Peg-modified arginine/lysine oxidoreductase

Abstract

The present invention is directed to an arginine/lysine oxidoreductase modified with polyethylene glycol and to a production method thereof and to methods of treating disorders responsive to a modification of amino acid levels reactive oxygen species and/or ammonium.

1 . A conjugate comprising an arginine/lysine oxidoreductase and at least one polyethylene glycol moiety. 
     
     
         2 . The conjugate of  claim 1 , wherein the at least one polyethylene glycol moiety has a weight average molecular weight of from about 1,000 Daltons to about 10,000 Daltons. 
     
     
         3 . The conjugate of  claim 1 , wherein the at least one polyethylene glycol moiety is covalently coupled to the arginine/lysine oxidoreductase via a linking group. 
     
     
         4 . The conjugate of  claim 3 , wherein the linking group is a succinimide group. 
     
     
         5 . The conjugate of  claim 3 , wherein the at least one polyethylene glycol moiety is coupled via a lysine residue to the arginine/lysine oxidoreductase. 
     
     
         6 . The conjugate of  claim 1  comprising from 1 to about 30 polyethylene glycol moieties, preferably from 1 to about 10 polyethylene glycol moieties. 
     
     
         7 . The conjugate of  claim 1 , wherein the arginine/lysine oxidoreductase is an L-arginine/L-lysine oxidoreductase. 
     
     
         8 . The conjugate of  claim 1 , wherein the arginine/lysine oxidoreductase is isolated from a natural source. 
     
     
         9 . The conjugate of  claim 1 , wherein the arginine/lysine oxidoreductase is a recombinant arginine/lysine oxidoreductase. 
     
     
         10 . The conjugate of  claim 1 , wherein the arginine/lysine oxidoreductase is selected from the group consisting of:
 (a) an arginine/lysine oxidoreductase having the sequence of SEQ ID NO: 2, SEQ ID NO:4, SEQ ID NO:6, and/or SEQ ID NO: 8,   (b) a sequence which is at least 70% identical to the sequence of (a), or/and   (c) a fragment of (a) or (b).   
     
     
         11 . The conjugate of  claim 10 , wherein the fragment has a length of at least about 30 amino acid residues. 
     
     
         12 . The conjugate of  claim 1 , wherein the conjugate is active in the circulation of a subject for at least 6 hours. 
     
     
         13 . The conjugate of  claim 1 , wherein the conjugate has an activity which is a factor of at least 30 larger than the activity of an arginine/lysine oxidoreductase not carrying a polyethylene glycol moiety. 
     
     
         14 . A pharmaceutical composition comprising the conjugate of  claim 1  in combination with one or more pharmaceutically acceptable carriers, adjuvants, diluents and/or additives. 
     
     
         15 - 16 . (canceled) 
     
     
         17 . A method for the prevention, alleviation and/or treatment of a disease responsive to reactive oxygen species and/or to ammonium, and/or which is responsive to modulation of amino acid levels in body fluids, comprising administering an effective amount of the conjugate of  claim 1  to a subject in need thereof. 
     
     
         18 . A method of producing the conjugate of  claim 1 , comprising the steps of:
 (a) recombinantly expressing the arginine/lysine oxidoreductase and/or isolating the arginine/lysine oxidoreductase from a natural source, and   (b) coupling at least one polyethylene glycol moiety to the arginine/lysine oxidoreductase of (a).   
     
     
         19 . The method of  claim 18 , wherein step (a) comprises recombinantly expressing a nucleic acid selected from the group consisting of:
 (i) a nucleic acid having the sequence SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, and/or SEQ ID NO:7,   (ii) a nucleic acid having a sequence complementary to the sequence of (i),   (iii) a nucleic acid having a sequence within the scope of the degeneracy of the genetic code of the sequence of (i) or (ii),   (iv) a nucleic acid having a sequence which is at least 70% identical to the sequence of (i), (ii) and/or (iii),   (v) a nucleic acid having a sequence which hybridizes with any of the sequences (i), (ii), (iii) and/or (iv) under stringent conditions, and   (vi) a nucleic acid comprising a fragment of any of the sequences of (i), (ii), (iii), (iv), and/or (v).   
     
     
         20 . The method of  claim 19 , wherein the fragment has a length of at least 90 nucleotide residues. 
     
     
         21 . A kit comprising the conjugate of  claim 1 . 
     
     
         22 . A method for enhancing the enzymatic activity and/or circulation time of an arginine/lysine oxidoreductase, comprising coupling at least one polyethylene glycol moiety to the arginine/lysine oxidoreductase. 
     
     
         23 . A method for protecting an arginine/lysine oxidoreductase against inactivation in body fluids comprising coupling at least one polyethylene glycol moiety to the arginine/lysine oxidoreductase. 
     
     
         24 . (canceled) 
     
     
         25 . Use of the conjugate of  claim 1  for the depletion of lysine and/or arginine in a liquid and/or for the production of hydrogen peroxide, ammonium and/or metabolites of lysine and/or arginine in a liquid. 
     
     
         26 . Use of  claim 25 , wherein the liquid is a body fluid of a mammal. 
     
     
         27 . Use of at least one polyethylene glycol group for increasing the activity and/or circulation time of an arginine/lysine oxidoreductase. 
     
     
         28 . Use of at least one polyethylene glycol group for protecting an arginine/lysine oxidoreductase against inactivation in body fluids. 
     
     
         29 . The method of  claim 17 , wherein the disease is selected from the group consisting of microbial infections, viral infections, and proliferative diseases, and wherein the proliferative disease can be cancer. 
     
     
         30 . The method of  claim 17 , wherein the amino acid levels in body fluids are lysine or/and arginine levels in plasma.