Pyy3 36

PYY_3–36 and GLP-1_{7–36 amide} were purchased from Bachem. Following initial high-fidelity synthesis, the peptide hormones underwent purification by high-resolution, high-performance liquid chromatography. Peptide compositions and purity were verified by quantitative amino acid analysis.
Sterile 0.9% (w/v) saline was purchased from Bayer. Using an aseptic technique in a laminar flow cabinet, PYY_3–36 and GLP-1_{7–36 amide} were separately dissolved in 0.9% saline, aliquoted into vials, and freeze dried. Representative PYY_3–36 and GLP-1_{7–36 amide} vials were sterile after culture for 7 days (Department of Microbiology, Hammersmith Hospital, London, United Kingdom), and endotoxin levels as measured by the Limulus Amoebocyte Lysate test (Associates of Cape Cod) were within the safe range for human infusion. Further representative vials of both PYY_3–36 and GLP-1_{7–36 amide} were randomly selected and sent for amino acid analysis by Alta Bioscience to calculate the actual peptide content of the vials. The bioactivity of the peptides was verified by measuring the suppression of food intake over 24 hours when injected sc into C57/BL6 mice (12 (link)), and by receptor-binding affinity assays using membranes prepared from HEK293 cells overexpressing recombinant human Y2 or GLP-1 receptor (25 (link)).

PYY_3-36 and ghrelin were purchased from Bachem (Bubendorf, Switzerland). NNC0165-1273 was identified from a series of modified PYY peptides and synthesized with the aim of increased selectivity for Y2 receptors vs the other Y receptors [peptide no. 29 in Østergaard et al. (9 (link))]. NNC0165-1273 showed a binding inhibitory constant (K_i) on the human Y₂ receptor of 2 nM and >10,000, 2500, and 1300 nM K_i values on the human Y1, Y4, and Y5 receptors, respectively. This compared with a K_i of PYY_3-36 of 0.40 nM on the Y2 receptor and 40, 13, and 3.2 nM on the Y1, Y4, and Y5 receptors, respectively, when tested in the same binding assays. This indicates a fivefold lower relative affinity of NNC0165-1273 for the Y2, but >250, 192-, and 400-fold selectivity, respectively, for the Y1, Y4, and Y5 receptors. Additionally, in in vitro metabolism studies, the peptide has as a half-life of 570 minutes vs 200 minutes for PYY _3-36 as measured in pig plasma. [For further information, description of the peptide and methods, see Østergaard et al. (9 (link)).]