Antinociception was determined using the radiant heat tail fiick technique using an Ugo Basile model 37360 instrument. The intensity was set to achieve a baseline between 2 and 3 s. Baseline latencies were determined before experimental treatments for all mice. Tail fiick analgesia was assessed quantally as a doubling or greater of the baseline latency, with a maximal 10 s latency to minimize damage to the tail. Data were also analyzed as percentage maximal percent efiect (%MPE), and similar results were observed. At each time point, MPE was calculated according to the formula: % MPE = 100 × (test latency – control latency)/(maximal latency – control latency). Compounds were injected subcutaneously (sc), and analgesia was assessed 30 min later. For antagonism studies, β-FNA (40 mg/kg, sc) and norbinaltorphimine (norBNI, 10 mg/kg, sc) were administered 24 h before 16 (MP1104). Naltrindole (NTI, 20 mg/kg, sc) was administered 15 min before 16 (MP1104). The 55 °C warm-water tail-withdrawal assay was additionally performed with C57BL/6J, MOR-1 KO, or KOR-1 KO mice as previously described.41 (link) Briefiy, water heated to 55 °C acted as a nociceptive stimulus with the latency to withdraw the tail taken as the end point. Mice showing no response within 5 s during the determination of baseline responses were excluded from the experiment. After determining baseline control responses, mice were administered vehicle or graded doses of morphine or 16 (MP1104). All samples were given as single subcutaneous (sc) injections with tail withdrawal latencies measured 30 and 40 min after administration unless otherwise stated. A cutofi of 15 s was used to avoid tissue damage; those mice failing to withdraw their tails within this time were assigned a maximal percent efiect (%MPE) of 100%. In the receptor selectivity studies, the DOR-1-selective antagonist naltrindole (20 mg/kg, ip) was administered 20 min prior to administration of 16 (MP1104). in vivo experiments were evaluated using GraphPad Prism, San Diego, CA. Statistical significance is indicated as follows: ns, p > 0.05; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001.