Physical Presence of Nor-Binaltorphimine in Mouse Brain over 21 Days after a Single Administration Corresponds to Its Long- Lasting Antagonistic Effect on k-Opioid Receptors

In the mouse 55°C warm-water tail-withdrawal assay, a single administration of nor-binaltorphimine (nor-BNI; 10 mg/kg i.p.) antagonized k-opioid receptor (KOR) agonist-induced antinociception up to 14 days, whereas naloxone (10 mg/kg i.p.)mediated antagonism lasted less than 1 day. In saturation binding experiments, mouse brain membranes isolated and washed 1 or 7 (but not 14) days after nor-BNI administration demonstrated a significant time-dependent decrease in maximal KOR agonist [H]U69,593 binding. To determine whether brain concentrations of nor-BNI were sufficient to explain the antagonism of KOR-mediated antinociception, mouse blood and perfused brain were harvested at time points ranging from 30 minutes to 21 days after a single administration and analyzed for the presence of nor-BNI using liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). Nor-BNI was detected in the perfused brain homogenate up to 21 days after administration (30 nmol i.c.v. or 10 mg/kg i.p.). Subsequent experiments in which nor-BNI was administered at doses estimated from the amounts detected in the brain homogenates isolated from pretreated mice over time demonstrated significant antagonism of U50,488 antinociception in a manner consistent with the magnitude of observed KOR antagonism. The dose (1.4 nmol) approximating the lowest amount of norBNI detected in brain on day 14 did not antagonize U50,488induced antinociception, consistent with the absence of U50,488 antagonism observed in vivo at this time point after pretreatment. Overall, the physical presence of nor-BNI in the mouse brain paralleled its in vivo pharmacological profile, suggesting physicochemical and pharmacokinetic properties of nor-BNI may contribute to the prolonged KOR antagonism.