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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 is a pancreatic hormone cosecreted with insulin that exerts unique roles in metabolism and glucose homeostasis. The therapeutic restoration of postprandial and basal amylin levels is highly desirable in diabetes mellitus. Protein conjugation with the biocompatible polymer polyethylene glycol (PEG) has been shown to extend the biological effects of biopharmaceuticals. We have designed a PEGylated human amylin by using the aminoreactive compound methoxylpolyethylene glycol succinimidyl carbonate (mPEGsc). The synthesis in organic solvent resulted in high yields of monoPEGylated human amylin, which showed large stability against aggregation, an 8 times increase in half-life in vivo compared to the non-conjugated amylin, and pharmacological activity as shown by modulation of cAMP production in MCF–7 cell line, decrease in glucagon and modulation of glycemia following subcutaneous administration in mice. Altogether these data reveal the potential use of PEGylated human amylin for the restoration of fasting amylin levels.

The amyloid precursor protein (APP) is well known for giving rise to the amyloid-β peptide and for its role in Alzheimer's disease. Much less is known, however, on the physiological roles of APP in the development and plasticity of the central nervous system. We have used phage display of a peptide library to identify high-affinity ligands of purified recombinant human sAPPα695 (the soluble, secreted ectodomain from the main neuronal APP isoform). Two peptides thus selected exhibited significant homologies with the conserved extracellular domain of several members of the semaphorin (Sema) family of axon guidance proteins. We show that sAPPα695 binds both purified recombinant Sema3A and Sema3A secreted by transfected HEK293 cells. Interestingly, sAPPα695 inhibited the collapse of embryonic chicken (Gallus gallus domesticus) dorsal root ganglia growth cones promoted by Sema3A (Kd≤8·10−9 M). Two Sema3A-derived peptides homologous to the peptides isolated by phage display blocked sAPPα binding and its inhibitory action on Sema3A function. These two peptides are comprised within a domain previously shown to be involved in binding of Sema3A to its cellular receptor, suggesting a competitive mechanism by which sAPPα modulates the biological action of semaphorins.

Amylin is a 37-aminoacid pancreatic protein that exerts control over several metabolic events such as glycemia and lacticemia. Amylin has long been shown to induce increases in arterial plasma glucose. We decided to investigate whether amylin plays additional roles in the glucose metabolism. We evaluated glucose homeostasis using whole blood from the tail tip of fasting, conscious, unrestrained normal and streptozotocyn-induced diabetic mice following subcutaneous administration of mouse amylin. Subcutaneous injection of 1 μg mouse amylin caused a transient decrease in whole blood glucose in both normal and diabetic mice in the absence of insulin. The blood glucose levels were lowest approximately 2 hours after amylin administration, after that they gradually recovered to the levels of the control group. The hypoglycemic effect followed a dose-dependent response ranging from 0.1 to 50 µg / mouse. These results reveal the ability for amylin in the direct control of glycemia at low doses in the absence of insulin.

We modified amylin chemically by conjugating methoxyl polyethyleneglycol succinimidyl carbonate (mPEGSC) of varying molecular weights (1 kDa, 2 kDa and 5 kDa). The reaction occurred within a few minutes, resulting in at least four distinct PEGylated products. The reaction products were separated by reversed-phase chromatography and identified by mass-spectrometry. The monoPEGylated and diPEGylated amylin products were generated rapidly through conjugation to the two amino groups of the N-terminal lysine residue. Both PEGylated amylin products bound to the receptor activity-modifying protein 1 (RAMP1). Pharmacological evaluation by subcutaneous administration in mice of monoPEGylated and diPEGylated amylin obtained with mPEG-SC 5 kDa revealed that both compounds modulated glycemia for longer times than unmodified amylin. Collectively, these data demonstrate the potential of bioconjugation with mPEG for the design of amylin therapeutics with sustained action.

Recebido em 2/8/11; aceito em 25/11/11; publicado na web em 28/2/12Here we report the validation of a derivatization method that makes use of fluorescamine as a selective reactant for the quantitative analysis of peptide and protein drugs in the dissolution profile from depot formulations. Typical current methods require separation of the nano/microparticles and time-consuming chromatographic runs. In this study we report a method which can be conducted without the need for complete physical separation of the particles or removal of the unreacted probe. This method was used here for the analysis of the release profile of octreotide in a depot formulation, with results in excellent agreement with reported chromatographic assays.