The relative efficacy was calculated as percent of the GLP-1(7-36)-amide Emax http://www.selleckchem.com/products/dinaciclib-sch727965.html value. The pharmacokinetics parameters were estimated using a non-compartmental model. The half-life ( t1/2) was calculated on the elimination phase of the log transformed pK curve. The maximal concentration ( Cmax) and time of maximal concentration ( Tmax) are the experimental data point corresponding to the maximum value of GLP-1 plasma concentration. The area under the curve (AUC) was calculated by using the

linear trapezoidal rule. The single glutamine residue naturally present in position 23 of GLP-1(7-36)-amide easily reacts with linear 5▒kDa and 20▒kDa alkylamino-monometoxy-PEG and a catalytic amount of bacterial transglutaminase to give the GLP-1(7-36)-amide-Q23-monopegylated derivative with yields of about 60%. However, the same enzymatic pegylation either does not occur or occurs with lower yields when a single glutamine residue substituted a native residue in other positions of the peptidic chain of GLP-1(7-36)- Screening Library nmr Q23N-amide mutant, as shown in Table 1. Monopegylated derivatives of natural GLP-1(7-36)-amide as well as of GLP-1(7-36)-Q23N-amide mutants were purified by cation-exchange chromatography with yields around 80% to obtain

homogenous products as demonstrated by analytical RP-HPLC (Fig. 1). As expected [14], the pegylation increased the apparent size of the conjugated peptides; for example, GLP-1(7-36)-amide-Q23-PEG 20▒kDa, which has a calculated molecular weight of around 23.3▒kDa, migrated in SDS-PAGE with an apparent molecular mass of 44–46▒kDa (Fig. 2). GLP-1-(7-36)-amide, its mutants and the derivatives mono-conjugated with 5, 20 or 50▒kDa PEG on Gln11, Gln23 and Gln30 were subjected to in vitro proteolysis by incubation with porcine DPP-IV. The results demonstrated a high degradation rate for both native Telomerase GLP-1-(7-36)-amide and its Q23N–A30Q double mutant, which showed 50% hydrolysis of N-terminal dipeptide after a few minutes and about 30▒min respectively. Conversely, GLP-1-(7-36)-amide-Q23-PEG 20▒kDa and GLP-1-(7-36)-amide-Q23-PEG 50▒kDa as well as

GLP-1-(7-36)-(Q23N–A30Q)-amide-Q30-PEG 20▒kDa, exhibited a similar slower degradation rate reaching about 50% hydrolysis after 8▒h incubation. GLP-1-(7-36)-amide-Q23-PEG 5▒kDa showed a lower stability, reaching 50% degradation after about 4▒h incubation, most likely due to a minor shielding effect of the shorter PEG chain. However, both PEG chain length and conjugation site were important determinants of proteolytic resistance, as demonstrated by the high stability of GLP-1-(7-36)-(T11Q–Q23N)-amide-Q11-PEG 5▒kDa (15% degradation after 8▒h incubation), where the 5▒kDa PEG chain is attached to a site closer to the N-terminus, which is cleaved by DPP-IV ( Fig. 3). The ability of the pegylated GLP-1 derivatives to activate the GLP-1 receptor was evaluated by measuring the stimulation of adenylyl cyclase in RIN-mf5 cell membranes.