Norbinaltorphimine

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.

Tail flick antinociception was determined using the radiant heat tail flick technique using an Ugo Basile model 37360 instrument as previously described.^{8 (link),9 (link)} The intensity was set to achieve a baseline between 2 and 3 s. Baseline latencies were determined before experimental treatments for all mice. Tail flick antinociception 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 analyzed as percent maximal effect, %MPE, and was calculated according to the formula: % MPE [(observed latency − baseline latency)/(maximal latency − baseline latency)] × 100. Compounds were injected subcutaneously (sc) or intracerebroventricularly (icv), and antinociception was assessed 15 min later at the peak effect. Intracerebroventricular dosing (icv) was carried out as previously described.⁸³ Briefly, the mice were anesthetized with isoflurane. A small incision was made, and synthetic opiate analog (2 ul/mouse) was injected using a 10 μL Hamilton syringe fitted to a 27 gauge needle. Injections were made into the right lateral ventricle at the following coordinates: 2 mm caudal to bregma, 2 mm lateral to sagittal suture, and 2 mm in depth. Mice were tested for antinociception 15 min post injection. For oral (po) studies, mice were fasted for 18 h with access to water before administering the drug by oral gavage. For the antagonism studies, β-FNA (40 mg/kg, sc) and norbinaltorphimine (norBNI, 10 mg/kg, sc) were administered 24 h before 3. Naltrindole (NTI, 0.5 mg/kg, sc) was administered 15 min before 3. Antinociception also was assessed using the hot plate test.⁵⁴ The hot plate (Ugo Basile 35100) consisted of a metal surface (55 °C) with a transparent plexiglass cylinder to contain the mouse. The latency to lick a hind paw or shake/flutter when the mouse was placed on the hot plate was measured, with a maximal latency of 30 s to avoid tissue damage. Baseline latencies were taken for each mouse prior to any drug administration. Mice were tested for analgesia with cumulative subcutaneous doses of the drug until the mouse can withstand the maximal latency. Once the mouse reached the maximal latency, the mouse was no longer given higher doses. In vivo experiments were evaluated using GraphPad Prism, San Diego, CA as described above.