001, n = 4) and EC50 values of 0.43 and 0.52▒nM, respectively ( Table 2). All the pegylated derivatives displayed Emax values equal to that of GLP-1-(7-36)-amide, conversely, important differences were observed in the EC50 values, as pegylation significantly reduced the potency in stimulating GLP-1 receptor signaling. It is noteworthy that the relative potency of GLP-1-(7-36)-amide pegylated on Gln23 with 5▒kDa PEG was higher than the corresponding derivative pegylated with 20▒kDa PEG, indicating a lower impact of the shorter PEG chain on the agonist activity. Additional experiments were conducted to characterize the ability of

GLP-1-(7-36)-amide, GLP-1(7-36)-amide mutants and the corresponding pegylated derivatives Selleck Erastin to induce insulin secretion from RIN-mf5 cells in Osimertinib nmr the presence of 2▒mM glucose (Table 2). All peptides and pegylated derivatives stimulated insulin release in a concentration-dependent manner within the range of 10⁻12–10⁻6▒M. As observed in the adenylyl cyclase assay, the major differences were observed in the EC50 values, with a loss of potency occurring for GLP-1 mutants and pegylated derivatives

in comparison with native GLP-1(7-36)-amide. Time-course of glucose stabilizing capabilities of GLP-1-(7-36)-amide, GLP-1-(7-36)-amide-Q23-PEG 5▒kDa and GLP-1-(7-36)-amide-Q23-PEG-20▒kDa were assessed in diabetic mice challenged with an oral glucose load administered 30▒min after a subcutaneous injection of 100▒µg/kg of pegylated GLP-1 derivatives or saline. As shown in Fig. 4, the glucose-lowering effect of GLP-1-(7-36)-amide-Q23-PEG 20▒kDa was maintained for about 8▒h while the effect of GLP-1-(7-36)-amide-Q23-PEG 5▒kDa lasted not more than 3.5▒h. The pharmacokinetic profiles of GLP-1-(7-36)-amide-Q23-PEG

5▒kDa and GLP-1-(7-36)-amide-Q23-PEG 20▒kDa administered by subcutaneous injections of 1▒mg/kg were studied not in adult rats (Fig. 5). GLP-1-(7-36)-amide-Q23-PEG 5▒kDa and GLP-1-(7-36)-amide-Q23-PEG 20▒kDa plasma concentrations peaked respectively at 3 and 9▒h, with Cmax values of 160 and 640▒ng/ml. GLP-1-(7-36)-amide-Q23-PEG 5▒kDa disappeared from plasma with a t½ value of 1.7▒h, while the circulating concentration of GLP-1-(7-36)-amide-Q23-PEG 20▒kDa decreased at a slower rate with a t½ value of 17.1▒h and after 96▒h was still around 5▒ng/ml. On the basis of their pharmacokinetic profiles, the calculated average AUC(0?96▒h) for subcutaneous administration of GLP-1-(7=36)-amide-Q23-PEG 5▒kDa and GLP-1-(7-36)-amide-Q23-PEG 20▒kDa was respectively 1408 and 16,800▒ng/ml × h ( Table 3). The significant contribution of 20▒kDa pegylation to the increase of circulating half-life of GLP-1 is clearly demonstrated by literature data of GLP-1-(7-36)-amide administered subcutaneously to rats displaying half-lives between 90 and 216▒min [15].