Method of treating certain indications associated with hyperglycemia

Abstract

The present invention relates to compositions and methods for inhibiting and reversing nonenzymatic cross-linking (protein aging). Accordingly, compositions are disclosed which comprise an agent capable of inhibiting the formation of advanced glycosylation endproducts of target proteins, and which additionally reverse pre-formed crosslinks in the advanced glycosylation endproducts by cleaving alpha-dicarbonyl-based protein crosslinks present in the advanced glycosylation endproducts. Certain agents useful are thiazolium salts. The method comprises contacting the target protein with the composition. Both industrial and therapeutic applications for the invention are envisioned, as food spoilage and animal protein aging can be treated. A novel immunoassay for detection of the reversal of the nonenzymatic crosslinking is also disclosed.

What is claimed:  
     
         1 . A method of determining abilities of test compounds to break advanced glycosylation endproducts (AGEs), comprising: 
 (a) incubating a test compound with a vessel coated with collagen to which coated vessel AGE-modified protein is bound; and    (b) detecting a concentration of AGE-modified protein remaining bound to the vessel following the incubating.    
     
     
         2 . The method of  claim 1 , comprising 
 (1) forming the coated vessel by incubating AGE-modified protein with a collagen-coated vessel; and    (b) washing the vessel with a wash buffer.    
     
     
         3 . The method of  claim 1 , wherein the incubation of step (a) comprises incubation at 37° C. for 4 hours.  
     
     
         4 . The method of  claim 1 , wherein the wash buffer of step (b) is PBS-Tween.  
     
     
         5 . The method of  claim 1 , wherein the incubation of step (c) comprises incubation at about 37° C. for 12-24 hours.  
     
     
         6 . The method of  claim 1 , wherein the AGE-modified protein is AGE-BSA.  
     
     
         7 . The method of  claim 6 , wherein the detecting comprises treatment with an antibody raised against BSA.  
     
     
         8 . The method of  claim 7 , further comprising treatment with a horseradish peroxidase-conjugated second antibody specific for the BSA-antibody.  
     
     
         9 . A method of determining abilities of test compounds to decrease amounts of IgG crosslinked to circulating red-blood cells in a diabetic rat, comprising: 
 (a) administering a test compound to the rat;    (b) thereafter collecting blood from the rat; and    (c) detecting the amount of IgG crosslinked to the deposited red blood cells.    
     
     
         10 . The method of  claim 9 , further comprising conducting the detecting with IgG-specific antibody, and removing unbound antibody by filtration.  
     
     
         11 . The method of  claim 9 , wherein the test compound is administered orally or intraperitoneally.  
     
     
         12 . The method of  claim 9 , wherein the IgG is detected with alkaline phosphatase-conjugated antibody specific for rat IgG.  
     
     
         13 . The method of  claim 9 , wherein the rat is treated with streptozotocin.