Compositions and methods for inhibiting angiogenesis

Abstract

The invention provides for methods of reducing or inhibiting angiogenesis, tumor growth and endothelial cell proliferation by the administration of compositions containing fragments, conformations, biological equivalent, or derivatives of antithrombin III. The invention also provides for pharmaceutical compositions comprising a fragment, conformation, biological equivalent, or derivative of antithrombin III and methods of identifying novel inhibitors of tumor growth, endothelial cell proliferation, and/or angiogenesis. The invention also relates to compositions and methods for altering angiogenesis in a mammal, as well as to methods of treatment for disorders associated with angiogenesis (e.g., cancer).

We claim:  
     
         1 . A method of reducing angiogenesis in a mammal comprising delivering to said mammal a composition comprising 
 at least one fragment, conformation, biological equivalent, or derivative of antithrombin III    wherein said fragment, conformation, biological equivalent, or derivative of antithrombin III reduces angiogenesis.    
     
     
         2 . A method of reducing angiogenesis in a mammal according to  claim 1  wherein said composition further comprises a physiologically acceptable vehicle.  
     
     
         3 . A method of reducing angiogenesis in a mammal according to  claim 1  wherein said at least one fragment, conformation, biological equivalent, or derivative of antithrombin III is chosen from 
 the L form of antithrombin III and the R form of antithrombin III.  
 
     
     
         4 . A method of reducing angiogenesis in a mammal according to  claim 1  wherein said at least one fragment, conformation, biological equivalent, or derivative of antithrombin III is chosen from 
 a synthesized fragment of antithrombin III that reduces angiogenesis,  
 a conformational variation of serpins that reduce angiogenesis,  
 an aggregate form of antithrombin III that reduces angiogenesis, and  
 a fusion protein of antithrombin III that reduces angiogenesis.  
 
     
     
         5 . A method of reducing angiogenesis in a mammal according to  claim 1  wherein said conformational variation of serpins is chosen from conformational variations of plasminogen activator inhibitor-1, α 2  antiplasmin, α1 proteinase inhibitor, heparin cofactor II, C1 inhibitor, α1 antichymotrypsin, protease nexin 1 or pigment epithelial derived factor.  
     
     
         6 . A method of reducing angiogenesis in a mammal according to  claim 1  wherein said at least one fragment, conformation, biological equivalent, or derivative of antithrombin III is produced transgenically, recombinantly, or purified from mammal antithrombin III.  
     
     
         7 . A method of reducing angiogenesis in a mammal according to  claim 1  wherein said at least one fragment, conformation, biological equivalent, or derivative of antithrombin III is produced in vivo by the delivery of an enzyme.  
     
     
         8 . A method of reducing endothelial cell proliferation in a mammal comprising delivering to said mammal a composition comprising 
 at least one fragment, conformation, biological equivalent, or derivative of antithrombin III    wherein said fragment, conformation, biological equivalent, or derivative of antithrombin III reduces endothelial cell proliferation.    
     
     
         9 . A method of reducing endothelial cell proliferation in a mammal according to  claim 8  wherein said composition further comprises a physiologically acceptable vehicle.  
     
     
         10 . A method of reducing endothelial cell proliferation in a mammal according to  claim 8  wherein said at least one fragment, conformation, biological equivalent, or derivative of antithrombin III is chosen from 
 the L form of antithrombin III and the R form of antithrombin III.  
 
     
     
         11 . A method of reducing endothelial cell proliferation in a mammal according to  claim 8  wherein said at least one fragment, conformation, biological equivalent, or derivative of antithrombin III is chosen from 
 a synthesized fragment of antithrombin III that reduces endothelial cell proliferation,  
 a conformational variation of serpins that reduce endothelial cell proliferation,  
 an aggregate form of antithrombin III that reduces endothelial cell proliferation, and  
 a fusion protein of antithrombin III that reduces endothelial cell proliferation.  
 
     
     
         12 . A method of reducing endothelial cell proliferation in a mammal according to  claim 8  wherein said conformational variation of serpins is chosen from conformational variations of plasminogen activator inhibitor-1, α 2  antiplasmin, α1 proteinase inhibitor, heparin cofactor II, C1 inhibitor, α1 antichymotrypsin, protease nexin 1 or pigment epithelial derived factor.  
     
     
         13 . A method of reducing endothelial cell proliferation in a mammal according to  claim 8  wherein said at least one fragment, conformation, biological equivalent, or derivative of antithrombin III is produced transgenically, recombinantly, or purified from mammal antithrombin III.  
     
     
         14 . A method of reducing endothelial cell proliferation in a mammal according to  claim 8  wherein said at least one fragment, conformation, biological equivalent, or derivative of antithrombin III is produced in vivo by the delivery an enzyme.  
     
     
         15 . A method of reducing tumor growth in a mammal comprising delivering to said mammal a composition comprising 
 at least one fragment, conformation, biological equivalent, or derivative of antithrombin III    wherein said fragment, conformation, biological equivalent, or derivative of antithrombin III reduces tumor growth.    
     
     
         16 . A method of reducing tumor growth in a mammal according to  claim 15  wherein said composition further comprises a physiologically acceptable vehicle.  
     
     
         17 . A method of reducing tumor growth in a mammal according to  claim 15  wherein said at least one fragment, conformation, biological equivalent, or derivative of antithrombin III is chosen from 
 the L form of antithrombin III and the R form of antithrombin III.  
 
     
     
         18 . A method of reducing tumor growth in a mammal according to  claim 15  wherein said at least one fragment, conformation, biological equivalent, or derivative of antithrombin III is chosen from 
 a synthesized fragment of antithrombin III that reduces endothelial cell proliferation,  
 a conformational variation of serpins that reduce endothelial cell proliferation,  
 an aggregate form of antithrombin III that reduces endothelial cell proliferation, and  
 a fusion protein of antithrombin III that reduces endothelial cell proliferation.  
 
     
     
         19 . A method of reducing tumor growth in a mammal according to  claim 15  wherein said conformational variation of serpins is chosen from conformational variations of plasminogen activator inhibitor-1, α 2  antiplasmin, α1 proteinase inhibitor, heparin cofactor II, C1 inhibitor, α1 antichymotrypsin, protease nexin 1 or pigment epithelial derived factor.  
     
     
         20 . A method of reducing tumor growth in a mammal according to  claim 15  wherein said at least one fragment, conformation, biological equivalent, or derivative of antithrombin III is produced transgenically, recombinantly, or purified from mammal antithrombin III.  
     
     
         21 . A method of reducing tumor growth in a mammal according to  claim 15  wherein said at least one fragment, conformation, biological equivalent, or derivative of antithrombin III is produced in vivo by the delivery of an enzyme.  
     
     
         22 . A method for identifying an agent that reduces tumor growth or angiogenesis comprising the steps of 
 a) inoculating a mammal with an inoculum of tumor cells in each of two inoculation sites substantially simultaneously;    b) identifying reduction of growth of a tumor at one inoculation site, wherein said tumor at the one inoculation site is a subordinate tumor, with concomitant growth of a tumor at the other inoculation site, wherein said tumor at the other inoculation site is a dominant tumor;    c) isolating cells from said dominant tumor; and    d) purifying a component which reduces endothelial cell proliferation, angiogenesis    or both endothelial cell proliferation and angiogenesis from the isolated dominant tumor cells.    
     
     
         23 . A method for identifying an agent that reduces tumor growth or angiogenesis according to  claim 22  wherein the tumor cells are derived from tumors selected from small cell lung cancers and hepatocellular carcinomas.  
     
     
         24 . A method for identifying an agent that reduces tumor growth or angiogenesis according to  claim 22  wherein the inoculation sites are the flanks of the mammal.  
     
     
         25 . A method for identifying an agent that reduces tumor growth or angiogenesis according to  claim 22  wherein the component of step (d) is purified from conditioned media from the cells of step (c).  
     
     
         26 . A method for identifying an agent that reduces tumor growth or angiogenesis according to  claim 22  wherein the agent that reduces tumor growth reduces endothelial cell proliferation.  
     
     
         27 . A method for identifying an agent that reduces tumor growth or angiogenesis according to  claim 22  wherein the agent that reduces tumor growth reduces angiogenesis.  
     
     
         28 . A method of reducing tumor growth comprising delivering to a mammal the agent that reduces tumor growth or angiogenesis identified by the method according to  claim 22 .  
     
     
         29 . A method of treating a disorder mediated by endothelial cell proliferation comprising delivering to a mammal a composition comprising at least one fragment, conformation, biological equivalent, or derivative of antithrombin III wherein said fragment, conformation, biological equivalent, or derivative of antithrombin III is delivered in an amount effective to reduce endothelial cell proliferation.  
     
     
         30 . A method of treating a disorder mediated by endothelial cell proliferation according to  claim 29  wherein said at least one fragment, conformation, biological equivalent, or derivative of antithrombin III is chosen from 
 the L form of antithrombin III and the R form of antithrombin III.  
 
     
     
         31 . A method of treating a disorder mediated by endothelial cell proliferation according to  claim 29  wherein said at least one fragment, conformation, biological equivalent, or derivative of antithrombin III is chosen from 
 a synthesized fragment of antithrombin III that reduces endothelial cell proliferation,  
 a conformational variations of serpins that reduce endothelial cell proliferation,  
 an aggregate form of antithrombin III that reduces endothelial cell proliferation, and  
 a fusion protein of antithrombin III that reduces endothelial cell proliferation.  
 
     
     
         32 . A method of treating a disorder mediated by endothelial cell proliferation according to  claim 29  wherein said conformational variation of serpins is chosen from conformational variations of plasminogen activator inhibitor-1, α 2  antiplasmin, α1 proteinase inhibitor, heparin cofactor II, C1 inhibitor, α1 antichymotrypsin, protease nexin 1 or pigment epithelial derived factor.  
     
     
         33 . A method of treating a disorder mediated by endothelial cell proliferation according to  claim 29  wherein said at least one fragment, conformation, biological equivalent, or derivative of antithrombin III is produced transgenically, recombinantly, or purified from mammal antithrombin III.  
     
     
         34 . A method of treating a disorder mediated by angiogenesis comprising delivering to a mammal a composition comprising at least one fragment, conformation, biological equivalent, or derivative of antithrombin III wherein said fragment, conformation, biological equivalent, or derivative of antithrombin III is delivered in an amount effective to reduce angiogenesis.  
     
     
         35 . A method of treating a disorder mediated by angiogenesis according to  claim 34  wherein said at least one fragment, conformation, biological equivalent, or derivative of antithrombin III is chosen from 
 the L form of antithrombin III and the R form of antithrombin III.  
 
     
     
         36 . A method of treating a disorder mediated by angiogenesis according to  claim 34  wherein said at least one fragment, conformation, biological equivalent, or derivative of antithrombin III is chosen from 
 a synthesized fragment of antithrombin III that reduces angiogenesis,  
 a conformational variation of serpins that reduce angiogenesis,  
 an aggregate form of antithrombin III that reduces angiogenesis, and  
 a fusion protein of antithrombin III that reduces angiogenesis.  
 
     
     
         37 . A method of treating a disorder mediated by angiogenesis according to  claim 34  wherein said conformational variation of serpins is chosen from conformational variations of plasminogen activator inhibitor-1, α 2  antiplasmin, α1 proteinase inhibitor, heparin cofactor II, C1 inhibitor, α1 antichymotrypsin, protease nexin 1 or pigment epithelial derived factor.  
     
     
         38 . A method of treating a disorder mediated by angiogenesis according to  claim 35  wherein said at least one fragment, conformation, biological equivalent, or derivative of antithrombin III is produced transgenically, recombinantly, or purified from mammal antithrombin III.  
     
     
         39 . A method of enhancing angiogenesis or endothelial cell proliferation comprising delivering a composition comprising at least one antagonist of a fragment, conformation, biological equivalent, or derivative of antithrombin III to a mammal wherein said fragment, conformation, biological equivalent, or derivative of antithrombin III reduces angiogenesis.  
     
     
         40 . A anti-angiogenic pharmaceutical composition comprising 
 a purified form of antithrombin III that reduces angiogenesis chosen from the (R) form of antithrombin III or the (L) form of antithrombin III, and    a physiologically acceptable carrier.