Peripheral opioid receptor antagonist NAP prevented the development of morphine-induced antinociceptive tolerance. (A) Chronic morphine exposure (75 mg) resulted in antinociceptive tolerance to morphine challenge (10 mg/kg) in the tail-immersion assay after 5 days of exposure. N = 5/group; ***P < 0.001 by two-way ANOVA with Bonferroni’s post hoc analysis. (B) Daily NAP injection prevented the development of antinociceptive tolerance in morphine-pelleted mice on day 7, indicated by the leftward shift in the cumulative morphine dose-response curve (1, 2, 4, 8, 16, and 32 mg/kg). N = 5/group; PP + VEH ED50 = 4.74 mg/kg; PP + NAP ED50 = 4.61 mg/kg; MP + VEH ED50 = 14.57 mg/kg (11.13–18.4, 95% confidence limit (C.L.)); MP + NAP ED50 = 6.95 mg/kg (5.54–8.92, 95% confidence limit (C.L.)) with nonlinear regression analysis (best-fit lines).