Consequence of Hapten Stereochemistry: An Efficacious Methamphetamine Vaccine

Article abstract of DOI:10.1021/jacs.9b07294

Recent trends in methamphetamine (METH) misuse and overdose suggest society is inadvertently overlooking a brewing METH crisis. In the past decade, psychostimulant-related lethal overdoses and hospitalizations have skyrocketed 127 and 245%, respectively. Unlike the opioid crisis, no pharmaceutical interventions are available for treating METH use disorder or reversing overdose. Herein, we report the first active vaccine that offers protection from lethal (+)-METH challenge in male Swiss Webster mice. This vaccine formulation of (S)MLMH-TT adjuvanted with CpG ODN 1826 + alum successfully raised anti-METH antibodies in high titers, reduced (+)-METH distribution to the brain, and lowered (+)-METH-associated stereotypies in a hyperlocomotion assay. A comparison of enantiomeric haptens and the racemate elucidated the importance of employing (S)-stereochemistry in METH hapten design for optimal protection.

Full text of DOI:10.1021/jacs.9b07294

Communication
pubs.acs.org/JACS
Cite This: J. Am. Chem. Soc. XXXX, XXX, XXX−XXX
Consequence of Hapten Stereochemistry: An Efficacious
Methamphetamine Vaccine
Margaret E. Olson,†,§ Takashi Sugane,‡,§ Bin Zhou, and Kim D. Janda*
Departments of Chemistry and Immunology, The Skaggs Institute for Chemical Biology, and Worm Institute of Research and
Medicine (WIRM), The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, California 92037, United States of
America
S
* Supporting Information
modulation, proves challenging. Alternatively, our laboratory
ABSTRACT: Recent trends in methamphetamine
(METH) misuse and overdose suggest society is
inadvertently overlooking a brewing METH crisis. In the
past decade, psychostimulant-related lethal overdoses and
hospitalizations have skyrocketed 127 and 245%,
respectively. Unlike the opioid crisis, no pharmaceutical
interventions are available for treating METH use disorder
or reversing overdose. Herein, we report the first active
vaccine that offers protection from lethal (+)-METH
challenge in male Swiss Webster mice. This vaccine
formulation of (S)MLMH-TT adjuvanted with CpG
ODN 1826 + alum successfully raised anti-METH
antibodies in high titers, reduced (+)-METH distribution
to the brain, and lowered (+)-METH-associated stereo-
typies in a hyperlocomotion assay. A comparison of
enantiomeric haptens and the racemate elucidated the
importance of employing (S)-stereochemistry in METH
hapten design for optimal protection.
has leveraged the field of immunopharmacotherapy with the
goal of developing an active vaccine for METH; an active
vaccine uses a METH analogue, the hapten, to generate high
concentration, high affinity antibodies that sequester drug in
circulation. To date, nine experimental METH vaccines have
been reported by our laboratory and others, but none have
advanced past in vivo, murine models. Herein, we report a key
advancement in METH vaccine development by designing the
first vaccine that imparts protection from lethal doses of
METH.
Three components are required in the design of a small
molecule conjugate vaccine: the hapten, the immunogenic
carrier protein, and an adjuvant system. Extensive efforts in our
laboratory previously identified optimal carrier protein and
adjuvant systems, tetanus toxoid (TT) and CpG ODN 1826 +
alum, respectively, both in the context of METH and other
drugs of abuse. With these components optimized, we focused
on hapten design in this work to deliver a clinically viable, anti-
METH vaccine candidate. The fundamental requirements in
hapten design aim to chemically append a linker through
which the immunogenic carrier can be covalently conjugated,
all while maintaining as much of the native drug structure as
possible.
Previous METH hapten design evaluated phenyl (red) and
N-methyl (blue) substituted haptens (Figure 1).4−12 Later
generation vaccines in both approaches generated antibodies
with nanomolar Kd affinity for (+)-METH, reduced
(+)-METH distribution to the CNS, and attenuated
(+)-METH-induced self-administration, hyperthermia, condi-
tioned place preference, and/or hyperlocomotion in murine
models. Despite these promising early results, no active vaccine
has reached clinical trials. The major limitation to currently
available METH vaccines is surmountable antibody protection,
a caveat common to all drugs of abuse vaccines. For example,
MH6, a Janda Laboratory vaccine that we advanced furthest in
preclinical development, lost efficacy in a self-administration
model.13 In an effort to address surmountability, the current
investigation sought to identify a vaccine efficacious against
lethal doses of (+)-METH. We propose that identifying a
vaccine candidate more equip to protect against the highest
concentrations of (+)-METH, a scenario representative of
ethamphetamine (METH), an illicit, psychostimulatory
Mphenethylamine, produces a highly addictive, euphoric
high by releasing high concentrations of dopamine, serotonin
and norepinephrine in the brain.1 Dopamine, in particular,
motivates and reinforces drug-taking behaviors that may
ultimately produce METH use disorder. The 2017 National
Survey on Drug Use and Health (NSDUH) reported 1.6
million past year users in the United States; included in this
number are nearly one million Americans with clinically
significant METH use disorder.2 Strikingly, this number
represents a 29% increase in the incidence of METH use
disorder compared to 2016.2
While the threat of METH-induced overdose is not as
widely publicized as the opioid crisis, rates of psychostimulant-
associated overdose death have risen since 2010. METH, now
being described as the “forgotten killer”, is responsible for
more overdose deaths in Texas and Colorado than opioids,
and in California, the number of psychostimulant-related lethal
overdoses rose by 127% from 2008 to 2013. For comparison,
the number of opioid-related overdose deaths rose by 8.4%.3
Unlike the opioid crisis, there are no pharmaceutical options
for the treatment of METH use disorder or overdose reversal.
Because the pharmacology of METH is complicated by the
involvement of multiple monoamine neurotransmitters, a
traditional approach to therapeutic design, namely receptor
Received: July 9, 2019
© XXXX American Chemical Society
A
DOI: 10.1021/jacs.9b07294
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Journal of the American Chemical Society
Communication
had yet to compare enantiomeric and racemic haptens head-to-
head.
The (S), (R), and (R/S) MLMH haptens were synthesized
as depicted in Scheme 1 from their respective N-Boc-
phenylalaninals (D, L, or racemic) with overall yields of 9.2%
(S), 10.5% (R), and 8.9% (R/S). To conjugate the haptens to
TT, carboxylic acids were first activated with N-hydroxysulfo-
succinimide (sulfo-NHS) using 1-ethyl-3-(3-(dimethylamino)-
propyl) carbodiimide (EDCI), then treated with TT to afford
the desired hapten-TT immunoconjugates.
As discussed, vide supra, the MLMH-TT immunoconjugates
were formulated with alum and CpG ODN 1826 adjuvants,
then administered intraperitoneally (i.p.) to Swiss Webster
mice at 0, 2, and 4 weeks. Antibody titers were assessed by
enzyme-linked immunosorbent assay (ELISA) against the
corresponding hapten-BSA conjugate, namely (S)MLMH,
(R)MLMH, or (R/S)MLMH-BSA (Figure 2A). Overall,
robust antibody titers were observed for the experimental
MLMH vaccines in comparison to vehicle (TT and alum +
CpG ODN 1826). Importantly, higher titers were observed as
the immunization study continued, evidence of a mounting
immune response. Notably, the increase in titers for the
(S)MLMH vaccine between the first and second bleeds was
more pronounced than the change associated with (R)MLMH
and (R/S)MLMH vaccines, possibly alluding to a stronger
immune response. To quantify antibody IC50’s for (+)-METH
raised by the (S)MLMH-TT, (R)MLMH-TT, and (R/
S)MLMH-TT vaccines, surface plasmon resonance (SPR)
was performed (Table S1). (S)MLMH-BSA, (R)MLMH-BSA,
and (R/S)MLMH-BSA were immobilized on a SPR chip, and
binding to (S)MLMH, (R)MLMH, and (R/S)MLMH serum
antibodies was measured in the competitive environment of
(+)-METH. As anticipated, hapten chirality was critical as
antibodies raised by (S)MLMH-TT exhibited 10-fold higher
affinity for (+)-METH compared to (R)MLMH-TT and (R/
S)MLMH-TT raised antibodies (∼0.2 vs ∼2 μM).
Figure 1. Structure of (+)-methamphetamine and the (S)-MLMH
hapten. (A) Previous efforts to develop an active METH vaccine have
evaluated conjugation to immunogenic proteins via the phenyl (red)
and N-methyl (blue) substructures of the drug. References for these
efforts are identified.4−10 (B) This work investigated modifications of
the methyl substructure for the first time.
drug seekers increasing dosages to achieve a high, will better
translate to clinical utility.
METH is a particularly challenging target for immunophar-
macotherapy because of its low molecular weight structure
(150 kDa), limited chemical-epitopes for antibody recognition,
and similarity to endogenous monoamines. In this study, we
reasoned that modifying the methyl substructure (Figure 1,
green), being the smallest chemical-epitope of the native
METH scaffold, would least influence the specificity of the
generated antibodies to the drug. In our initial design, three
linker identities were considered, 7-propionamidoheptanoic
acid, (3-propionamidopropanoyl)glycine], and 5-oxo-5-((2-
oxo-2-((5-propionamidopentyl)amino)ethyl)amino)pentanoic
acid (Figure S1), before ultimately arriving at 7-propionami-
doheptanoic acid to garnish the optimal series of methyl-linked
METH haptens (MLMHs), (S)MLMH, (R)MLMH, and (R/
S)MLMH. This work marks the first comparison of
enantiomeric and racemic haptens. METH exists in two
stereoisomers; the dextrorotatory isomer [(+)-METH/(S)-
METH] is significantly more potent than (−)-METH.14 Yet,
despite this knowledge, the field of immunopharmacotherapy
As a stimulant, METH induces a quantifiable increase in
rodent locomotor activity upon exposure with a ceiling of
stereotypy. Stereotypies are repetitive movements that may
present as adverse events in response to high concentrations of
drug challenge. Colloquially, these behaviors would be referred
Scheme 1. Synthesis of METH Haptens
a
Reagents and Conditions: (a) Ph2P = CHCO2Et, DCM, 0 °C to rt, (S) 90%, (R) 90%, (R/S) 76%; (b) H2, Pd/C, EtOH, rt, (S) 85%, (R) 98%,
(R/S) 95%; (c) TFA, DCM, rt, then NsCl, DIPEA, DCM, 0 °C to rt, (S) 54%, (R) 52%, (R/S) 86%; (d) MeI, Cs2CO3, DMF, rt, (S) 91%, (R)
79%, (R/S) 81%; (e) NaOH, MeOH, rt, (S) quant., (R) 94%, (R/S) quant.; (f) ethyl 7-aminoheptanoate, HATU, DIPEA, DMF, rt, (S) 94%, (R)
89%, (R/S) 93%; (g) 4-tBuPhSH, Cs2CO3, DMF, then NaOH, MeOH, (S) 26%, (R) 34%, (R/S) 19%.
B
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Figure 2. Evaluation of MLMH vaccines. (A) Anti-METH antibody midpoint titers measured by ELISA using serum from vaccinated mice (n = 6/
group) on days 21 (bleed 1) and 35 (bleed 2). Bars denote means SEM (B) (+)-METH-induced hyperlocomotor activity in vaccinated mice (n
= 6/group) at 2 and 4 mg/kg. Bars denote means SEM *P < 0.05 determined by one-way ANOVA. (C) Concentration of (+)-METH in the
blood and brains of vaccinated and naive mice (n = 6/group). Bars denote means SEM ***P < 0.001 determined by one-way ANOVA. (D)
Kaplan−Meier curve for survival in vaccinated groups (n = 5 or 6/group). The efficacy of MH6-TT, an anti-METH vaccine previously reported by
the Janda Laboratory, was evaluated and shown for comparison.
to as “tweaking”. Using locomotor behavior as a model,
vaccinated mice were challenged with (+)-METH at 1, 2, and
4 mg/kg concentrations and hyperlocomotor activity was
evaluated using ANY-Maze video tracking software (Stoelting
Co.). At low concentrations, vaccine efficacy can be measured
by an ability to block METH-induced hyperlocomotion;
whereas at high concentrations, vaccine efficiency is gauged
by an ability to prevent stereotypy. The control group of mice
vaccinated with TT and alum + CpG ODN 1826 alone
exhibited elevated hyperlocomotor activity upon (+)-METH
challenge at 1 mg/kg and the appearance of minor stereotypy
and dramatic stereotypy at 2 mg/kg and 4 mg/kg, respectively
(Figures 2B and S2). METH induced stereotypy presents as a
freezing behavior in mice, where the animals rear on their hind
legs and aggressively clean their face. Counterintuitively, this
behavior is opposite to stimulant-induced hyperlocomotor
activity and appears as a sharp decline in hyperlocomotion
after drug exposure. Mice experiencing normal elimination of
(+)-METH show a gradual return to baseline. Analysis of
experimental vaccine cohorts demonstrated that all vaccines
convey minimal protection to (+)-METH at low 1 mg/kg
doses as animals exhibited equivalent locomotor activity to
vehicle immunized mice. However, at 2 mg/kg and especially 4
mg/kg, (R)MLMH-TT, (R/S)MLMH-TT and vehicle immu-
nized mice exhibited stereotypy, whereas (S)MLMH-TT
immunized mice exhibited minimal to no adverse effects.
Taken together, this finding is significant in that (S)MLMH-
TT vaccinated mice are protected from high, potentially lethal
(+)-METH concentrations.
mice. Analogously, though not significant, reduced levels of
(+)-METH were detected in the brains of (S)MLMH-TT,
(R)MLMH-TT, and (R/S)MLMH-TT vaccinated mice versus
vehicle. This biodistribution data demonstrated that vaccine-
generated antibodies effectively scavenged free drug in the
blood (Figure 2C). Specifically, mice vaccinated with
(S)MLMH-TT, (R)MLMH-TT, and (R/S)MLMH-TT ap-
propriated nearly 3.9, 4.4, and 4.7 times more (+)-METH in
the blood than unvaccinated mice.
Based on these results and the findings of the hyper-
locomotor activity assay, we probed the ability of these MLMH
vaccines to protect against (+)-METH lethal challenge. To
date, no report has described the successful use of an active
vaccine to protect against METH overdose. Vehicle mice
dosed i.p. with 20 mg/kg of (+)-METH exhibited a 20%
survival rate at 48 h, while (R)MLMH-TT vaccinated mice
exhibited 16.7% surivial, (R/S)MLMH-TT vaccinated mice
exhibited 33.3% survival, and (S)MLMH-TT vaccinated mice
exhibited 83.3% survival (Figure 2D). The MLMH vaccines
significantly outperformed MH6-TT, an anti-METH vaccine
that had previously exhibited promising results in modifying
(+)-METH induced behaviors.7,13,15,16 Importantly, this report
marks the first direct comparison of two anti-METH vaccines
of unique classes, namely N-methyl linked and methyl-linked
METH haptens, and the first evaluation of MH6-TT in a
lethality study (see SI for a complete set of MH6 data vs the
MLMH series).
Taken together, the results of ELISA, SPR, behavioral
testing, biodistribution and lethal challenge revealed that
conjugating METH immunogens via the methyl substructure
(MLMH) is a successful strategy in METH hapten design.
Importantly, a direct comparison of (S) and (R) enantiomers
and the racemate unequivocally defined (S) stereochemistry as
a fundamental requirement of hapten design. While each
vaccine, (S)MLMH-TT, (R)MLMH-TT, and (R/S)MLMH-
TT, generated antibodies and affected blood/brain partition-
ing, only the (S)MLMH-TT vaccine effectively prevented
To confirm these findings, biodistribution of (+)-METH
̈
was investigated. In this study, vaccinated and naive mice were
treated with 4 mg/kg (+)-METH, then sacrificed after 15 min
by decapitation. Drug concentrations in the blood and brain
were quantified by liquid chromatography mass spectrometry.
Ultimately, significantly elevated levels of (+)-METH were
observed in the plasma of (S)MLMH-TT, (R)MLMH-TT, and
(R/S)MLMH-TT vaccinated mice versus vehicle vaccinated
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(7) Moreno, A. Y.; Mayorov, A. V.; Janda, K. D. Impact of distinct
chemical structures for the development of a methamphetamine
vaccine. J. Am. Chem. Soc. 2011, 133 (17), 6587−95.
(8) Shen, X. Y.; Kosten, T. A.; Lopez, A. Y.; Kinsey, B. M.; Kosten,
T. R.; Orson, F. M. A vaccine against methamphetamine attenuates its
behavioral effects in mice. Drug Alcohol Depend. 2013, 129 (1−2),
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amine CNS Distribution through Vaccine Linker Design. ACS Chem.
Neurosci. 2017, 8 (3), 468−472.
(+)-METH-associated stereotypy and lethality. These results
suggest that (S)MLMH-TT presents a viable candidate for
further consideration in more advanced rodent and nonhuman
primate preclinical models.
ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/jacs.9b07294.
Experimental procedures, NMR spectra, supplementary
figures (PDF)
(11) Ruedi-Bettschen, D.; Wood, S. L.; Gunnell, M. G.; West, C. M.;
Pidaparthi, R. R.; Carroll, F. I.; Blough, B. E.; Owens, S. M.
Vaccination protects rats from methamphetamine-induced impair-
ment of behavioral responding for food. Vaccine 2013, 31 (41),
4596−4602.
AUTHOR INFORMATION
■
Corresponding Author
*kdjanda@scripps.edu
(12) Stevens, M. W.; Gunnell, M. G.; Tawney, R.; Owens, S. M.
Optimization of a methamphetamine conjugate vaccine for antibody
production in mice. Int. Immunopharmacol. 2016, 35, 137−141.
(13) Miller, M. L.; Aarde, S. M.; Moreno, A. Y.; Creehan, K. M.;
Janda, K. D.; Taffe, M. A. Effects of active anti-methamphetamine
vaccination on intravenous self-administration in rats. Drug Alcohol
Depend. 2015, 153, 29−36.
(14) Harris, J. E.; Baldessarinig, R. J. Uptake of (3H)-catecholamines
by homogenates of rat corpus striatum and cerebral cortex: effects of
amphetamine analogues. Neuropharmacology 1973, 12 (7), 669−679.
(15) Miller, M. L.; Moreno, A. Y.; Aarde, S. M.; Creehan, K. M.;
Vandewater, S. A.; Vaillancourt, B. D.; Wright, M. J., Jr.; Janda, K. D.;
Taffe, M. A. A methamphetamine vaccine attenuates methamphet-
amine-induced disruptions in thermoregulation and activity in rats.
Biol. Psychiatry 2013, 73 (8), 721−8.
ORCID
Margaret E. Olson: 0000-0003-1865-9615
Kim D. Janda: 0000-0001-6759-4227
Present Addresses
†M.E.O.: Assistant Professor of Medicinal Chemistry,
Roosevelt University, College of Pharmacy, 1400 N. Roosevelt
Blvd., Schaumburg, IL 60173
‡T.S.: Astellas Pharma Inc., Modality Research Lab, 21,
Miyukigaoka, Tsukuba-shi, Ibaraki 305-8585, Japan
Author Contributions
§These authors contributed equally.
Notes
The authors declare no competing financial interest.
(16) Nguyen, J. D.; Bremer, P. T.; Hwang, C. S.; Vandewater, S. A.;
Collins, K. C.; Creehan, K. M.; Janda, K. D.; Taffe, M. A. Effective
active vaccination against methamphetamine in female rats. Drug
Alcohol Depend. 2017, 175, 179−186.
ACKNOWLEDGMENTS
■
Research was supported by the National Institutes of Health
grants R01 DA024705 (K.D.J.) and F32 DA044692 (M.E.O.),
and the Skaggs Institute for Chemical Biology (K.D.J.).
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