Preparation of long-acting superoxide dismutase using high molecular weight polyethylene glycol (41,000-72,000...

Superoxide dismutase (SOD) has demonstrated therapeutic potential for treating a variety of conditions including radiation injury, oxygen toxicity, reperfusion injury, and inflammation, especially arthritis. However, the native enzyme's short half-life in plasma (6 minutes in mice, 25 minutes in man) limits the enzyme's effectiveness in many applications, or requires infusion of large doses. High doses of SOD derived from either natural or rDNA sources may increase the potential for immunologic sensitization. One effective use of native SOD is intra-articular administration for treatment of arthritis, where injection of SOD into joints retards elimination (15 hour terminal half-life), allowing the effective use of lower doses. To overcome the limitations resulting from rapid clearance, various researchers have increased the persistence of SOD by cross-linking SOD or by attaching polymeric substances, including dextrans, albumin, Ficoll, polyvinyl alcohol or polyethylene glycol (PEG). PEG is relatively safe; however, the amount of modification by PEG, is the MW range 1,900-5,000 daltons, which is necessary to optimally increase serum persistence and reduce immunogenicity, results in the loss of much of the enzymatic activity. In this report we describe the preparation of SOD adducts containing 1 to 4 strands of high MW PEG (41,000-72,000 daltons). The MW range of these adducts, measured by steric exclusion HPLC based on protein standards, is 200,000 to over 1,000,000 daltons. The number of PEG strands attached per SOD dimer (32,000 daltons) was measured by HPLC. Because of the low degree of protein modification required to produce very high MW products, these PEG-SODs retain 90%-100% of the SOD activity of the native enzyme. Additionally, these very large adducts demonstrate longer persistence and lower immunogenicity and antigenicity compared to the more highly modified PEG-SODs containing low MW PEG (i.e., 7-16 strands of 5,000 dalton methoxy-PEG).