Preparation and Characterization of PEGylated Amylin

Abstract

Abstract. Amylin is a pancreatic hormone that plays important roles in overall metabolism and in glucose homeostasis. The therapeutic restoration of postprandial and basal amylin levels is highly desirable for patients with diabetes who need to avoid glucose excursions. Protein conjugation with polyethylene glycol (PEG) has long been known to be a convenient approach for extending the biological effects of biopharmaceuticals. We have investigated the reactivity of amylin with methoxy polyethylene glycol succinimidyl carbonate and methoxy polyethylene glycol succinimidyl propionate, which have an average molecular weight of 5 kDa. The reaction, which was conducted in both aqueous and organic (dimethyl sulfoxide) solvents, occurred within a few minutes and resulted in at least four detectable products with distinct kinetic phases. These results suggest a kinetic selectivity for PEGylation by succinimidyl derivatives; these derivatives exhibit enhanced reactivity with primary amine groups, as indicated by an evaluation of the remaining amino groups using fluorescamine. The analysis of tryptic fragments from mono-and diPEGylated amylin revealed that conjugation occurred within the 1-11 amino acid region, most likely at the two amine groups of Lys 1 . The reaction products were efficiently separated by C-18 reversed phase chromatography. Binding assays confirmed the ability of mono-and diPEGylated amylin to interact with the amylin co-receptor receptor activity-modifying protein 2. Subcutaneous administration in mice revealed the effectiveness of monoPEG-amylin and diPEG-amylin in reducing glycemia; both compounds exhibited prolonged action compared to unmodified amylin. These features suggest the potential use of PEGylated amylin to restore basal amylin levels.

Amylin structure–function relationships and receptor pharmacology: implications for amylin mimetic drug development

“…The longer duration of action of these conjugates also validate this technique in improving the half‐life of amylin (Guterres et al ., ). These conjugates were also effective in reducing glycaemia when subcutaneously administered in rats with consistently prolonged durations of action over that of unmodified amylin (Guerreiro et al ., ).…”

Amylin: Pharmacology, Physiology, and Clinical Potential

“…Given the short half-life of amylin, another strategy to improve the molecule has been to improve its half-life through the addition of molecular scaffolds such as polyethylene glycol (PEG). A recent preparation has been described for the PEGylation of amylin (Guerreiro et al, 2013). Subcutaneous administration of PEGylated amylin in mice revealed the effectiveness of monoPEGamylin and diPEG-amylin in reducing glycemia, with both compounds exhibiting prolonged action relative to unmodified amylin (Guerreiro et al, 2013).…”

“…A recent preparation has been described for the PEGylation of amylin (Guerreiro et al, 2013). Subcutaneous administration of PEGylated amylin in mice revealed the effectiveness of monoPEGamylin and diPEG-amylin in reducing glycemia, with both compounds exhibiting prolonged action relative to unmodified amylin (Guerreiro et al, 2013). Further testing of these molecules is warranted.…”

The Effect of Protein PEGylation on Physical Stability in Liquid Formulation

“…Numerous studies have investigated the PEGylation process, its effect on biological activity and circulation time, and the effect of PEGylation on the protein thermal stability . There are, however, surprisingly few studies that report the impact of the PEGylation on the pharmaceutical formulation development.…”

“…6 Although our choice of a 5-kDa PEG is smaller than the latest favorites of the industry, protein drugs such as Adagen R , Oncaspar R , and Somavert R are modified using 5 kDa PEGs. 4,7 Numerous studies have investigated the PEGylation process, its effect on biological activity and circulation time, [8][9][10][11][12][13][14] and the effect of PEGylation on the protein thermal stability. 8,15,16 There are, however, surprisingly few studies that report the impact of the PEGylation on the pharmaceutical formulation development.…”