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. 2022 Jun 23;13:3590. doi: 10.1038/s41467-022-31342-z

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© The Author(s) 2022

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 6 — a Hot plate latency test to measure analgesia. Male Sprague-Dawley rats were first administered with saline or a single intravenous (IV) dose of morphine (35 µmol/kg; equivalent to 10 mg/kg). After 5 min, the morphine treated animals were administered with a single intravenous dose of vehicle or the opioid antagonists. Saline group: saline + vehicle; control group: 35 µmol/kg morphine + vehicle; all other groups: 35 µmol/kg morphine + specified dose of antagonists. The 35 µmol/kg dose of antagonists represent 11.5 mg/kg, 23 mg/kg, 12.5 mg/kg, and 30 mg/kg for naloxone, naloxegol, methylnaltrexone (MNTX), and AG10-L2-Nal, respectively. The hot plate withdrawal latency to heat exposure (withdrawal or shaking of the hind paw, sharp withdrawal, licking of fore or hind paw, or attempting to escape by jumping) was recorded 1 h after the morphine dose before the rats were removed from the hot plate. Statistical differences were determined using Kruskal–Wallis test followed by Dunn’s multiple comparisons test, H = 77.02, P < 0.0001. All data are presented as mean (±s.d.) (n = 6 rats per group, *P < 0.05). b Gastrointestinal (GI) transit assay at 1 h after different IV bolus doses of the test compounds. The dosing schedule is similar to the hot plate assay with an additional oral gavage of charcoal meal 30 min after the saline or morphine dose. The significance of differences was measured by two-way ANOVA followed by Tukey’s post hoc test (n = 6 rats per group, *P < 0.05), dose F(2,90) = 262.3, P < 0.0001, compound F(5,90) = 670.8, P < 0.0001. Source data are provided as a Source Data file.