Pharmaceutical Preparation and Method of Treatment of Human Malignancies with Arginine Deprivation

Abstract

The present invention provides an isolated and substantially purified recombinant human arginase having sufficiently high enzymatic activity and stability to maintain Adequate Arginine Depletion in a patient. The present invention also provides a pharmaceutical composition comprising the modified invention enzyme and method for treatment of diseases using the pharmaceutical composition.

1 . An isolated recombinant human arginase I, comprising substantially the same amino acid sequence as set forth in SEQ ID NO: 9 and having a purity of 80-100%. 
     
     
         2 . The recombinant human arginase I according to  claim 1  further comprising six histidines attached to the amino terminal end thereof. 
     
     
         3 . The recombinant human arginase I according to  claim 1  having a specific activity of at least 250 I.U./mg. 
     
     
         4 . The recombinant human arginase I according to  claim 3  having a specific activity of 500 to 600 I.U./mg. 
     
     
         5 . The recombinant human arginase I according to  claim 4 , comprising a modification that results in an in vitro plasma half-life of at least approximately 3 days. 
     
     
         6 . An isolated recombinant human arginase I according to  claim 1 , having a purity of at least 90%. 
     
     
         7 . The recombinant human arginase I according to  claim 5 , wherein said modification is pegylation. 
     
     
         8 . The recombinant human arginase I according to  claim 7 , wherein said pegylation results from covalently attaching at least one polyethylene glycol (PEG) moiety to said arginase using a coupling agent. 
     
     
         9 . The recombinant human arginase I according to  claim 8 , wherein said coupling agent is selected from the group consisting of 2,4,6-trichloro-s-triazine (cyanuric chloride, CC) and succinimide propionic acid (SPA). 
     
     
         10 . A pharmaceutical composition comprising an isolated and substantially purified arginase. 
     
     
         11 . The pharmaceutical composition according to  claim 10  wherein said recombinant human arginase is human arginase I. 
     
     
         12 . The pharmaceutical composition according to  claim 10  wherein said recombinant human arginase is human arginase I, further comprising six additional histidines attached to the amino terminal end thereof. 
     
     
         13 . The pharmaceutical composition according to  claim 10 , wherein said composition is further formulated in a pharmaceutically acceptable carrier. 
     
     
         14 . The pharmaceutical composition according to  claim 10 , wherein the formulation of said pharmaceutical composition is in a form suitable for oral use, for a sterile injectable solution or a sterile injectable suspension. 
     
     
         15 . The pharmaceutical composition according to  claim 10 , wherein said recombinant human arginase I has a specific enzyme activity of at least 250 I.U./mg. 
     
     
         16 . The pharmaceutical composition according to  claim 15 , wherein said recombinant human arginase I has a specific enzyme activity of 500 to 600 I.U./mg. 
     
     
         17 . The pharmaceutical composition according to  claim 16 , wherein said recombinant human arginase I has a half-life in patient plasma of at least 3 days. 
     
     
         18 . The pharmaceutical composition according to  claim 17 , wherein said recombinant human arginase I has a half-life in patient plasma of approximately at least 1 day.