Fig. 6: Reduction in fentanyl-induced antinociception and brain tissue distribution in dmLT and LTA1 vaccinated animals compared to alum or antigen alone groups.

Groups of Balb/c mice (n = 5–20) were left naïve or immunized on weeks 0, 3, and 6 with FEN-CRM IM or with adjuvant and route combinations as performed previously. These included 1500 μg alum IM, 0.1–1 μg dmLT IM, 0.1 μg dmLT IM + 5 μg dmLT SL boosters (dmLT SL), or 5 μg LTA1 IM + 5 μg LTA1 IN boosters (LTA1 IN). FEN-CRM was administered at 5 μg for IM delivery and 9–10 μg for mucosal delivery. Blood was collected on week 6, 8, and 10 for serum analyses and mice challenged with fentanyl doses during weeks 9–12. Nociception tests were conducted using tail flick and hotplate assays with tissue taken for drug quantification. a Schematic of immunization schedule. b Compiled serum anti-FEN (FEN-BSA) serum IgG ELISA from week 6, 8, and 10 tailbleeds. c % maximum possible effect (MPE) from tail flick antinociception evaluation. d % MPE from hotplate antinociception evaluation. e Fentanyl concentrations in brain and serum after a final 0.1 mg/kg fentanyl challenge. F Correlation between brain fentanyl levels or tail flick antinociception %MPE vs anti-FEN serum IgG for immunized groups. Bars at mean + s.e.m. with significance determined by ANOVA paired with Bonferroni post-hoc test as shown (*P < 0.05, **P < 0.01, ***P < 0.001). Spearman correlations P values and correlation coefficient (r) indicated on relevant graph.