Plasma Clearance and Immunologic Properties of Long-Acting Superoxide Dismutase Prepared Using 35,000 to 120,000 Dalton Poly-Ethylene Glycol

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Abstract
Some biological properties of bovine and recombinant human Cu, Zn superoxide dismutase (bSOD and rhSOD)-poly-ethylene glycol (PEG) adducts prepared by coupling 1–9 strands of high molecular weight PEG (35,000–120,000 daltons) are compared to SOD adducts coupled with 7 or 15 strands of low molecular weight PEG (5,000 daltons). Plasma clearance after i.v. injection was measured in mice and dogs. Conjugates of bSOD with 2 strands of PEG 40,000, 3 strands of PEG 72,000 or 1 strand of PEG 100,000 demonstrated half-lives of about 36 hours in mice, whereas the half-life of a conjugate with 7 strands of PEG 5,000 was about 24 hours. A PEG-bSOD with an average of 3.3 strands of PEG 41,000 was cleared from plasma with a terminal half-life of 36 to 48 hours after intraperitoneal injection in mice. PEG-SODs prepared from bSOD and rhSOD with 3 strands of PEG 50,000 each had plasma half-lives of approximately five days in dogs. An enzyme immunoassay (ELISA) was employed to measure cross-reactivity with a rabbit antibody directed against bSOD. A series of bSOD adducts with 2 to 9 strands of PEG 35,000–120,000 were compared to PEG-bSODs with 7 or 15 strands of PEG 5,000. Attaching larger PEG strands was at least 3 times more effective in reducing antigenicity, compared to PEG 5,000. Ability to induce sensitizing antibodies was measured using subcutaneous sensitization followed by i.v. or s.c. challenge in mice. Some bSOD conjugates with either 7 or 15 strands of PEG 5,000 induced sensitization reactions before the sixth challenge. Fewer than 1 % of the animals tested with bSOD or rhSOD adducts with 3 or 4 strands of PEG 65,000, or 3 strands of PEG in the 30,000–50,000 molecular weight range, showed signs of anaphylaxis during six or seven challenges. In a passive cutaneous anaphylaxis test (optimized to measure mouse IgE), neither bSOD coupled to 2 strands of PEG 120,000 nor bSOD coupled to 9 strands of PEG 35,000 induced detectable antibodies against either of these PEG-bSOD preparations or against bSOD; however, the adduct with 2 strands of PEG 120,000 reacted weakly with pre-formed antibodies to bSOD. The high molecular weight PEG-bSODs tested were not immunogenic, but were weakly antigenic, compared to bSOD.