A Fentanyl Vaccine Alters Fentanyl Distribution and Protects against Fentanyl-Induced Effects in Mice and Rats

Abstract

Fentanyl is an extremely potent synthetic opioid that has been increasingly used to adulterate heroin, cocaine, and counterfeit prescription pills, leading to an increase in opioid-induced fatal overdoses in the United States, Canada, and Europe. A vaccine targeting fentanyl could offer protection against the toxic effects of fentanyl in both recreational drug users and others in professions at risk of accidental exposure. This study focuses on the development of a vaccine consisting of a fentanyl-based hapten (F) conjugated to keyhole limpet hemocyanin (KLH) carrier protein or to GMP-grade subunit KLH (sKLH). Immunization with F-KLH in mice and rats reduced fentanyl-induced hotplate antinociception, and in rats reduced fentanyl distribution to the brain compared with controls. F-KLH did not reduce the antinociceptive effects of equianalgesic doses of heroin or oxycodone in rats. To assess the vaccine effect on fentanyl toxicity, rats immunized with F-sKLH or unconjugated sKLH were exposed to increasing subcutaneous doses of fentanyl. Vaccination with F-sKLH shifted the dose-response curves to the right for both fentanyl-induced antinociception and respiratory depression. Naloxone reversed fentanyl effects in both groups, showing that its ability to reverse respiratory depression was preserved. These data demonstrate preclinical selectivity and efficacy of a fentanyl vaccine and suggest that vaccines may offer a therapeutic option in reducing fentanyl-induced side effects.

Fig. 1.

Structure of fentanyl (A) and F (B).

Fig. 2.

Experiment 1. F-KLH reduces fentanyl-induced antinociceptive effects and increases serum fentanyl concentrations in mice. Vaccination with F-KLH significantly reduced fentanyl-induced hotplate antinociception by 60% (mean ± S.E.M.) (A) and increased serum fentanyl concentrations (mean ± S.D.) (B) compared with controls 30 minutes after a 0.05 mg/kg, s.c., dose of fentanyl. Numbers above bars represent the percent difference from controls. **P < 0.01; ***P < 0.001 compared with controls using unpaired t tests with Welsh’s correction. n = 6/group.

Fig. 3.

Experiment 2. Selectivity and pharmacokinetic efficacy of F-KLH in rats. (A) Vaccination with F-KLH significantly reduced fentanyl-induced hotplate antinociception by 93% 30 minutes after a 0.035 mg/kg, s.c., dose of fentanyl. (B and C) F-KLH had no effect on heroin- or oxycodone-induced antinociception 30 minutes after a 1 or 2.25 mg/kg dose of heroin or oxycodone, respectively. Serum fentanyl concentrations were significantly increased (D) and brain fentanyl concentrations were significantly decreased (E) compared with controls 4 minutes after a 1-minute 0.05 mg/kg, i.v., infusion of fentanyl. Numbers above bars represent the percentage difference from controls. *P < 0.05; - *P < 0.01; ***P < 0.001 compared with controls. Mean ± S.D. (D and E), mean ± S.E.M. (A–C); n = 12/group (A, D, and E) and n = 6/group (B and C).

Fig. 4.

Experiments 3 and 4. Fentanyl dose-response and F-sKLH effects on hotplate antinociception, respiratory depression, and bradycardia in rats. Fentanyl was administered subcutaneously every 15 minutes at increasing doses in nonimmunized rats, and the doses listed are the cumulative dose received. (A) Effect of fentanyl on hotplate antinociception. Latency to respond is capped at 60 seconds. Naloxone (0.1 mg/kg, s.c.) was administered 15 minutes after the final fentanyl dose. (B) Effect of fentanyl on respiratory depression measured as SaO₂. (C) Effect of fentanyl on heart rate. **P < 0.01; ***P < 0.001 for the difference between values compared with baseline. (D) Vaccine effects on fentanyl-induced antinociception. (E) Vaccine effects on fentanyl-induced respiratory depression measured as SaO₂. (F) Vaccine effects on fentanyl-induced decreases in heart rate. *P < 0.05; **P < 0.01; ***P < 0.001 for differences from baseline within groups. #P < 0.05; ##P < 0.01; ###P < 0.001 for the difference between groups at each dose. There were no differences between groups in latency to respond, SaO₂, or heart rate after naloxone treatment. Mean ± S.D.; n = 8/group.

Fig. 5.

Experiment 4; F-sKLH alters fentanyl distribution in serum and to the brain. F-sKLH vaccination increases serum (A) and decreases brain fentanyl (B) distribution by 73% 30 minutes after receiving a cumulative 0.1 mg/kg, s.c., fentanyl dose. Numbers above bars represent the percent difference from controls. Mean ± S.D., ***P < 0.001 compared with controls using unpaired t tests with Welch’s correction.

Scheme 1.

Synthetic pathway to hapten [8]. (a–g) Reactants: 2-(Boc-amino)ethylbromide, K₂CO₃, acetonitrile, 80°C, 88% (a); aniline, AcOH, NaBH₃CN, CH₂Cl₂, 80°C, 86% (b); propionyl chloride, DIPEA, CH₂Cl₂, rt, 95% (c); TFA/CH₂Cl₂ (2/8-v/v), rt, quantitative (d); glutaric anhydride, pyridine, CH₂Cl₂, rt, quantitative (e); (Gly)₄-OtBu, HBTU, DIPEA, CH₂Cl₂, rt, 74%; TFA/CH₂Cl₂ (2/8-v/v), rt, 92% (f); TFA/CH₂Cl₂ (2/8-v/v), rt, 92% (g).