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

Article abstract of DOI:10.1002/anie.201511654

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.

Full text of DOI:10.1002/anie.201511654

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International Edition: DOI: 10.1002/anie.201511654
German Edition: DOI: 10.1002/ange.201511654
Vaccines
Combatting Synthetic Designer Opioids: A Conjugate Vaccine Ablates
Lethal Doses of Fentanyl Class Drugs
Paul T. Bremer+, Atsushi Kimishima+, Joel E. Schlosburg, Bin Zhou, Karen C. Collins, and
Kim D. Janda*
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.
F
entanyl is an effective synthetic opioid that is used legally as
a schedule II prescription pain reliever. However, fentanyl
presents a significant abuse liability owing to the euphoric
feeling it elicits by activating m-opioid receptors (MOR) in the
brain, the same pharmacological targets as the illegal sched-
ule I opioid, heroin.[1] Excessive MOR activation results in
respiratory depression, which can be fatal.[2] Fentanyl is > 10-
fold more potent than heroin, and > 80-fold more potent than
morphine. As a result, fentanyl poses a significant risk for
overdosing when it is consumed from unregulated sources.[3]
Furthermore, the ease of fentanyl synthesis enables illegal
production and the creation of designer drug analogues.[4] The
fact that the pharmacology of these analogues has yet to be
properly characterized makes them particularly dangerous,
especially when certain modifications, even methyl additions,
can increase potency, notably at the 3-position (Figure 1).[5]
Last July, the National Institute on Drug Abuse (NIDA)
reported an alarming surge in fentanyl overdose deaths:[6] the
latest update in a long stream of overdose cases starting with
a-methylfentanyl (also known as “China White”) in the late
Figure 1. Construction of fentanyl immunoconjugate and structures of
fentanyl analogues recognized by polyclonal antibodies.
1980s.[7] A newer designer analogue, acetylfentanyl, further
exacerbates the opioid epidemic because of its deceptive sale
as heroin or as a heroin mixture,[8] and it has been linked to
a number of overdose deaths.[9] In addition to the US, fentanyl
abuse is on the rise across Europe; while the most overdose
deaths occurred in Estonia, the highest consumption of
fentanyl per capita was reported in Germany and Austria.[10]
To combat the harmful and addictive effects of fentanyl
and its analogues, we pursued an immunopharmacotherapeu-
tic approach, similar to previous campaigns for addiction
therapeutics against cocaine,[11] nicotine,[12] methampheta-
mine,[13] and heroin.[14] The basis of this strategy involves
active vaccination of a protein–drug conjugate to generate an
in vivo immunoantagonist, which effectively minimizes con-
centrations of the target drug at the sites of action. As a result,
the vaccine reduces the addiction liability and overdose
potential of the specific drug. In this work, we report the first
instance of an efficacious fentanyl-conjugated vaccine. Upon
immunization, this vaccine successfully stimulated endoge-
nous generation of IgG antibodies with specificity for fentanyl
class drugs. Moreover, mouse antiserum showed nanomolar
affinity for a variety of fentanyl analogues by SPR analytical
[*] P. T. Bremer,[+] Dr. A. Kimishima,[+] Dr. J. E. Schlosburg, Dr. B. Zhou,
Dr. K. C. Collins, Prof.Dr. K. D. Janda
Departments of Chemistry and Immunology
The Skaggs Institute for Chemical Biology
Worm Institute for Research and Medicine (WIRM)
The Scripps Research Institute
10550 N Torrey Pines Rd BCC-582, La Jolla, CA 92037 (USA)
E-mail: kdjanda@scripps.edu
[+] These authors contributed equally to this work.
Supporting information for this article can be found under http://dx.
doi.org/10.1002/anie.201511654.
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methods. When mice were dosed with potentially lethal
quantities of fentanyl analogues, the vaccine imparted
significant protection. No other vaccines to date have
demonstrated blockade of the acutely lethal effects of any
drugs of abuse. Importantly, our research efforts have yielded
significant progress for mitigating the pharmacodynamic
effects of fentanyl class drugs.
In developing a fentanyl vaccine, hapten design presented
the initial and possibly the most crucial challenge. As we have
reported previously, small molecule haptens must faithfully
preserve the natural structure of the target molecule to make
a successful immunoconjugate.[15] Confronted not only with
fentanyl, but also designer analogues, our hapten incorpo-
rated the core N-(1-phenethylpiperidin-4-yl)-N-phenylaceta-
mide scaffold to achieve broad immune specificity for
virtually all of the fentanyl derivatives. With this mindset,
the propanoyl group in fentanyl was selected as the point of
linker attachment because it would not sterically encumber
the core structure (Figure 1). Ultimately, hapten design was
accomplished by replacing the propanoyl group in fentanyl
with a glutaric acid moiety. The added carboxyl group enabled
N-hydroxysuccinimide (NHS) ester formation for bioconju-
gation of the hapten[16] to an immunogenic carrier protein
(Figure 1). This reliable amide coupling reaction is a standard
method for generating hapten–protein conjugates,[12c,17] facil-
itating high loading of fentanyl onto bovine serum albumin
(BSA) and tetanus toxoid (TT) proteins through surface
lysine residues: 38 and 40 copies were obtained, respectively,
as assessed by MALDI-TOF spectra (Figure S1a,b). Of the
two conjugates, the fentanyl-TT conjugate (Fent-TT;
Figure 1) was chosen for immunization because TT is
a component of clinically approved tetanus and glycoconju-
gate vaccines. For vaccine formulation, Fent-TT was com-
bined with the adjuvants Al(OH)3 (alum) and CpG oligode-
oxynucleotide (ODN) 1826, which are effective in boosting
IgG antibody responses to a heroin conjugate vaccine.[14a]
When mice were immunized with the Fent-TT vaccine, it
induced very high anti-fentanyl antibody midpoint titers by
ELISA of > 100,000 (Figure 2), providing ample in vivo
neutralization capacity for a variety of fentanyls (Figure 1).
To assess vaccine performance, we employed antinoci-
ception assays, which are a standard method for measuring
the analgesic potential of opioid drugs in rodent models.[14a,18]
Opioids such as fentanyl increase pain thresholds in a dose-
responsive manner, and these thresholds can be quantified by
measuring animal latency to nociception induced by a hot
surface. A drug vaccine will blunt the pharmacological action
of the target drug through serum-antibody-mediated immu-
noantagonism of an administered dose. Therefore, a successful
vaccine should shift the drug dose-response curve in anti-
nociception assays to higher concentrations. Comparison of
drug ED50 doses in vaccinated and non-vaccinated mice
serves as a useful metric for drug vaccine efficacy. Previously,
we have reported vaccine-induced shifts of about 5 to 10-fold,
which caused heroin-dependent rats to extinguish drug self-
administration.[14a,b] In the current study, we observed large
fentanyl ED50 shifts of over 30-fold. Remarkably, during the
initial week-6 testing session, fentanyl dosing was incapable of
overriding the protective capacity of the vaccine (Figure S2).
Figure 2. Timeline of experiments and anti-fentanyl antibody titers.
Fent-TT (50 mg) was formulated with alum (750 mg)+CpG ODN 1826
(50 mg) and administered i.p. to each mouse (N=6). IgG titers were
determined by ELISA against a fentanyl-BSA conjugate. Points denote
meansÆSEM. Key: i=vaccine injection, a=antinociception assay,
f=affinity determination by SPR, b=blood/brain biodistribution study.
One month later (week-10), anti-drug titers in vaccinated
mice had decreased to a point where smaller fentanyl doses
could be used to generate full dose-response curves for ED50
determination; large vaccine-mediated shifts were observed
(33-fold in the tail immersion test, Figure 3). Strikingly, at the
two largest doses that were safely administered to vaccinated
mice (2.2 and 4.4 mgkgÀ1), untreated mice experienced an 18
and 55% fatality rate, respectively, thus demonstrating the
ability of the vaccine to block lethal fentanyl doses.
As a testament to the ability of the vaccine to neutralize
other fentanyl analogues, Fent-TT-immunized mice showed
protection from two of the most common illegal fentanyl
derivatives,
3-methylfentanyl
and
a-methylfentanyl
(Figure 1). The a-Me analogue was equipotent with the
parent compound, and the vaccine was able to shift the a-Me
ED50 by about 8-fold (Figure 3). On the other hand, the 3-Me
analogue was extraordinarily potent (about 10-fold greater
than fentanyl), yet the vaccine still produced a 4-fold ED50
shift (Figure 3). Overall, our behavioral results indicate that
the Fent-TT vaccine provided ample attenuation of large
fentanyl doses in vivo while demonstrating a therapeutically
useful level of cross-reactivity to fentanyl analogues.
Clinically, these results implicate Fent-TT as an effective
addiction therapy for curbing fentanyl abuse and overdose-
induced lethality.
From a pharmacokinetic standpoint, we investigated the
effect of the Fent-TT vaccine on the biodistribution of
a fentanyl dose. Following administration of a fentanyl dose,
we sacrificed both control and vaccinated mice at roughly the
tmax (time at peak drug plasma concentrations) and measured
fentanyl concentrations in both brain and blood samples by
LCMS (Figure 4). Our results clearly show how serum
antibodies in vaccinated mice act as a depot to bind 45-
times the amount of fentanyl relative to serum proteins in
control mice. This translated to a significant reduction in
fentanyl brain concentrations of vaccinated mice, lending to
a pharmacological explanation of how the vaccine attenuates
fentanyl psychoactivity.
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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 meansÆSEM 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.
binding.[20] Our results from the SPR competition experiment
(Figure 5a) indicated that antibodies from Fent-TT-immu-
nized mice have high affinity for fentanyl derivatives,
generating binding curves with low nanomolar IC50 values
and limits of detection in the pM range (Figure 5a; Support-
ing Information, Figure S3). Relative affinities between
analogues with different R1 alkyl groups were very similar,
likely owing to the fact that the R1 position was used as
a linker attachment point. As expected, methylation at other
positions resulted in lower affinity, but the IC50 values were
still < 100 nm. Furthermore, the SPR IC50 values mirrored the
results in behavioral assays, and in both cases followed a trend
of Fent > a-Me > cis-3-Me. Because the Fent-TT vaccine gave
broad specificity to fentanyl class drugs, two clinically used
opioids, methadone (MeD) and oxycodone (Oxy), were
Figure 4. Biodistribution of fentanyl in blood and brain samples.
tested to ensure minimal cross reactivity. Indeed, affinities
for these opioids were > 7,500 times lower compared to
fentanyl (Figure 5a), demonstrating that they could still be
used in Fent-TT-vaccinated subjects.
Vaccinated and control mice (N=6 each) were dosed with 0.2 mgkgÀ1
fentanyl and tissue was harvested 15 min post-injection. Fentanyl
quantification was performed by LCMS analysis. Bars denote mean-
s+SEM. ***p<0.001, unpaired t-test.
Further validation of the SPR method was pursued to
confirm that the generated IC50 values were representative of
the actual KD; hapten affinity does not always reflect free
drug affinity. To address this problem, we affinity purified
anti-fentanyl antibodies and loaded them onto an SPR chip
for direct measurement of free fentanyl binding kinetics. As
shown in the sensorgrams (Figure 5b), the fentanyl KD of
purified antibodies (2 nm), is in close agreement with the IC50
value determined by the competition method (5 nm). Thus, we
have demonstrated the SPR competition method as an
accurate way to measure drug affinities of polyclonal serum
antibodies. A crucial aspect of immunopharmacotherapy is
proper characterization of anti-drug antibodies, and the SPR
method could help to facilitate this facet of drug vaccine
development. Additionally, the method could be used to
Behavioral and pharmacokinetic results were corrobo-
rated with thorough biochemical analysis of antiserum
derived from Fent-TT-vaccinated mice. To achieve this, we
employed surface plasmon resonance (SPR) spectroscopy,
a highly sensitive technique for investigating protein–protein
or protein–small-molecule binding interactions.[19] In a new
application of SPR, we measured binding affinities of
polyclonal antibodies in vaccinated mouse serum for various
fentanyl analogues. Diluted mouse serum was preincubated
with a series of concentrations of selected fentanyl derivatives
and then injected into a Biacore 3000 containing a Fent-BSA-
coated chip. Essentially, this method is a more sophisticated
version of competitive ELISA in which serially diluted free
drug competes with an immobilized drug hapten for antibody
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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. 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 258C on a BiOptix 404pi. Original experimental sensorgrams are shown in black and fitted curves are traced in white.
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Acknowledgements
We thank the National Institute on Drug Addiction for
funding (grants R21DA039634, F31DA037709) and Dr.
Michael Taffe for providing DEA licensing (RT0485537).
This is manuscript number 29249 from TSRI.
Keywords: antibody · biosensors · conjugate vaccines ·
fentanyls · immunology
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Received: December 25, 2015
Revised: January 22, 2015
Published online: February 15, 2016
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