Pharmacological characterisation of selective Y4 and dual Y2/Y4 receptor agonists:novel targets for putative anti-obesity therapies

摘要

The GIPIO (Gastrointestinal Peptides in Obesity) collaboration was set up with EU (FP7) funding in 2008, with the aim to develop a series of peptide analogues for the treatment of obesity. Based on the dual Y2/Y4 agonist, Obinepitide ([Q34]hPP), and the Y4 agonist, TM30339 (hPP2-36), novel peptides were developed and subsequently modified by either PEGylation or lipidation to improve their relatively short half-life and pharmacokinetic profile. This thesis presents a detailed study of the in vitro pharmacological data performed to aid the progression of the most promising peptide candidates. An assessment of Y2Y4 potency and activity was made, using optimised Y4 (human colonic epithelial monolayers) and Y2Y4 (human colon mucosae) bioassays, where preparations were mounted in Ussing chambers and changes in short-circuit current (Isc) recorded. In addition, receptor specificity of the most promising agonists was dissected using Y1 and Y2 antagonists. Furthermore, an assessment of Y4 receptor desensitisation was made by application of a subsequent addition of the native hormone, hPP. With a lysine linker at position 30 or 22, chemical modification of Obinepitide with either PEGylation or Palmitoylation resulted in prolonged reductions in Isc in both preparations compared with the transient response seen with their respective predecessors. Increasing the PEG size resulted in greater reductions in Isc, though a PEG22 modification resulted in a loss of functional activity. Mid to long chain lipidation of TM30339 (lauric acid and palmitic acid) also resulted in sustained reductions in Isc. Interestingly, lipidation with a short chain fatty acid (caprylic acid) caused a biphasic response, with an initial transient drop in Isc followed by a sustained anti-secretory response. Importantly, PEG and lipid side chains had no effect upon Isc. Pre-treatment of either human mucosa or epithelial monolayers with PEG2-, or 5-ylated Obinepitide did not cause significant subsequent Y4 desensitisation, an effect that was greater with the larger PEGylated peptide. Contrary to this, long chain lipidated peptides seemed to facilitate rapid and sustained desensitisation, revealed by the loss of further Y4 signalling even after nM lipidated agonist. This divergence in receptor signalling was also observed in fluorescent imaging studies performed by our collaborators. The data presented herein provides the first functional evidence of prolonged activity at the Y2 and Y4 receptors and these modified peptides have the potential to act for longer in vivo as anti-obesity treatments.