An Immunconjugate Vaccine Alters Distribution and Reduces the Antinociceptive, Behavioral and Physiological Effects of Fentanyl in Male and Female Rats

Abstract

Fentanyl (FEN) is a potent synthetic opioid associated with increasing incidence of opioid use disorder (OUD) and fatal opioid overdose. Vaccine immunotherapy for FEN-associated disorders may be a viable therapeutic strategy. Here, we expand and confirm our previous study in mice showing immunological and antinociception efficacy of our FEN vaccine administered with the adjuvant dmLT. In this study, immunized male and female rats produced significant levels of anti-FEN antibodies that were highly effective at neutralizing FEN-induced antinociception in the tail flick assay and hot plate assays. The vaccine also decreased FEN brain levels following drug administration. Immunization blocked FEN-induced, but not morphine-induced, rate-disrupting effects on schedule-controlled responding. Vaccination prevented decreases on physiological measures (oxygen saturation, heart rate) and reduction in overall activity following FEN administration in male rats. The impact of FEN on these measures was greater in unvaccinated male rats compared to unvaccinated female rats. Cross-reactivity assays showed anti-FEN antibodies bound to FEN and sufentanil but not to morphine, methadone, buprenorphine, or oxycodone. These data support further clinical development of this vaccine to address OUD in humans.

Keywords: adjuvant; analgesia; anti-fentanyl vaccine; antibodies; conjugate; opioids; overdose; vaccines.

Figure 1

Experimental design sequence and immunogenicity of the FEN-CRM+dmLT vaccine formulation. Timeline and sequence of the experiments are presented in (A) and was created with BioRender.com. Rats (N = 15) were vaccinated at weeks 0, 3 and 6 and blood samples obtained at 6, 8, 10 and blood and brain samples taken at 20 weeks. IgG antibody levels were determined using ELISA with FEN-BSA as the coating antigen. Anti-FEN IgG antibody levels over weeks in male Sprague Dawley rats are presented in (B). Anti-FEN IgG antibody levels over weeks in female Sprague Dawley rats are shown in panel (C). Data are presented as serum EU (ELISA Units)/mL (log 10, mean ± SEM). * p > 0.05, ** p < 0.01; Male vs. Female ^## p < 0.01, ^#### p < 0.0001.

Figure 2

Vaccine efficacy against FEN-induced anti-nociception and FEN distribution in male Sprague Dawley rats. Rats were vaccinated with PBS (CONTROL, open bars, N = 15) or (FEN-CRM, gray bars, N = 15) at day 0, then boosted at 3 and 6 weeks. At 8–12 weeks post initial vaccination, rats were administered one of two doses of FEN (0.05 mg/kg and 0.1 mg/kg, SC) then tested in the tail flick (A,B) and hotplate (C,D) nociception assays. Data are presented as mean ± SEM %MPE (Maximal Possible Effect). Vaccinated rats showed no response to FEN-induced analgesia in the tail flick and hotplate assays whereas unvaccinated rats showed robust analgesic effects with the high FEN dose. Brain (E) FEN levels were obtained following administration of FEN (0.1 mg/kg, SC) at approximately week 20 post initial vaccination in the same rats. FEN was prevented from penetrating the brain in vaccinated rats but not in unvaccinated rats. **** p < 0.0001; CONTROL male vs. CONTROL female groups: ^# p < 0.05, ^## p < 0.01, ^### p < 0.001, ^#### p < 0.0001.

Figure 3

Vaccine efficacy against FEN-induced anti-nociception and FEN distribution in female Sprague Dawley rats. Rats were vaccinated with PBS (CONTROL, open bars, N = 15) or (FEN-CRM, gray bars, N = 15) at day 0, then boosted at 3 and 6 weeks. At 8–12 weeks post initial vaccination rats were administered one of two doses of FEN (0.05 mg/kg and 0.1 mg/kg) then tested in the tail flick (A,B) and hotplate (C,D) nociception assays. Data are presented as mean ± SEM %MPE (Maximal Possible Effect). Vaccinated rats showed no response to FEN-induced analgesia in the tail flick and hotplate assays whereas unvaccinated rats showed robust analgesic effects at the high FEN dose. Brain (E) FEN levels were obtained following administration of FEN (0.1 mg/kg, SC) at approximately week 20 post initial vaccination in the same rats that underwent analgesic testing. FEN was prevented from penetrating the brain in vaccinated rats but not unvaccinated rats. **** p < 0.0001.

Figure 4

Effects of FEN and MOR on schedule-controlled responding in male and female Sprague Dawley rats. Rats (N = 13–14 males (A,B); N = 12, females (C,D)) were trained to lever press for food under an FR15 (frequency ratio) schedule of responding. Once a rat met testing criteria, it was administered one of four doses of FEN (A,C) or MOR (B,D). Rats were then vaccinated at 0, 3 and 6 weeks and retested with both compounds between weeks 8–10 post-initial vaccination. Data are presented as mean ± SEM % control rate of responding. Vaccination significantly blocked the rate decreasing effects of FEN but did not alter the influence of MOR on response rates in both male and female rats. * p < 0.05, *** p < 0.001, **** p < 0.0001.

Figure 5

Vaccine efficacy against FEN-induced physiological effects in male and female Sprague Dawley rats. Sprague Dawley rats (male, CONTROL, N = 6, FEN-CRM, N = 8, (A–C); female, CONTROL, N = 9, FEN-CRM, N = 6, (D–F)). Groups were vaccinated with PBS or FEN-CRM+dmLT (day 0, 3 and 6 weeks, IM) and at approximately 10 weeks post initial vaccination rats were administered 0.1 mg/kg FEN (SC) and oxygen saturation (%), heart rate (beats per minute, bpm), and activity (counts) were measured using a pulse oximetry system. FEN significantly decreased all measures in unvaccinated male rats and these effects were attenuated by the vaccine. FEN significantly decreased oxygen saturation in unvaccinated female rats that was reversed by vaccination. CONTROL vs. FEN-CRM male and female groups: ** p < 0.01, *** p < 0.001,**** p < 0.0001; CONTROL male vs. CONTROL female groups: ^# p < 0.05, ^## p < 0.01, ^### p < 0.001, ^#### p < 0.0001.

Figure 6

Anti-FEN antibody specificity in samples from vaccinated rats. Specificity of anti-FEN IgG antibodies generated by FEN-CRM+dmLT was determined using ELISA by coating the plate with different target antigens and processing serum collected at week 20 from unvaccinated control and vaccinated male rats. As shown, anti-FEN antibodies bound only to the FEN-BSA and SUFENTANIL-BSA antigens and not to other opioids tested. Data are presented as Serum EU (ELISA Units)/mL subtracted from control. **** p < 0.0001.