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. 2022 Apr 20;5(5):331–343. doi: 10.1021/acsptsci.1c00260

Efficacy and Selectivity of Monovalent and Bivalent Vaccination Strategies to Protect against Exposure to Carfentanil, Fentanyl, and Their Mixtures in Rats

Bethany Crouse †,‡, Mariah M Wu †,‡, Valeria Gradinati †, Andrew J Kassick §, Daihyun Song †, Rajwana Jahan ∥, Saadyah Averick §, Scott Runyon ∥, Sandra D Comer ⊥, Marco Pravetoni †,∇,⊗,*
PMCID: PMC9112413  PMID: 35592436

Abstract

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Drug-related fatal overdoses have significantly increased in the past decade due to the widespread availability of illicit fentanyl and other potent synthetic opioids such as carfentanil. Deliberate or accidental consumption or exposure to carfentanil, fentanyl, and their mixture induces respiratory depression and bradycardia that can be difficult to reverse with the opioid receptor antagonist naloxone. Vaccines offer a promising strategy to reduce the incidence of fatalities associated with fentanyl-related substances, as well as treatment for opioid use disorder (OUD). This study reports monovalent and bivalent vaccination strategies that elicit polyclonal antibody responses effective in protecting against the pharmacological actions of carfentanil, fentanyl, or carfentanil/fentanyl mixtures. Rats were prophylactically immunized with individual conjugate vaccines containing either carfentanil- or fentanyl-based haptens, or their combination in bivalent vaccine formulations, and then challenged with carfentanil, fentanyl, or their mixture. First, these studies identified a lead vaccine protective against carfentanil-induced antinociception, respiratory depression, and bradycardia. Then, efficacy against both carfentanil and fentanyl was achieved through bivalent vaccination strategies that combined lead anti-carfentanil and anti-fentanyl vaccines via either heterologous prime/boost or co-administration immunization regimens. These preclinical data support the development of vaccines as a viable strategy to prevent toxicity from exposure to excessive doses of carfentanil, fentanyl, or their mixtures.

Keywords: opioid use disorder, vaccine, multivalent, overdose, carfentanil, fentanyl

The widespread accessibility of synthetic opioids coupled with high rates of relapse has demonstrated the need for new interventional strategies to treat and manage opioid use disorder (OUD) and overdose. In the 12 months ending in April 2021, there were 75 673 opioid-related deaths in the U.S., representing a large proportion of the 100 306 fatal drug overdoses.1 Fentanyl and other potent synthetic opioids accounted for the majority of these deaths,1 consistent with the previously reported trend of increasing incidence of fentanyl-related mortalities from June 2019 to May 2020 (>38.4%).2 The COVID-19 pandemic further complicated and exacerbated the opioid public health crisis, as shown by increased rate of fatal and non-fatal overdoses attributed to synthetic opioids alone or in combination with other drugs.3−6 Carfentanil is a synthetic opioid that is 80–100× more potent than its parent compound fentanyl and 10,000× more potent than morphine.7 Both fentanyl and carfentanil were placed in Schedule II of the Controlled Substances Act, meaning that they have “high potential for abuse” and are “considered dangerous”.8 Carfentanil-induced toxicity is reported in non-human primates at a dose of 0.8 μg/kg,9 which would translate to a dose of only 56 μg (approximately the mass of a grain of salt) in an average-sized human. Carfentanil’s only approved clinical use is in veterinary medicine as a large-animal tranquilizer;7 however, illicitly manufactured carfentanil has been found in street mixtures intended for recreational use by humans. While the exact number of deaths due to carfentanil is unknown given its extremely high potency and low tissue concentration, which complicates detection by most currently available testing methods,7 it has been speculated to contribute significantly to the increased incidence of fatal opioid-related overdoses.10

Fentanyl and carfentanil have been found as adulterants in street drug mixtures of heroin, cocaine, benzodiazepines, and methamphetamine,7,11,12 which can lead to unintentional consumption and accidental overdose in individuals diagnosed with a substance use disorder (SUD) and occasional drug users. While it has often been hypothesized that first responders, law enforcement, paramedics, and customs agents may be accidentally exposed to fentanyl and its analogues as an occupational hazard,13 the risk of overdose is largely anecdotal and thought to be low with the proper precautions.13 Nevertheless, due to carfentanil’s extremely high potency, the possibility of symptoms may be greater after exposure compared to other synthetic opioids. Carfentanil may also be of concern to civilian and military personnel exposed to chemical attacks or deliberate poisoning. In 2002, Russian Special Forces introduced aerosolized carfentanil and remifentanil through the ventilation system of a Moscow theater to subdue Chechen terrorists who were holding hostages. Of the 800 people who were exposed, 127 died and over 650 were hospitalized.12 This has led to increased concerns that carfentanil may be used as a chemical weapon during wartime or in terrorist attacks.

While the mu-opioid receptor (MOR) antagonist naloxone is an approved treatment for opioid overdose, its relatively short duration of action can lead to a return of opioid overdose symptoms several hours after apparent recovery, a phenomenon known as re-narcotization.7,12,14 Additionally, naloxone is only useful if it is administered soon after the overdose occurs, requiring an individual to have access to the medication and someone available to deliver it. Respiratory deficits are further complicated by the propensity of fentanyl-like drugs to induce airway closure and muscle rigidity in the chest, known as wooden chest syndrome, which are mediated by α1-adrenergic and cholinergic signaling and therefore partially refractory to naloxone.15−17 Despite the availability of effective medications for treating OUD (MOUDs), including methadone, buprenorphine, and naltrexone, opioid-related overdoses continue to increase, which highlights the need for novel, long-lasting, and more selective strategies to treat OUD and prevent overdose from highly potent fentanyl analogues.

One such treatment modality is active immunization via conjugate vaccines to stimulate the production of opioid-specific polyclonal IgG antibodies. Antibodies bind to the target drug in circulation and prevent the distribution of unbound (free) drug to the brain, which prevents drug-induced rewarding effects as well as lethal symptoms such as bradycardia and respiratory depression.18−25 Vaccine-induced polyclonal antibodies have significantly longer half-lives compared to traditional MOUDs or overdose reversal agents,26 reducing the risk of re-narcotization after opioid exposure. Additionally, because antibodies selectively neutralize the target opioid itself, without interfering with MOR’s activity, this treatment strategy may be effective at preventing the pharmacological effects of target opioids regardless of the underlying signaling mechanism. Finally, vaccines can be administered both therapeutically and prophylactically, making them a desirable choice for individuals at high risk of opioid overdose and/or developing OUD.

In this study, two novel carfentanil-based haptens, F11 and F13, were synthesized, conjugated to a carrier protein, and tested in rats in comparison to a previously described vaccine containing a fentanyl-based (F1) hapten.21 Vaccines composed of F1 conjugated to diphtheria cross-reactive material-197 (CRM) were previously shown to be effective in preventing fentanyl-induced antinociception, respiratory depression, bradycardia, and intravenous self-administration.21 Evaluation of F1-CRM alongside other conjugates containing a series of next-generation haptens (F2–6, and F7–10) provided further evidence for this approach against fentanyl and selected analogs such as sufentanil, alfentanil, and acetylfentanyl.21,27 In this series, the fentanyl-based hapten F12 was synthesized but discarded early on from further screening (unpublished data). The F11 and F13 haptens are structurally different than other carfentanil-targeting haptens reported in the literature,26,28 as the pendant phenethyl substituent has been replaced with linear amide-based linkers (Figure 1). Compounds F11 and F13 retain the characteristic 4-anilidopiperidine core structure of both fentanyl and carfentanil, but also possess the sterically crowded methyl ester-bearing quaternary carbon center of the latter. This strategy was designed with the goal of preserving antibody affinity for fentanyl while also extending cross-reactivity to carfentanil. Haptens F11 and F13 were conjugated to the CRM carrier protein and tested for their efficacy in blocking the effects of carfentanil, fentanyl, or their mixture in rats. To achieve protection against both target drugs, the efficacy of bivalent vaccination strategies to target both fentanyl and carfentanil was also assessed. Previous studies reported the use of bivalent vaccines against fentanyl and its analogues consisting of either divalent presentation of two haptens sharing the same linker conjugated to the same carrier or co-administration of individual conjugates.19,29,30 Similar studies reported the possibility of co-administration of individual conjugate vaccines to target heroin and oxycodone mixtures25,31 or heroin and fentanyl mixtures.19,29,30,32,33 The current study further extends the concept of bivalent vaccination by testing co-administration of individual conjugate vaccines in comparison to a heterologous prime/boost immunization regimen commonly used in vaccines for infectious diseases (reviewed in ref (34)). Results support further development of multivalent vaccine formulations to protect against the potentially lethal effects of structurally related and distinct drug substances in both mono- or poly-drug use or overdose scenarios.

Figure 1.

Figure 1

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Haptens targeting fentanyl (F1) and carfentanil (F11 and F13).

Materials and Methods

Synthesis of Fentanyl- and Carfentanil-Based Haptens

General Information

N-Benzylnorcarfentanil oxalate (1) was prepared according to published procedures.35 All other reagents and solvents were obtained from commercial sources and used according to manufacturer’s instructions. 1H and 13C NMR spectra were measured in chloroform-d (CDCl3), dimethyl sulfoxide-d6 (DMSO-d6), or methanol-d4 (CD3OD) on a Bruker Avance 500 MHz spectrometer. Chemical shifts are reported in ppm employing the residual solvent resonance as the internal standard. Liquid chromatography paired with mass spectrometry (LC-MS) was performed using a Dionex Ultimate 3000 ultra-high-performance liquid chromatography (UHPLC) system coupled to a Thermo Scientific TSQ Quantum Access MAX triple-quadrupole mass spectrometer. Reverse-phase chromatographic separation was accomplished on an Agilent ZORBAX Eclipse Plus C18 column (3.5 μm, 100 × 4.6 mm) with acetonitrile (CH3CN) and water (H2O), modified with 0.1% formic acid, as the mobile phase solvents. Standard HPLC method consisted of a linear gradient from 20 to 95% CH3CN over 5 min followed by a hold at 95% CH3CN for 1 min and then a re-equilibration at 20% CH3CN for 2.5 min. Total run time was 10 min, flow rate was 0.400 mL/min, and the injection volume was 10 μL.

Fentanyl-Based Hapten (F1)

The fentanyl-based hapten containing a tetraglycine linker (F1, or F(Gly)4) was synthesized as previously described20 and belongs to a series of Fn haptens currently under development.21,27

Carfentanil-Based Hapten (F11)

The synthesis of F11 (Scheme 1) was initiated with the preparation of norcarfentanil from the previously described compound, N-benzylnorcarfentanil oxalate, 1.35 Michael addition of norcarfentanil and tert-butyl acrylate in CH3CN solvent at room temperature provided tert-butyl ester intermediate 2. Treatment of the resulting 1,4-addition product 2 with neat trifluoroacetic acid (TFA) then afforded the corresponding carboxylic acid TFA salt 3 in excellent yield. Acid 3 was then converted to amide 4 following a standard EDC coupling with N-Boc-ethylenediamine and excess diisopropylethylamine. Subsequent acid-mediated deprotection of Boc-carbamate 4 with 85% H3PO4 then furnished the amino-functionalized carfentanil hapten (F11) for conjugation to the CRM carrier protein. Detailed experimental procedures and corresponding compact spectral data for the synthesis of F11 and its intermediate precursors are available in the Supporting Information.

Scheme 1.

Scheme 1

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Reagents and conditions: (a) HCOONH4, 10% Pd/C, MeOH, reflux, 3 h; (b) tert-butyl acrylate, CH3CN, room temperature (RT), 20 h; (c) TFA, RT, 1.5 h; (d) N-Boc-ethylenediamine, EDC, DMAP, CH2Cl2, 0 °C → RT; 20 h; (e) 85% H3PO4, CH2Cl2, RT, 1 h.

Carfentanil-Based Hapten (F13)

Synthesis of F13 (Scheme 2) was initiated by a previously reported aniline-based Bargellini reaction to arrive at the sterically hindered carboxylic acid 5.36 A one-pot transformation of compound 5 involving N-acylation with propionic anhydride and subsequent acid to ester interconversion then furnished N-Boc norcarfentanil 6 in good yield. Standard acidic hydrolysis of carbamate 6 revealed amine 7 which was then treated with N-Boc-2-aminoacetaldehyde in the presence of Na(OAc)3BH to yield the corresponding reductive amination product 8. TFA-mediated Boc-deprotection of carbamate 8 followed by N-acylation of the resultant primary amine 9 with 5-(benzyloxy)-5-oxopentanoyl chloride 11 afforded benzyl ester 12 in moderate yield. Compound 12 was subsequently converted into the corresponding carboxylic acid 13 via palladium-catalyzed hydrogenolysis. The remainder of the peptide linker was installed with a benzotriazole-mediated amide coupling protocol employing gly4Cbz arriving at ester intermediate 14. Benzyl ester hydrogenation with 10% palladium on carbon at atmospheric pressure then arrived at the desired carfentanil-targeting hapten F13. Supporting Information.

Scheme 2.

Scheme 2

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Reagents and conditions: (a) N-Boc-4-piperidone, NaOH, CHCl3, THF, 0 °C → RT, overnight; (b) propionic anhydride, TEA, EtOAc, MeOH, reflux, 5 h; (c) HCl (4 M in dioxane), DCM, 0 °C → RT, 20 h; (d) N-Boc-2-aminoacetaldehyde, Na(OAc)3BH, 1,2-DCE, 0 °C → RT, 20 h; (e) TFA, DCM, 0 °C → RT, overnight; (f) 5-(benzyloxy)-5-oxopentanoic acid, oxalyl chloride, DCM, DMF (cat.), 0 °C → RT, 3 h; (g) benzyl 5-chloro-5-oxopentanoate 11, TEA, DCM, 0 °C → RT, overnight; (h) Pd (10% on C), H2 (40 psi), MeOH, RT, overnight; (i) gly4Cbz, BOP, TEA, DMF, 0 °C → RT, 16 h; (j) Pd (10% on C), H2 (1 atm), MeOH, RT, overnight.

Conjugation and Formulation of Vaccines

All haptens were conjugated to either the CRM (Pfenex, San Diego, CA) carrier protein for immunization studies or bovine serum albumin (BSA) for antibody analysis by ELISA. The F1 hapten was conjugated to proteins via carbodiimide chemistry as described before.21 The novel F11 and F13 haptens were conjugated following procedures previously described for other haptens in the Fn series.21 Briefly, haptens were dissolved at a concentration of 5.2 mM in 0.1 M 2-(N-morpholino)ethanesulfonic acid (MES) buffer pH 5.0 containing 10% DMSO (final) and activated with 208 mM N-ethyl-N′-(3 dimethylaminopropyl)carbodiimide hydrochloride (EDAC, Sigma-Aldrich, St. Louis, MO). The reaction mixture was stirred for 10 min at room temperature, and then CRM or BSA were added to a final concentration of 2.8 mg/mL and stirred for 3 h at room temperature. To terminate the reaction, MES buffer was exchanged with PBS buffer pH 7.2 using an Amicon filter unit (MilliporeSigma, Merck, Burlington, MA) with 50 kDa molecular weight cutoff, and resuspended to a final concentration of 2.5 mg/mL prior to storage at 4 °C. A final concentration of 250 mM sucrose was added to the MES and PBS buffers for CRM stabilization during the conjugation reaction. The haptenation ratios of CRM conjugates were measured by MALDI-TOF (AB SCIEX 5800, Foster City, CA) and estimated according to the following formula,

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as previously described.21 The unconjugated protein and the conjugate vaccines were formulated with aluminum hydroxide adjuvant (Alhydrogel 85, 2%, Brenntag Biosector, Denmark) prior to in vivo immunization studies.

Drugs

Fentanyl citrate was obtained from the University of Minnesota Boynton Pharmacy, and carfentanil hydrochloride was obtained from the NIDA Drug Supply at RTI International (Research Triangle Park, NC). Naloxone hydrochloride was obtained through Sigma-Aldrich (St. Louis, MO). Drug doses are expressed as concentration of the free base.

Animal Subjects

All animal studies were approved by the University of Minnesota Animal Care and Use Committee and conducted in AAALAC-approved facilities. Male Sprague–Dawley rats (Envigo, Indianapolis, IN) were 8 weeks old at arrival. Rats were housed under standard conditions with a 14/10 light/dark cycle and provided with food and water ad libitum.

In Vivo Efficacy: Antinociception, Respiratory Depression, and Bradycardia

Opioid-induced behavioral and pharmacological effects were measured via the hot plate test and pulse oximetry as previously described.21 Briefly, antinociception was assessed by placing rats on a hot plate set to 54 °C (Columbus Instruments, Columbus, OH) at baseline and at 15 min intervals up to 60 min post-drug challenge. Rats were removed from the hot plate when a lift or flick of the hind paw was observed, or by reaching the maximal cutoff of 30 s to avoid thermal tissue damage. Data are displayed as latency to respond. To assess the effects of opioids on respiratory depression and bradycardia, a MouseOx Plus pulse oximeter (Starr Life Sciences, Oakmont, PA) was used to measure oxygen saturation (SaO2) and heart rate (bpm, beats per minute) on the same schedule. Drug doses for in vivo challenges were chosen based on previous studies.21,28

Antibody Analysis

Serum hapten-specific IgG antibody titers were analyzed via indirect ELISA using blood collected from tail veins.21 Determination of hapten-specific IgG antibody titers in lung and heart tissue was performed on supernatant obtained after centrifuging homogenized tissue for 20 min at 10000xg at 4 °C. Ninety-six-well plates were coated with 5 ng/well of the corresponding F1/11/13-BSA conjugate, or unconjugated BSA as a control, diluted in 50 mM Na2CO3, pH 9.6 (Sigma-Aldrich, St. Louis, MO). Plates were blocked with 1% porcine gelatin for 1 h at room temperature. Plates were then incubated with diluted serum samples for 90 min at room temperature with shaking, then an additional 30 min at room temperature without shaking. Plates were washed and incubated with an HRP-conjugated goat anti-rat IgG overnight at 4 °C (1:50,000, Jackson ImmunoResearch Laboratories). The following day, plates were developed with enzyme substrate o-phenylenediamine (OPD; SigmaFast tablet set, Sigma-Aldrich, St. Louis, MO). After 30 min of incubation with OPD, 2% oxalic acid was added to stop the enzymatic reaction. Plates were read at 492 nm on a Tecan Infinite M1000 PRO microplate reader. Titers are reported as the dilution producing 50% maximal binding (EC50).

Analysis of Antibody-Bound and -Unbound (Free) Fentanyl and Carfentanil Concentration in Serum and Tissue

After the last drug challenge, rats were euthanized for collection of serum, brain, heart, and lungs for post mortem analysis of carfentanil or fentanyl concentrations. To obtain drug concentrations (total, bound and unbound), tissue extraction and analysis by LCMS/MS were performed as described previously.21 Additional steps were performed to obtain unbound (free) drug concentrations in lung and heart tissue. Nanosep filter units (10 kDa cutoff, Pall Life Sciences, Port Washington, NY) were pre-treated with 5% Tween-20 in distilled water for 1 h at room temperature and then rinsed with distilled water to minimize hydrophobic interactions. Lung or heart homogenate samples were then centrifuged in the filter units at 10000xg at room temperature for 1 h. Unfiltered samples (total opioid) and flow through from the filter unit (unbound opioid) were analyzed via LCMS/MS. Drug concentrations were determined using an Agilent G6470A triple-quadrupole LCMS/MS system consisting of an Infinity II 1290 G7116B multicolumn thermostat, G7120A high-speed quad pumps, and a G7267B multisampler. Data acquisition and peak integration were analyzed using Mass Hunter software (Tokyo, Japan). Percent unbound was calculated as (unbound opioid/total opioid) × 100%.

Statistical Analysis

Mean latency to respond on the hot plate, oxygen saturation, respiratory rate, and heart rate were analyzed by two-way ANOVA with Geisser–Greenhouse correction followed by Dunnett’s or Bonferroni’s multiple comparisons post-hoc test. Drug concentration in tissue, antibody bound/unbound analysis, and antibody titers were analyzed by one-way ANOVA paired with Tukey’s multiple comparisons post-hoc test. Correlations between antibody titers and drug concentrations were performed via Pearson correlation. Statistical analyses were performed using Prism v.9 (GraphPad, La Jolla, CA).

Results

A Carfentanil-Based Hapten Protects against Carfentanil-Induced Respiratory Depression

To assess the efficacy of vaccine formulations containing the novel carfentanil-based haptens F11 and F13, rats were immunized intramuscularly (i.m.) on days 0, 21, and 42 with 60 μg CRM, F1-CRM, F11-CRM, or F13-CRM adsorbed on 90 μg alum. All active vaccine formulations induced detectable serum IgG antibody titers for the cognate hapten, measured one week after third immunization (Figure S1). Two weeks after the third immunization, rats were challenged s.c. with carfentanil (0.02 mg/kg) and assessed for drug-induced antinociception, respiratory depression, and bradycardia. Rats immunized with F13-CRM were the only group to show any attenuation of antinociception after carfentanil challenge, although this difference is not statistically significant (Figure 2A). All other immunized groups were identical to the control. When assessing protection against other opioid-induced pharmacological effects, F13-CRM prevented carfentanil-induced respiratory depression relative to control at most time points (Figure 2B). In contrast, rats immunized with F11-CRM attained significantly attenuated respiratory depression only at 30 min post-challenge. No differences were seen between any groups when assessing carfentanil-induced bradycardia (Figure 2C). While F1-CRM has been previously shown to protect against fentanyl and sufentanil,21 it did not protect against carfentanil. These data suggest that the F13-CRM conjugate vaccine protects against selected pharmacological and behavioral effects of carfentanil.

Figure 2.

Figure 2

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Immunization with F13-CRM protected against carfentanil- and fentanyl-induced respiratory depression. Following a baseline measurement, immunized male Sprague–Dawley rats (n = 6 per group) were challenged s.c. with 0.02 mg/kg carfentanil and tested every 15 min for (A) antinociception via latency to respond on a hot plate, (B) respiratory depression reported as the percent (%) oxygen saturation measured by pulse oximetry, and (C) bradycardia reported as heart rate (beats per minute, bpm). The same cohort was challenged s.c. with 0.1 mg/kg fentanyl and tested every 15 min for (D) antinociception, (E) respiratory depression, and (F) bradycardia. Finally, rats were challenged s.c. with a mixture of 0.05 mg/kg fentanyl and 0.01 mg/kg carfentanil delivered as a single bolus and tested every 15 min for (G) antinociception, (H) respiratory depression, and (I) bradycardia. Naloxone (0.1 mg/kg) was administered at 60 min and its effects were assessed 15 min later. Data are presented as mean ± SEM. Statistical analysis was performed via two-way ANOVA paired with Dunnett’s (B, C, E–I) or Bonferroni’s (A, D) multiple comparisons post-hoc tests. Statistical symbols: **** p < 0.0001 ** or **(red) p < 0.01, * or *(red) or #(red) p < 0.05. Placement of * indicates significance between F1-CRM and CRM, #(red) indicates significance between F11-CRM and CRM, and *(red) indicates significance between F13-CRM and CRM.

A Carfentanil-Based Hapten May Cross-Protect against Fentanyl

Because of the increasing involvement of fentanyl analogues in drug-related overdoses, it is important to identify possible cross-reactivity of lead haptens within the fentanyl-like chemical family to assess the potential of individual vaccines for cross-protection. Previous studies have demonstrated the feasibility of targeting both fentanyl and carfentanil with the same hapten structure.28 Here, the same rats were challenged s.c. with 0.1 mg/kg fentanyl, and immunization with F1-CRM protected against fentanyl-induced antinociception, respiratory depression, and bradycardia (Figure 2D–F). Rats immunized with F13-CRM also showed significant protection against fentanyl-induced respiratory depression at 15 min post-drug challenge (Figure 2E). These results indicate that the F13 hapten may target both carfentanil and fentanyl, warranting further investigation.

Vaccine Efficacy against a Mixture of Fentanyl and Carfentanil

Because of the potential efficacy of F13 against both fentanyl and carfentanil, rats were then re-challenged s.c. with a mixture of fentanyl (0.05 mg/kg) and carfentanil (0.01 mg/kg) delivered as a single bolus. While no groups were significantly protected against the behavioral effects of the fentanyl/carfentanil mixture, rats immunized with F13-CRM trended toward protection as reported by the hot plate test (p = 0.0612 at t = 45 min; p = 0.1090 at t = 60 min) and oximetry measures (p = 0.0544 at t = 45 min and p = 0.0453 at t = 60 min) relative to all other groups (Figure 2G,H). Significant differences in heart rate were found only at 60 min post-challenge (Figure 2I). After 60 min, rats were rescued with naloxone (0.1 mg/kg, s.c), and all immunized groups returned to baseline in all parameters tested, indicating that fentanyl- and carfentanil-specific antibodies do not interfere with naloxone rescue of opioid-induced effects.

Anti-carfentanil Vaccines Prevent Distribution of Carfentanil to the Brain

To assess the dose–response relationship of carfentanil and its effects on antinociception and respiratory depression, rats were administered carfentanil (0.005 mg/kg, s.c.) every 15 min up to a cumulative dose of 0.02 mg/kg. Antinociception and pulse oximetry were measured at each time point before the subsequent injection. Both F11- and F13-CRM offered protection against carfentanil-induced antinociception at 0.005 mg/kg carfentanil when compared to CRM immunized controls, with F13-CRM also protecting against doses up to 0.015 mg/kg (Figure 3A). At 0.015 mg/kg, groups immunized with either F11-CRM or F13-CRM had significantly higher oxygen saturation compared to control (Figure 3B), and F13-CRM immunized animals also displayed significantly higher heart rate (Figure 3C). Fifteen minutes after the final carfentanil dose, animals were euthanized and serum, brain, heart and lungs were collected to determine the concentration of carfentanil in each organ. All immunized groups showed a significantly lower brain:serum carfentanil ratio compared to control, indicating that antibodies produced from active immunization retained the drug in the serum and prevented its distribution to the brain. Groups immunized with F11 and F13 displayed significantly lower brain:serum ratios of carfentanil compared to the F1-CRM group. The group immunized with F13-CRM displayed an increased carfentanil concentration in the heart and lungs compared to control or F1-CRM immunized groups, likely due to an increase in the antibody-bound fraction of drug in either serum, extracellular fluids, or parenchyma. Together, these data show that active immunization alters the distribution of carfentanil to the brain, and possibly other target organs such as the heart and lungs, which attenuates carfentanil-induced pharmacological effects.

Figure 3.

Figure 3

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Vaccination alters the distribution of carfentanil to key organs. Following a baseline measurement, immunized male Sprague–Dawley rats (n = 4–6 per group) were repeatedly challenged s.c. with 5 μg/kg carfentanil every 15 min and tested every 15 min for (A) antinociception, (B) oxygen saturation (%), and (C) heart rate (bpm). At 15 min after the final dose, rats were euthanized, and organs were collected to analyze the distribution of carfentanil. (D) Ratio of carfentanil in the brain versus the serum, and concentration of carfentanil in the (E) heart, and (F) lungs. Data are presented as mean ± SEM. Statistical analysis was performed via one- (D−F) or two-way (A−C) ANOVA paired with Tukey’s (D-F) Dunnett’s (B,C), or Bonferroni’s (A) multiple comparisons post-hoc test. Statistical symbols: **** or ****(red) or ####(red) p < 0.0001, ***(red) p < 0.001, ** or ## or **(red) p < 0.01,* or # or *(red) or #(red) p < 0.05. Placement of * above columns indicates significance compared to control, # above columns indicates significance compared to F1-CRM, #(red) indicates significance between F11-CRM and CRM, and *(red) indicates significance between F13-CRM and CRM.

Comparison of Bivalent Vaccination Strategies in Protecting against the Behavioral and Pharmacological Effects of Carfentanil, Fentanyl, and Their Mixture

An independent follow-up study conducted in a second cohort of rats tested the hypothesis that bivalent vaccination with F1-CRM and F13-CRM would protect against carfentanil, fentanyl, and their mixture to a greater degree than each individual vaccine. In this study, rats were immunized on days 0, 14, 28, and 42 with 60 μg of either individual F1-CRM or F13-CRM conjugates (monovalent), or both F1- and F13-CRM co-administered as individual vaccinations on either hind leg (bivalent co-administration, or Co). The co-administration strategy consisted of immunization with half doses (30 μg) of each individual vaccine to equal the total conjugate and alum dose used in monovalent vaccination. An additional treatment group tested whether a heterologous vaccination strategy, consisting of alternating full doses of either F1-CRM or F13-CRM every 2 weeks (bivalent heterologous, or Het), would enhance protection against the pharmacological effects of carfentanil, fentanyl, or their mixture. The monovalent, co-administered, and heterologous vaccinations strategies provided rats with an equal total dose of protein. Antibody titers against both F1 and F13 were detected on day 49 (Figure S1) and showed that co-administration did not interfere with the generation of antibodies to either hapten. Rats were then challenged with multiple drugs over the next 3 weeks, with drugs administered each week randomized among groups to reduce the potential confounding effects of tolerance. As expected, rats vaccinated with F1-CRM were significantly protected against the antinociceptive effects of fentanyl (0.1 mg/kg, s.c.). Interestingly, rats that received the co-administered immunization with both F1- and F13-CRM were protected against fentanyl-induced antinociception at 30, 45, and 60 min post-drug challenge, and trended toward protection (p = 0.0798) at 15 min (Figure 4A). Rats immunized with F1- and F13-CRM on a heterologous immunization schedule were only protected against the antinociceptive effects of fentanyl at 45 and 60 min post-exposure. When assessing fentanyl-induced respiratory depression (Figure 4B) and bradycardia (Figure 4C), co-administered vaccination afforded protection against fentanyl-induced respiratory depression at 30 min post-drug exposure, similar to rats that received F1-CRM. However, both bivalent immunization strategies trended toward protection at all time points [at t = 15 min, p = 0.0567 (Het) and p = 0.0570 (Co); at t = 45 min, p = 0.0684 (Het) and p = 0.0634 (Co); at t = 60 min, p = 0.1010 (Het) and p = 0.1634 (Co)]. Following a carfentanil challenge (0.02 mg/kg, s.c), rats immunized with F13-CRM were protected against carfentanil-induced respiratory depression, as expected (Figure 4E). While not statistically significant, the co-administered immunized group displayed early trends in protection (p = 0.1513 at t = 15 min) against carfentanil-induced respiratory depression (Figure 4E). No differences in carfentanil-induced antinociception (Figure 4D) or bradycardia were detected (Figure 4F).

Figure 4.

Figure 4

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Bivalent vaccination strategies may enhance protection against the pharmacological effects of both carfentanil and fentanyl. Rats were vaccinated with F1-CRM, F13-CRM, or both in either a co-administered (Co) or heterologous (Het) vaccination schedule every 2 weeks for a total of four vaccinations. Following a baseline measurement, rats (n = 4–6 per group) were challenged s.c. with 0.1 mg/kg fentanyl and tested every 15 min for (A) antinociception, (B) oxygen saturation (%), and (C) heart rate (bpm). The same cohort of rats was challenged s.c. with 0.02 mg/kg carfentanil and tested every 15 min for (D) antinociception, (E) respiratory depression and (F) bradycardia. Finally, rats were challenged with a combined s.c. bolus dose of fentanyl (0.05 mg/kg) and carfentanil (0.01 mg/kg). Every 15 min, rats were tested for (G) antinociception, (H) respiratory depression and (I) bradycardia. Data are presented as mean ± SEM. Statistical analysis was performed via two-way ANOVA paired with Dunnett’s (A–C, E, F, H, I) or Bonferroni’s (D, G) multiple comparisons post-hoc test. Statistical symbols: ****(red) or ****p < 0.0001, ##(red) or **(red) or **p < 0.01, # or #(red) or *(red) or * p < 0.05. Placement of * indicates significance between F1-CRM and CRM, # indicates significance between F13-CRM and CRM, #(red) indicates significance between heterologous and CRM, and *(red) indicates significance between co-administration and CRM.

After challenge with fentanyl (0.05 mg/kg, s.c) combined with carfentanil (0.01 mg/kg, s.c), only the co-administered bivalent group was significantly protected against drug-induced antinociception at all time points post-exposure (Figure 4G). When assessing respiratory depression, rats immunized with F13-CRM or the co-administered bivalent regimen were significantly protected from respiratory depression at most time points, while the heterologous immunized group trended toward protection (p = 0.0804 at t = 15 min) at early time points (Figure 4H). Similar to the carfentanil challenge, no differences were found between any group when measuring heart rate, except for the latest time point (Figure 4I). These data suggest that bivalent immunization strategies can protect against the pharmacological effects of carfentanil, fentanyl, or their mixture.

Bivalent Immunization Strategies Alter the Distribution of Higher Doses of Fentanyl and Carfentanil to Key Organs

Rats were challenged with a final mixture of 0.1 mg/kg fentanyl and 0.02 mg/kg carfentanil (2× dose from the original dual challenge). Oxygen saturation and heart rate were measured at 30 min post-challenge, and then blood, brain, lungs, and heart were collected to measure opioid concentrations in each compartment. At this higher dose, no differences in respiratory depression and bradycardia were detected across groups (Figure S2), but there was a significant reduction in brain:serum ratio of carfentanil or fentanyl in all groups immunized with either the F13 hapten or the F1 hapten, respectively (Figure 5A,B). Carfentanil and fentanyl concentrations were also increased in the heart (Figure 5C,D) and lungs (Figure 5G,H) in groups immunized with either the F13 or F1 haptens, which correlated with increased opioid-specific antibody titers (Figure S4). In these organs, there was a significant decrease in unbound (free circulating) fentanyl in the F1 monovalent and bivalent immunized groups and a decrease in unbound carfentanil in the F13 monovalent and bivalent immunized groups, indicating that most of the drug circulating in these organs is antibody-bound (Figure 5E,F,I,J). While it is not clear whether antibody-bound complexes would reside in blood, extracellular fluids, or parenchyma, these data are consistent with previous reports of antibody-bound nicotine in the heart, lungs, and other tissues.37,38 These results indicate that a bivalent vaccination strategy that combines fentanyl- and carfentanil-based haptens may enhance protection against both drugs alone and in combination when compared to monovalent vaccination strategies.

Figure 5.

Figure 5

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Bivalent immunization strategies alter the distribution of carfentanil and fentanyl to key organs. Immunized rats (n = 4–6 per group) were challenged with a combined s.c. bolus dose of 0.1 mg/kg fentanyl and 0.02 mg/kg carfentanil. After 30 min, serum, brain, lungs, and heart were collected. Shown: (A) brain:serum ratio of carfentanil and (B) brain:serum ratio of fentanyl. Hearts were analyzed for (C) total carfentanil concentration (bound + unbound), (D) total fentanyl concentration (bound + unbound), (E) unbound (free circulating) carfentanil as a percent of total carfentanil, and (F) unbound fentanyl as a percent of total fentanyl. Lungs were analyzed for (G) total carfentanil concentration (H) total fentanyl concentration, (I) unbound carfentanil as a percent of total carfentanil, and (J) unbound fentanyl as a percent of total fentanyl. Data are presented as mean ± SEM. Statistical analysis was performed via one-way ANOVA paired with Tukey’s multiple comparisons post-hoc test. Symbols: #### or ****p < 0.0001, ###p < 0.001, ## or **p < 0.01, # or *p < 0.05. Placement of * above columns indicates significance compared to control or as indicated by brackets, and # indicates significance compared to F1-CRM.

Discussion

Vaccines may offer a viable approach to prevent overdose related to carfentanil, fentanyl, and their mixtures. Previous studies showed that vaccines containing carfentanil-based haptens are effective at reducing antinociception, respiratory depression, and distribution of carfentanil to the brain after drug challenge.28 This study reports the development of vaccines containing two novel carfentanil-based F11 and F13 hapten structures (Figure 1), which were conjugated to CRM, a carrier protein suitable for further product development,39 and adsorbed to a commercially available aluminum adjuvant commonly used in marketed vaccine formulations. Similar to F1, both F11 and F13 haptens lack the N-phenethyl substituent, and thus are considered unscheduled by the Drug Enforcement Agency (Pravetoni, personal communication). F11 contains a 5-atom linker displaying an amino terminal group suited for conjugation to aspartic and glutamic acid residues on the carrier protein, while F13 contains an 18-atom long tetraglycine linker displaying a carboxyl group suitable for lysine-reactive conjugations (Figure 1). Both haptens were conjugated to CRM by carbodiimide chemistry, which yielded similar haptenation ratios (F11-CRM = 10; F13-CRM = 12), minimizing concerns for direct comparison due to their similar molecular weight, number of haptens displayed on the carrier, or relative conjugation efficiency. Given that F13 outperformed F11in vivo, it is likely that the longer linker length enabled the accessibility of the carfentanil moiety and the carrier structure to be recognized by hapten-specific B and cognate carrier-specific CD4+ T cell lymphocytes after immunization. The importance of the linker length has been noted with other vaccines against OUD and SUD40−42 and may inform design of future haptens derived from other opioids, psychostimulants, or designer drugs.

Antibodies elicited by F13 had significant cross-reactivity with the F1 hapten, while the F1 hapten had almost no cross-reactivity to the F13 hapten, which is consistent with the in vivo efficacy data for F1-CRM and F13-CRM. The F13 hapten produced significantly higher antibody titers than F1 when each was tested against its cognate coating antigen (Figure S1). While comparisons of ELISA data resulting from different coating antigens should be interpreted with caution, this may suggest that the F13 hapten is generally more immunogenic than F1, perhaps due to the increased number of polar residues within the hapten itself, which could potentially result in greater engagement of hapten-specific B cell receptors (BCRs). Although in the context of carfentanil, F13 proved superior to F1, F1 was still the more effective hapten against fentanyl, and possibly other analogues.21 Hence, in an attempt to target fentanyl and carfentanil simultaneously, this study tested the efficacy of bivalent vaccine formulations that combine F1-CRM and F13-CRM.

Multivalent immunization is an appealing strategy to address multiple drug targets simultaneously, bypassing a limitation of current pharmacotherapies for SUDs. To date, bivalent or trivalent immunization strategies have shown proof of principle against nicotine,43,44 heroin/fentanyl,19,29,33 and heroin/oxycodone,31,45 but our understanding of how to best deploy multivalent vaccines in the context of OUD and other SUDs is still limited. For instance, it is not clear if divalent presentation of structurally unrelated haptens on the same carrier protein is a viable strategy to circumvent the need for admixing two individual conjugate vaccines in the same formulation.30,32 The current study showed that when comparing antibody titers from bivalent vaccination strategies, co-administration produced significantly higher F13-specific antibody titers compared to heterologous immunization, while F1-specific titers were similar between the two groups. This discrepancy may be because a bivalent co-administration strategy simultaneously engages both overlapping and non-overlapping B cell populations specific for one antigen or the other, while a heterologous prime/boost strategy introduces one antigen at a time, which may refine the antibody specificity toward those that are specific for both fentanyl and carfentanil at the expense of higher titers. While F1 was used for the initial antigen followed by an F13 boost in the heterologous immunization strategy, it would be of interest to test whether priming with an antigen of potentially higher immunogenicity (e.g., F13) would further increase the antibody concentration or narrow the selectivity of the polyclonal antibody responses toward an immunodominant epitope or antigen. Despite the differences seen in antibody titers, in vivo results suggest that either strategy could be effective at reducing carfentanil’s centrally mediated effects. Future studies will determine whether multiple individual vaccines could be co-administered in the same vehicle without interfering with their respective properties.

The in vivo pharmacokinetics of carfentanil are relatively understudied. One published study showed that administration of high doses of carfentanil (>0.01 mg/kg) cause nonlinear accumulation and impaired clearance in rats.46 The authors postulated this to be one of the main causes of the toxicity and life-threatening effects associated with carfentanil. Consistently, the behavioral effects of carfentanil were longer-lasting than fentanyl in non-human primate models of self-administration,47 which provides additional evidence of the longer half-life of fentanyl analogues. The carfentanil doses used for the current study ranged from 0.01 to 0.02 mg/kg in most cases, exceeding the limit needed for nonlinear accumulation. Despite this, rats immunized with the carfentanil-based hapten F13 showed attenuation of respiratory depression after carfentanil challenge, indicating that immunization and subsequent neutralization of carfentanil in the serum via antibody binding may prevent the toxic effects associated with nonlinear accumulation. Published literature shows that opioids in immunized animals persist in the serum for much longer than controls,23 suggesting that antibody-bound opioid is protected from metabolism. In this case, antibody-bound carfentanil may prevent impaired clearance by neutralizing a portion of the serum carfentanil and allowing for linear accumulation and clearance similar to what is observed at lower carfentanil doses. As the potentially unusual pharmacokinetics of carfentanil are studied in further detail, it will also be necessary to determine how the addition of carfentanil-specific antibodies through active or passive immunization change these pharmacokinetic properties. For instance, we have previously reported that immunization with OUD vaccines increases antibody-bound opioid in the serum, reducing the free circulating opioid.25,48,49 This study extended these findings by showing that vaccination resulted in a decrease in free circulating fentanyl and carfentanil in the heart and lungs, which may contribute to protection against drug-induced effects on respiratory and cardiovascular functions. Previous reports showed that nicotine vaccines elicit functional drug-specific antibody IgG and IgA responses in various organs including the lungs.37,38 Consistently, the current results suggest that vaccine-induced antibodies may be effective in decreasing the unbound fentanyl and carfentanil circulating within the lungs and heart vasculature or parenchyma.

Despite showing considerable efficacy at attenuating carfentanil-induced antinociception and respiratory depression, the vaccines tested remained largely ineffective at reducing carfentanil-induced bradycardia. While most opioids have been shown to induce some level of bradycardia after administration, published literature shows that the relative doses needed to induce bradycardia can vary between opioids.50 There are few papers investigating the dose–response relationships of different pharmacological effects of carfentanil,46,51 but our current data may suggest that bradycardia is induced preferentially and/or at lower doses as compared to analgesia or respiratory depression, making it resistant to attenuation by active immunization. Comprehensive studies detailing carfentanil’s dose-dependent pharmacological effects, including its reinforcing effects, will be needed in order to fully understand and develop effective medications against this extremely potent opioid.

As the illicit use of carfentanil and other highly potent opioids increases, development of effective therapeutics is needed to prevent toxicity or overdose due to accidental or deliberate exposure in drug users, medical workers, first responders, or military personnel. While the approval of naloxone for reversal of opioid overdose has saved countless lives, naloxone may not be as effective in reversing the effects of highly potent opioids. Development of vaccines that can induce the production of antibodies that prevent opioids from entering the brain is a novel strategy that may be more effective with highly potent opioids due to the favorable molar ratio of drug molecules to antibody binding sites. Here, the development and testing of a pair of novel anti-carfentanil conjugate vaccines are described. We found that F13-CRM was more effective than F11-CRM at attenuating the effects of carfentanil, fentanyl, or their mixture. We then tested the effectiveness of F13-CRM to be administered in tandem with a lead fentanyl vaccine (F1-CRM) and found that either co-administered or heterologous immunization strategies further increased protection against both drugs. These studies inform and justify further development and clinical testing of F13-CRM as a lead anti-carfentanil vaccine in single and multivalent formulations to reduce the incidence of carfentanil overdose in subjects at risk of exposure.

Acknowledgments

This work was supported by the National Institute on Drug Abuse (NIDA) and the National Institute of Neurological Disorders and Stroke (NINDS) under award numbers UG3DA048386 (M.P.), T32DA007097 (M.M.W. and B.C.), and F31DA054760 (B.C.), and the University of Minnesota Graduate Program in Pharmacology (D.S.). We thank our Scientific Officer Dr. Jason Sousa from the Division of Therapeutics and Medical Consequences from NIDA.

Glossary

Abbreviations

OUD

opioid use disorder

MOUD

medications for opioid use disorder

BSA

bovine serum albumin

SUD

substance use disorder

MOR

mu-opioid receptor

CRM

cross-reactive material

SaO2

oxygen saturation

bpm

beats per minute

s.c.

subcutaneous

i.m.

intramuscular

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsptsci.1c00260.

  • Synthesis methods for F11 and F13 (Schemes S1 and S2); opioid-specific antibody titers in the serum, heart, and lungs of rats after immunization (Figure S1); pharmacological effects of a high-dose fentanyl/carfentanil challenge in rats (Figure S2); total combined fentanyl/carfentanil molar concentration in serum, heart, and lungs after fentanyl/carfentanil challenge (Figure S3); and correlation between opioid-specific IgG titers and concentration of opioid in the brain, serum, heart, and lungs (Figure S4) (PDF)

Author Present Address

# Medtronic, Neuromodulation Division, St. Paul, Minnesota

Author Contributions

B.C. and M.M.W. are co-first authors. B.C. designed and performed experiments, analyzed data, and wrote the manuscript. M.M.W. designed and performed experiments, analyzed data, and wrote the manuscript. V.G. synthesized materials for experiments. A.J.K. synthesized materials for experiments and edited the manuscript. D.S. performed experiments and analyzed data. R.J. synthesized materials for the experiments. S.A. synthesized materials for experiments and edited the manuscript. S.R. synthesized materials for experiments and edited the manuscript. S.D.C. reviewed and edited the manuscript. M.P. designed experiments, analyzed data, and wrote the manuscript.

The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

The authors declare the following competing financial interest(s): Pravetoni is the inventor of Fentanyl hapten-conjugates and methods for making and using same, Application No. 62/932,757. Pravetoni, Averick, and Runyon are inventors of Fentanyl haptens, fentanyl hapten conjugates, and methods for making and using the same, Application No. 62/989,417. The other authors declare no conflict of interest.

Special Issue

Published as part of the ACS Pharmacology & Translational Science virtual special issue “New Drug Modalities in Medicinal Chemistry, Pharmacology, and Translational Science”.

Supplementary Material

pt1c00260_si_001.pdf (727.9KB, pdf)

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