Pharmacological characterization of the bifunctional opioid ligand H-Dmt-Tic-Gly-NH-Bzl (UFP-505)†  N. Dietis, J. McDonald, S. Molinari, G. Calo, R. Guerrini, D.J. Rowbotham, D.G. Lambert  British Journal of Anaesthesia  Volume 108, Issue 2, Pages 262-270 (February 2012) DOI: 10.1093/bja/aer377 Copyright © 2012 The Author(s) Terms and Conditions

Fig 1 Saturation-binding assays performed on CHOhKOP, CHOhDOP, CHOhMOP, and CHOhNOP cell membranes, with increasing concentrations of tritiated diprenorphine (3H-DPN) or tritiated UFP-101 (3H-UFP-101). Log-transformed specific binding isotherms are shown and were used to determine the maximum receptor binding capacity (Bmax) and equilibrium dissociation constant (pKd) in the respective membranes (see also Table 2 and Supplementary material Fig. S1). Single representative curves are presented here (from n=5). Radioligand binding affinity (pkd) and receptor density (Bmax; fmol/mg protein) values are summarized in Table 2. British Journal of Anaesthesia 2012 108, 262-270DOI: (10.1093/bja/aer377) Copyright © 2012 The Author(s) Terms and Conditions

Fig 2 Displacement of tritiated diprenorphine (3H-DPN) by UFP-505 and reference ligands at (a) CHOhMOP, (b) CHOhDOP, (c) CHOhKOP and displacement of tritiated UFP-101 (3H-UFP-101), and nociceptin/orphanin FQ (d) at CHOhNOP cell membranes. Receptor binding affinity of UFP-505 and reference ligands (pKi) was calculated using the Cheng–Prusoff equation and are also summarized in Table 2. Data are means (sem) of five experiments for each cell line. Fentanyl and morphine binding curves at CHOhMOP (a) are included for comparison, where error bars are omitted for clarity and can be seen in Table 2 [pKi; fentanyl 7.35 (0.33), morphine 8.55 (0.14)]. Reference ligands: EM1, endomorphin-1; nBNI, norbinaltorphimine; N/OFQ, nociceptin/orphanin FQ. British Journal of Anaesthesia 2012 108, 262-270DOI: (10.1093/bja/aer377) Copyright © 2012 The Author(s) Terms and Conditions

Fig 3 Effects of UFP-505 at MOP and DOP receptors. Ligand-mediated GTPγ35S binding was measured in membranes prepared from (a) CHOhMOP and (b) CHOhDOP cells (agonist experiments). Data are presented as percentage stimulation of GTPγ35S binding from basal (un-stimulated) and are represented as means (sem) of three (a) or five (b) independent experiments. (c) At CHOhDOP, the addition of 10 nM UFP-505 shifts the agonist DPDPE (control) curve to the right. Data are mean (sem) of five independent experiments. These data are also summarized in Table 2. British Journal of Anaesthesia 2012 108, 262-270DOI: (10.1093/bja/aer377) Copyright © 2012 The Author(s) Terms and Conditions

Fig 4 Ligand-stimulated GTPγ35S binding by UFP-505 and reference ligands measured in membranes of (a) rat spinal cord tissue and (b) rat cerebral cortex. Data are presented as percentage stimulation of GTPγ35S binding from basal (un-stimulated) and are represented as means (sem) of three (a) or four (b) independent experiments. Data are also presented in Table 2. British Journal of Anaesthesia 2012 108, 262-270DOI: (10.1093/bja/aer377) Copyright © 2012 The Author(s) Terms and Conditions

Fig 5 Effects of dermorphin and UFP-505 in the electrically stimulated guinea pig ileum (MOP receptor-rich), presented as percentage of control twitch in the absence and presence of 10 nM naloxone and 100 nM of the MOP-selective antagonist CTOP. (a) The dermorphin curve was shifted to the right by naloxone and CTOP. (b) Similarly, the UFP-505 curve was shifted to the right by naloxone and CTOP. All data are mean (sem) from at least five experiments and are also summarized in Table 2. British Journal of Anaesthesia 2012 108, 262-270DOI: (10.1093/bja/aer377) Copyright © 2012 The Author(s) Terms and Conditions

Fig 6 Effects of DPDPE in the electrically stimulated mouse vas deferens (DOP receptor-rich), in the absence and presence of increasing concentrations of UFP-505 and 10 nM naloxone. (a) The DPDPE curve is shifted in parallel to the right by increasing concentrations of UFP-505 (1, 10, 100 nM). Data presented as percentage of control twitch. (b) Schild analysis of (a) produces a regression line with a slope of 0.96 (0.04) which represents competitive antagonism, whereas the extrapolation of the line when y=0 represents the antagonist potency pA2 (logarithmic) 9.15. (c) In a similar preparation, naloxone shifted the DPDPE curve to the right. Data presented as percentage of control twitch. All data were produced from at least five experiments for each preparation and are summarized in Table 2. British Journal of Anaesthesia 2012 108, 262-270DOI: (10.1093/bja/aer377) Copyright © 2012 The Author(s) Terms and Conditions