Combatting Synthetic Designer Opioids: A Conjugate Vaccine Ablates Lethal Doses of Fentanyl Class Drugs

Abstract

Fentanyl is an addictive prescription opioid that is over 80 times more potent than morphine. The synthetic nature of fentanyl has enabled the creation of dangerous "designer drug" analogues that escape toxicology screening, yet display comparable potency to the parent drug. Alarmingly, a large number of fatalities have been linked to overdose of fentanyl derivatives. Herein, we report an effective immunotherapy for reducing the psychoactive effects of fentanyl class drugs. A single conjugate vaccine was created that elicited high levels of antibodies with cross-reactivity for a wide panel of fentanyl analogues. Moreover, vaccinated mice gained significant protection from lethal fentanyl doses. Lastly, a surface plasmon resonance (SPR)-based technique was established enabling drug-specificity profiling of antibodies derived directly from serum. Our newly developed fentanyl vaccine and analytical methods may assist in the battle against synthetic opioid abuse.

Keywords: antibody; biosensors; conjugate vaccines; fentanyls; immunology.

Figure 1

Construction of fentanyl immunoconjugate and structures of fentanyl analogues recognized by polyclonal antibodies.

Figure 2

Timeline of experiments and anti-fentanyl antibody titers. Fent-TT (50 μg) was formulated with alum (750 μg) + CpG ODN 1826 (50 μg) and administered i.p. to each mouse (N=6). IgG titers were determined by ELISA against fentanyl-BSA conjugate. Points denote means ± SEM. Key: i=vaccine injection, a=antinociception assay, f=affinity determination by SPR b=blood/brain biodistribution study.

Figure 3

Fentanyl analogue dose-response curves and ED50 values in antinociception assays. Vaccinated and control mice (N=6 each) were cumulatively dosed with the specified drug and latency to nociception was measured by tail immersion (top) and hot plate (bottom) tests. Points denote means ± SEM expressed as a percentage of the maximum possible drug effect (%MPE). For all three drugs, p-values were <0.001 in comparing control vs. vaccine groups by an unpaired t test.

Figure 4

Biodistribution of fentanyl in blood and brain samples. Vaccinated and control mice (N=6 each) were dosed with 0.2 mg/kg fentanyl and tissue was harvested 15 min post-injection. Fentanyl quantification was performed by LCMS analysis. Bars denote means + SEM. *** p < 0.001, unpaired t test.

Figure 5

Antiserum opioid binding curves and SPR sensorgrams. a) Diluted mouse serum from week 6 was incubated with serial dilutions of the listed opioids and injected into a Biacore 3000 containing a Fent-BSA loaded sensor chip. Signal produced by antibody binding to the SPR chip without drug present was used as a reference for 100% binding. Fentanyls used were racemic and 3-Me was cis. See Figure 1 for structures. b) Overlaid plots of sensorgrams obtained for the interaction between fentanyl (1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.9, 1.95 and 0 nM) and immobilized anti-fentanyl antibodies at 25 °C on a BiOptix 404pi. Original experimental sensorgrams are shown in black and fitted curves are traced in white.