was determined using the 32 -P-HMK-labelled VIM 133 and by indirect immunologic methods.
  • Optimal coating of VIM 133 was achieved at a coating concentration of 10 ⁇ g/ml (>400 ng bound/well at approx. 80% coating eff.) and used throughout the subsequent studies.
  • Human Vn derived from outdated human plasma was purified by affinity chromatography using a resin coupled to affinity-purified sheep anti-human Vn IgG and was iodinated by the Bolton-Hunter method.
  • the predominantly monomeric form of Vn was converted to the predominantly multimeric form by incubation with 6 M urea (urea-treated Vn or denatured Vn) overnight at room temperature, dialyzed, and assessed by reduced and non-reduced SDS-PAGE and native polyacrylamide get electrophoresis.
  • Time course binding experiments indicate that, in contrast to multimeric Vn (Fig. 16A) , the affinity- purified monomeric Vn demonstrated no significant specific binding even after 2 hr incubation with VIM 133 coated surfaces (Fig. 16B) .
  • the monomeric form of 125 I-labelled Vn has little or no affinity for vimentin.
  • the multimeric form of Vn binds with a calculated association constant of approximately 0.5 nM (Fig. 17). This could be pathophysiologically and pharmacologically relevant if the multimeric form only represents approximately 3-6% of the total plasma Vn pool.
  • the relative concentration of Vn in a blood clot is approximately 2- to 3-fold greater than in whole blood.
  • the following tests were run to determine the nature and characteristics of Vn from the plasma and/or platelet pool incorporated into the clot. Immunofluorescence and three- dimensional confocal scanning laser microscopic image analysis of platelet-rich clots indicates that the majority of PAI-l is co-localized with Vn and the vimentin cytoskeleton of lysed platelets or fibrin fragments. However, there are also significant deposits of Vn associated with fibrin strands which is not always co-localized with PAI-l.
  • Vn antigen levels in serum corresponds to a 40-50% decrease in plasminogen levels, >95% decrease in fibrinogen levels and ⁇ 5% decrease in albumin (Fig. 18) .
  • Vn like plasminogen
  • fibrinogen is immobilized within the clot on specific binding sites which are generated during fibrin formation.
  • various concentrations of unlabelled Vn were incubated with excess fibrinogen (1 mg/ l) in the presence or absence of thrombin/calcium, the fibrin clot pelleted by centrifugation, and the Vn antigen levels in the pre- and post-clotting supernatants determined by an ELISA.
  • the amount of Vn bound to fibrin indicates dose-dependent binding which saturates at approximately 85 nM with dissociation constant of approximately 40 nM (Fig. 19) .
  • Aliquots of the pre- and post- clotting supernatants were subjected to non-reducing native, polyacrylamide gel electrophoresis followed by immunoblot analysis in order to visualize the monomeric and multimeric forms of Vn.
  • the starting solution of Vn was a mixture of conformations, although the monomeric Vn constituted approximately 30%-50% of the total (Fig. 20, "Pre" Panel) .
  • Vn conforms in the supernatants of clotted samples illustrate that the monomeric Vn conforms are depleted from the clot supernatant and that the excess predominately multimeric conform left unbound to fibrin in the fluid-phase (Fig. 9, "Post" Panel) is reduced.
  • fibrin in contrast to vimentin binds the monomeric conform of Vn, which represents >95% of the total plasma Vn pool.
  • Vn binding to fibrin 100,000 cpm of 25 I-labelled Vn was incubated with fibrinogen in a microfuge test tube containing TBS buffer (0.125 ml), a clot was formed by the addition of thrombin and the time-dependent diffusion of 125 I-labelled Vn from the hanging clot into the buffer containing either normal saline (Fig. 21, solid symbols) or 2.0 M NaCl (Fig. 21, open circles) was determined in aliquots by a gamma counter.
  • FIG. 22A A microtiter plate assay to detect the binding of Vn and Vn-PAI-1 complexes to fibrin clots via the specific binding of biotinylated Vn to fibrin coated wells is illustrated in Fig. 22A. Saturable binding is observed at 35 nM with a calculated half-maximal binding concentration of 15-20 nM. These numbers are generally in agreement with those obtained by t e hanging clot assay (above) ,and the differences observed may be due to the nature of the assays (i.e., fluid- phase (hanging clot) vs. solid-phase (microtiter wells)).
  • Vn binding to clots is increased in the presence of PAI-l, and significantly greater amounts of active PAI-l can be incorporated into the clot in the presence of Vn.
  • 2.0 M NaCl had little significant effect on the displacement of Vn from fibrin-coated microtiter wells.
  • the fibrin-binding region appears to be located on the amino-terminal half of Vn, close to the PAI-l binding site(s) .
  • Biotinylated monomeric Vn 50 nM was preincubated with various concentrations of normal mouse IgG (NM-IgG) , mAB 8E6, mAB 153 or mAB 1244 IgG followed by incubation in fibrin coated wells.
  • NM-IgG normal mouse IgG
  • mAB 8E6 mAB 153 or mAB 1244 IgG
  • Fig. 24 illustrates the displacement of fibrin- bound, biotinylated Vn following 1 hr incubation with the VIM 133 and 28-mer peptides.
  • PAI-l PAI-l
  • maximal displacement (reduction in MOD/min) of 40% bound Vn was achieved with 2.0 nM of the VIM 28-mer peptide while the VIM 133-mer displaced slightly greater Vn than the control, scrambled 27-mer peptide (SCR) .
  • SCR scrambled 27-mer peptide
  • both the VIM 133 and 27-mer peptides displaced maximum of 40-60% of the biotinylated Vn.
  • fibrin coated wells were preincubated with buffer containing PAI-l alone (Fig. 25A, open bars) or preformed Vn-PAI-1 complexes (Fig. 25A, solid bars) , the unbound ligands washed away, and the wells incubated for 1 hr with various concentrations of the VIM 133 peptide.
  • the residual PAI-l bound to fibrin was directly detected using 125 I-labelled MAI 12 IgG. In the presence of Vn and no peptide, there is approximately two-fold greater PAI-l bound to the clots as compared to PAI-l alone.
  • Figure 26A illustrates that the presence of plasma alone results in an approximately 25% decrease in the bound Vn and that the VIM 28-mer peptide displaced additional amounts of Vn in the presence of plasma compared to the buffer only controls. These data indicate that the vimentin peptide is more specific for the clot-bound Vn and that plasma Vn has a minimal effect on the ability of vimentin to bind clot-bound Vn.
  • Fig. 26B illustrates the dose-dependent prothrombolytic effects of the vimentin 133 mer peptide (VM- 133) on whole platelet-rich plasma clot lysis in 96-well microtiter plates.
  • Various doses of purified VM-133 mer peptide was preincubated in citrated platelet-rich human plasma containing tracer amounts of 125 I-fibrinogen, the plasma samples (total volume 0.085 ml) were then recalcificied and allowed to clot for one hour prior to the initiation of exogenous tPA mediated lysis as measurement of the release of soluble 125 I-fibrin degradation fragments into the surrounding buffer.
  • plasma samples were pretreated with MAI-12 IgG, an inhibitory mAB directed against the reactive center of PAI-l and, the negative controls were plasma samples pretreated with buffer only.
  • FIG. 27 illustrates a model of the proposed interactions between different conformations of Vn with PAI-l, fibrin and vimentin.
  • vimentin has a high affinity for multimeric Vn, particularly multimeric Vn-PAI-1 complexes.
  • fibrin appears to have a higher affinity for monomeric Vn but once this form of Vn is bound to fibrin, it can begin the multimerization process which may include PAI-l. This model may explain why the vimentin peptides can displace fibrin-bound Vn or Vn-PAI-1 complexes.
  • PAI-l inhibitor which enhances fibrinolytic activity by binding adjacent to the reactive center and negatively charged, carboxy-terminal strained-loop of PAI-l to inhibit PAI-l interactions with t-PA was developed as follows.
  • the VR-1 region of the t-PA light chain (residues 296-308) may be modified to eliminate inhibition of tPA by PAI-l. Madison et al. (1989) NATURE 339-721 and (1990) PROC. NATL. ACAD. SCI. USA 87:3530-3533.
  • a synthetic t-PA exosite I peptide (14 mer residues 296-308) and its scramble control peptide were synthesized. These peptides were tested using a plasminogen-dependent, tPA chromogenic substrate (S-2288) combined with the microtiter well-bound fibrin plate lysis assay. Fibrin-coated wells were pre-incubated in the presence of absence of Vn/PAI-1 complexes prior to the addition of tPA, plasminogen, the synthetic peptides, mAB-12, and S-2288.
  • S-2288 plasminogen-dependent, tPA chromogenic substrate
  • Vn i.e., monomeric versus multimeric
  • PAI-l a binding substrate for PAI-l
  • Vn Monoclonal antibodies were used to characterize various functional conformations of PAI-l and Vn.
  • the site of Vn which binds to the proposed heparin binding region of PAI-l has been reported to lie in both (1) the N-terminus somatomedian B domain and (2) an area contained within the middle region of the molecule at or near the peparin binding domain.
  • mABs 153 and 8E6 bound equivalent amounts of urea-treated Vn which in turn were found to bind equivalent amounts of PAI-l (Figs. 31A and 31B) .
  • mABs 153 and 8E6 were shown to recognize distinct PAI-l binding sites since PAI-l binding to mAB 153-immobilized Vn was inhibited 85 ⁇ 2% by addition of mAB 8E6, and PAI-l binding to mAB 8E6-immobilized Vn was inhibited 87 ⁇ 2% in the presence of mAB 153 (Fig. 32) .
  • Lys lie Leu Leu Ala 130

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Abstract

On inhibe la formation et l'extension de thrombus en administrant des substances qui diminuent l'accumulation de l'inhibiteur 1 des activateurs du plasminogène aux emplacement des lésions vasculaires. On a découvert que cet inhibiteur s'accumule dans une structure ternaire composée de vitronectine, de cet inhibiteur et d'une protéine fibrillaire choisie dans le groupe constitué par la vimentine et la fibrine. Des complexes circulants binaires de cet inhibiteur et de vitronectine peuvent entrer dans le caillot et/ou les cellules endothéliales ayant subi une lésion et se fixer à la protéine fibrillaire. L'inhibiteur, lorsqu'il est présent, inhibe les activateurs endogènes du plasminogène, ce qui permet une accumulation de fibrine et l'extension du caillot. Une diminution de la quantité d'inhibiteur présent permet donc aux substances fibrinolytiques endogènes ou administrées d'inhiber la formation de la fibrine et par voie de conséquence de limiter la formation et/ou l'extension de thrombus et de caillots.
PCT/CA1995/000279 1994-05-10 1995-05-10 Methodes et compositions pour augmenter l'activite fibrinolytique endogene WO1995030438A2 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6767540B2 (en) * 2000-01-14 2004-07-27 Tanox, Inc. Use of antagonists of plasminogen activator inhibitor-1 (PAI-1) for the treatment of asthma and chronic obstructive pulmonary disease
EP2566890A2 (fr) * 2010-05-03 2013-03-13 AbbVie Inc. Anticorps anti-pai-1 et leurs procédés d'utilisation

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EP0589181A3 (fr) * 1992-07-30 1995-02-22 Yeda Res & Dev Dérivés synthetiques de vitronectine et leur compositions pharmaceutiques.

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6767540B2 (en) * 2000-01-14 2004-07-27 Tanox, Inc. Use of antagonists of plasminogen activator inhibitor-1 (PAI-1) for the treatment of asthma and chronic obstructive pulmonary disease
EP2566890A2 (fr) * 2010-05-03 2013-03-13 AbbVie Inc. Anticorps anti-pai-1 et leurs procédés d'utilisation
EP2566890A4 (fr) * 2010-05-03 2013-11-20 Abbvie Inc Anticorps anti-pai-1 et leurs procédés d'utilisation

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