High molecular weight PEGylation of human pancreatic polypeptide at position 22 improves stability and reduces food intake in mice.

BACKGROUND AND PURPOSE: Human pancreatic polypeptide (hPP) is known to suppress appetite and food intake, thereby representing a potential therapeutic approach against obesity and associated metabolic disorders. The aim of this study was to improve hPP stability by covalent PEGylation with diverse molecular weight polyethylene glycols (PEGs) at two positions using promising lead structures while maintaining target activity. EXPERIMENTAL APPROACH: Modified peptides were synthesized by combined solid-phase and solution-phase peptide synthesis. Their potency was investigated in constitutively expressing human epithelial cells and isolated human colonic mucosa as well as receptor-transfected artificial cell lines. Human blood plasma and porcine liver homogenates were used to examine the in vitro stability of the analogues. The most promising variants were injected s.c. in C57BL/6JRj mice to monitor fasting-induced food intake and bioavailability. KEY RESULTS: In human epithelia and colonic mucosal preparations, activity of the modified hPP peptides depended on the core sequence and latency of the peptides was related to PEG size. Peptides modified with a 22 kDa PEG (PEG22) remained intact in blood plasma and on incubation with liver homogenates for more than 96 h. Finally, hPP2-36 , [K22 (PEG22)]hPP2-36 and [K22 (PEG22),Q34 ]hPP significantly reduced cumulative food intake in mice over 16 h after s.c. administration. CONCLUSIONS AND IMPLICATIONS: Modification with PEG22 at position 22 stabilizes hPP significantly while extending its biological activities and could be used in drug development prospectively.

Pancreatic polypeptide and its central Y4 receptors are essential for cued fear extinction and permanent suppression of fear.

BACKGROUND AND PURPOSE: Avoiding danger and finding food are closely related behaviours that are essential for surviving in a natural environment. Growing evidence supports an important role of gut-brain peptides in modulating energy homeostasis and emotional-affective behaviour. For instance, postprandial release of pancreatic polypeptide (PP) reduced food intake and altered stress-induced motor activity and anxiety by activating central Y4 receptors. EXPERIMENTAL APPROACH: We characterized [K(30) (PEG2)]hPP2-36 as long-acting Y4 receptor agonist and injected it peripherally into wildtype and Y4 receptor knockout (Y4KO) C57Bl/6NCrl mice to investigate the role of Y4 receptors in fear conditioning. Extinction and relapse after extinction was measured by spontaneous recovery and renewal. KEY RESULTS: The Y4KO mice showed impaired cued and context fear extinction without affecting acquisition, consolidation or recall of fear. Correspondingly, peripheral injection of [K(30) (PEG2)]hPP2-36 facilitated extinction learning upon fasting, an effect that was long-lasting and generalized. Furthermore, peripherally applied [K(30) (PEG2)]hPP2-36 before extinction inhibited the activation of orexin-expressing neurons in the lateral hypothalamus in WT, but not in Y4KO mice. CONCLUSIONS AND IMPLICATIONS: Our findings suggests suppression of excessive arousal as a possible mechanism for the extinction-promoting effect of central Y4 receptors and provide strong evidence that fear extinction requires integration of vegetative stimuli with cortical and subcortical information, a process crucially depending on Y4 receptors. Importantly, in the lateral hypothalamus two peptide systems, PP and orexin, interact to generate an emotional response adapted to the current homeostatic state. Detailed investigations of feeding-relevant genes may thus deliver multiple intervention points for treating anxiety-related disorders.