Impact of distinct chemical structures for the development of a methamphetamine vaccine

Article abstract of DOI:10.1021/ja108807j

(+)-Methamphetamine (METH) use and addiction has grown at alarming rates over the past two decades, while no approved pharmacotherapy exists for its treatment. Immunopharmacotherapy has the potential to offer relief through producing highly specific antibodies that prevent drug penetration across the blood-brain barrier thus decreasing reinforcement of the behavior. Current immunotherapy efforts against methamphetamine have focused on a single hapten structure, namely linker attachment at the aromatic ring of the METH molecule. Hapten design is largely responsible for immune recognition, as it affects presentation of the target antigen and thus the quality of the response. In the current paper we report the systematic generation of a series of haptens designed to target the most stable conformations of methamphetamine as determined by molecular modeling. On the basis of our previous studies with nicotine, we show that introduction of strategic molecular constraint is able to maximize immune recognition of the target structure as evidenced by higher antibody affinity. Vaccination of GIX⁺ mice with six unique METH immunoconjugates resulted in high antibody titers for three particularly promising formulations (45-108 μg/mL, after the second immunization) and high affinity (82, 130, and 169 nM for MH2, MH6, and MH7 hapten-based vaccines, respectively). These findings represent a unique approach to the design of new vaccines against methamphetamine abuse.

Full text of DOI:10.1021/ja108807j

ARTICLE
pubs.acs.org/JACS
Impact of Distinct Chemical Structures for the Development of a
Methamphetamine Vaccine
Amira Y. Moreno, Alexander V. Mayorov, and Kim D. Janda*
Departments of Chemistry and Immunology, the Skaggs Institute for Chemical Biology, and the Worm Institute of Research and
Medicine (WIRM), The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, California 92037, United States
S Supporting Information
b
ABSTRACT: (þ)-Methamphetamine (METH) use and addiction
has grown at alarming rates over the past two decades, while no
approved pharmacotherapy exists for its treatment. Immunopharma-
cotherapy has the potential to offer relief through producing highly
specific antibodies that prevent drug penetration across the bloodꢀ
brain barrier thus decreasing reinforcement of the behavior. Current
immunotherapy efforts against methamphetamine have focused on a
single hapten structure, namely linker attachment at the aromatic ring
of the METH molecule. Hapten design is largely responsible for
immune recognition, as it affects presentation of the target antigen and
thus the quality of the response. In the current paper we report the systematic generation of a series of haptens designed to target the
most stable conformations of methamphetamine as determined by molecular modeling. On the basis of our previous studies with
nicotine, we show that introduction of strategic molecular constraint is able to maximize immune recognition of the target structure
as evidenced by higher antibody affinity. Vaccination of GIXþ mice with six unique METH immunoconjugates resulted in high
antibody titers for three particularly promising formulations (45ꢀ108 μg/mL, after the second immunization) and high affinity (82,
130, and 169 nM for MH2, MH6, and MH7 hapten-based vaccines, respectively). These findings represent a unique approach to the
design of new vaccines against methamphetamine abuse.
’ INTRODUCTION
antibody concentration (titer). Small molecules, such as METH,
require appendage to carrier macromolecules in order to elicit an
immune response. The chemical positioning of a linker to the
target antigen has proven to be crucial for proper immune
stimulation both in terms of amount of antibody elicited and
antibody specificity.10 Thus, proper hapten design is critical for
immune recognition, as it affects presentation of the target
antigen and thus quality of the response.10,11 Immunotherapy
efforts against methamphetamine have largely focused on the use
of a single scaffold, i.e., linker attachment at the aromatic ring of
the parent molecule (Figure 1).12 Variations of linker identity
and length have allowed for some immune regulation, yet
vaccination of this structure has proven largely ineffective during
behavioral testing.9 The sole exception is a METH vaccine based
on a self-adjuvanting peptide construct wherein efficacy was
independent of hapten design and was determined by the
presence of an additional T cell epitope from tetanus toxin.13
Based on the poor response obtained from active vaccination,
the bulk of the literature to date has focused on the use of anti-
METH monoclonal antibodies, i.e., passive vaccination, which
when administered has shown reduction of METH-associated
behavior.14 Despite this potential efficacy, the expense of passive
vaccination is of concern. Active vaccination generates
(þ)-Methamphetamine (METH) use and addiction in the
United States has grown at alarming rates over the past two
decades,1 burdening the US economy with an estimated medical,
lost productivity, and law enforcement cost of $23.4 billion.2 The
effect of methamphetamine on the dopaminergic signaling path-
way is largely responsible for its powerful rewarding3 as well as
addictive properties. Furthermore, the high rate of relapse in the
patients undergoing methamphetamine withdrawal underscores
the level of challenge in development of an effective therapy for
methamphetamine addiction.4 Currently, psychosocial and be-
havioral management is the only available treatment.
Development of an efficacious pharmacotherapy is of pressing
concern, yet the complexity of drug action on the brain circuitry
has presented a significant challenge.5 Immunopharmacotherapy
uses an alternative approach wherein antibodies are used to
prevent drug distribution to brain receptors, thus decreasing
reinforcement of the behavior. Previous immunotherapy efforts
have targeted various drugs of abuse6 and importantly, antinicotine7
and anticocaine vaccines8 have shown titer dependent efficacy
during clinical trials. Active vaccination efforts against METH
have largely proven ineffective during behavioral testing,9 pro-
viding an impetus for development of more effective approaches
to a vaccine against methamphetamine addiction.
The success of any small molecule active vaccine is intimately
determined by three factors: antibody specificity, affinity, and
Received: September 30, 2010
Published: April 07, 2011
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conformation that positioned the largest substituents anti to one
another (Φ ≈ 180°, Ψ ≈ 180°) with two separate gauche-anti
conformers (Φ ≈ 180°, Ψ ≈ ( 60° and Φ ≈ ( 60°, Ψ ≈ 180°)
also identified as potential energy sinks (Figure 2). These findings
allowed us to identify two approaches to constructing conforma-
tionally constrained methamphetamine haptens: (1) C1ꢀC4 con-
straint and (2) C1-phenyl ring constraint (Figure 3). The former
approach was convenient to achieve by using a commercially
available, appropriately ornamented piperazine template, while the
tetrahydroisoquinoline (THIQ) template was found to be suitable
for establishing the latter type of dihedral constraint, both by
matching the dihedral angles of the energetically favored (ꢀ)
gauche-anti conformer (Φ ≈ ꢀ60°, Ψ ≈ 180°) and by possessing
a sufficient basicity of the requisite secondary amine nitrogen. Thus,
the designed haptens fall into one of three categories as determined
by the identity of their core structures. MH1 and MH2 present an
inherently anti-anti constrained piperazine nucleus, MH3 and MH5
are derivatized tetrahydroisoquinolines, and finally MH6 and MH7
are functionalized versions of the unconstrained methamphetamine
molecule (Figure 4).
Figure 1. Previously reported haptens for active anti-METH vaccines.
immunological memory to repeated exposure of the drug con-
jugate. Thus, the cost effectiveness of treatment is increased,
allowing for longer sustained protection with minimal compli-
ance and could be a viable approach for relapse prevention.
As a path forward for the development of a METH vaccine, we
report the systematic generation of a series of unique chemical
structures designed to target the most stable conformations of
methamphetamine in solution as determined by molecular
modeling. We present the serological analysis of GIXþ mice
following vaccination with six unique METH haptens, with three
of them being particularly promising, and elaborate on the
impact of these findings on the design of future vaccines against
methamphetamine abuse.
The global minima of (þ)-methamphetamine and the hapten
core structures MH1ꢀ3, MH5ꢀ7 (in their respective protonated
forms) were obtained using the hybrid Monte Carlo/low frequency
mode simulations (MCMM/LMCS) procedure as implemented in
Macromodel17 using the energy minimization routine as described
above. To simplify the computational experiments and the subse-
quent comparison of structures, the alkylsulfhydryl linker HS-(CH2)4
was removed in the simulations of all hapten structures, except
for MH6, where only the terminal sulfhydryl group was removed.
Superpositions of the minimized parent molecule with all haptens
were performed by alignment of four key loci: the amine nitrogen,
the N-Me, the C2-carbon atom, and the C3-carbon atom. Super-
positions of the constrained haptens MH1(R) and MH2(R), as
well as unconstrained haptens MH6(S) and MH7(S), with the
global minimum conformation of methamphetamine showed an
’ RESULTS AND DISCUSSION
Theoretical Calculations. Vital to constrained hapten design, the
conformational profile of the protonated form of (þ)-methamphet-
amine was examined using MacroModel 9.1 equipped with
Maestro 7.5 graphical interface (Schr€odinger, Portland, OR).
Structures were minimized using the OPLS_2005 force field15
and the PolakꢀRibiere conjugate gradient. Aqueous solution con-
ditions were simulated using the continuum dielectric water solvent
model (GB/SA).16 The key dihedral angles in these simulations
were denoted as Φ (C1ꢀNꢀC2ꢀC3) and Ψ (NꢀC2ꢀC3ꢀPh).
As expected, two dihedral drive simulations on the global minimum
of the methamphetamine structure showed the lowest energy
Figure 2. The conformational profile of N-methylamphetamine (Φ/ψ), as determined by dihedral drive/OPLS_2005-GB/SA simulations. Potential
energy is expressed in relative terms to the following minima: ꢀ148.208 kJ/mol (Φ = 196°) and ꢀ148.197 kJ/mol (Ψ = 178°).
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Figure 5. Stereoview of superposition of the global minima of (þ)-
methamphetamine (red), MH1 (blue) and MH2 (gold). rmsd
(MH1ꢀMETH) = 0.15, rmsd (MH2ꢀMETH) = 0.11. Hydrogens
are omitted for clarity.
Figure 3. Two strategies toward conformationally constrained
methamphetamine haptens.
Figure 6. Stereoview of superposition of the global minima of (þ)-
methamphetamine (red), MH6 (green), and MH7 (blue). rmsd
(MH6ꢀMETH) = 0.01, rmsd (MH7ꢀMETH) = 0.001. Hydrogens
are omitted for clarity.
Figure 7. Stereoview of superposition of the (ꢀ) gauche/anti con-
formation of (þ)-methamphetamine (red), and the global minima of
two regioisomers of MH3 (green and gold) and MH5 (blue). rmsd
(MH3ꢀMETH) = 0.06, rmsd (MH5ꢀMETH) = 0.02. Hydrogens are
omitted for clarity.
Figure 4. Designed hapten structures targeting (þ)-methamphetamine.
excellent fit (Figures 5 and 6). Similarly, MH3(S) and MH5(R)
structures were determined to share a matching core conform-
ation with the (ꢀ) gauche-anti conformer of methamphetamine
(Figure 7). Thus, each of the two constrained core categories
mimics a distinct low energy conformation of the target structure.
Deviations in conformation for all superpositions were calculated
as a root-mean-square (rms) and were found to be reasonably
low at <0.15, indicating a good fit.
It is important to point out that we considered the presence of basic
secondary amine functionality in our haptens to be vital for the
success of the methamphetamine vaccine. While the basicity of the
acyl-piperazine core-based haptens MH1 and MH2 (pKa = 11.12 for
piperidine, 9.82 for piperazine)18 was expected to be similar to that of
methamphetamine (pKa = 10.1),19 the ionization constant of the
THIQ template has been shown be somewhat lower (pKa ≈ 9.30).20
We hypothesized that both core structures were basic enough to
ensure sufficient protonated state population at the physiological pH
(7.4), giving rise to antibodies targeting the methamphetamine
structure in its protonated form. A possible complication from using
a basic amine linker in the MH5 hapten was recognized, due to the
uncertainty of its protonation behavior. Using the NMR work of
Beaumont and co-workers on the structurally related aminomethyl-
THIQ analogues20 as a guide, we concluded that the linker nitrogen
should be expected to be somewhat more basic than the THIQ core
nitrogen (pKa (dimethylamine) = 10.73 vs pKa (THIQ) = 9.3).18
Computational pKa prediction as implemented in Schrodinger Epik
program21 supported this hypothesis, yielding the pKa estimates at
9.62 for the linker amino group, and 6.72 for the THIQ core.
Regardless, comparison of the global minima of both protonated
forms showed near identical conformations (rmsd =0.055) (not
shown). In addition, as pointed out by Beaumont and co-workers,20
intramolecular hydrogen bonding between the protonated and the
free amino groups is likely and was in fact found by the computer
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carboxylic acid haptens for attachment to free amino groups in the
lysine residues of carrier proteins.9 Yet, we argue that this type of
chemistry may not be optimal when dealing with a structure, such
as methamphetamine, which itself presents a reactive secondary
amine. This could lead to loss of material to oligomerization-type
reactions as well as unreliable conjugation. We have opted instead
for an orthogonal conjugation technique using a maleimide spacer
with pH-modulated thiol selectivity. The robustness of this
conjugation method is highlighted by similar conjugation efficien-
cies for all haptens tested, determined by the number of hapten
copies on the carrier, which we suspected to have an impact on the
efficacy of immune stimulation toward a particular target. Efficacy
of conjugation was monitored using MALDI-TOF MS for hapten
coupling to bovine serum albumin (BSA) under the same condi-
tions, and all haptens were found to produce similar coupling rates
at 24ꢀ29 copies per BSA molecule.
Figure 8. Stereoview of the global minimum of the protonated form of
MH5, showing the putative intramolecular hydrogen bonding. Hydro-
gens are omitted for clarity.
simulations in both protonated forms (Figure 8), which suggested a
certain degree of proton sharing between the amino groups, which we
deemed both acceptable and interesting to pursue as a structural
feature of a methamphetamine hapten.
Hapten Design. Our strategy for hapten design consisted of
three main veins: (1) to focus the response on the lowest energy
conformations of methamphetamine as elucidated by molecular
modeling, (2) to mimic the most psychoactive enantiomer of the
parent molecule, and (3) to maximize hapten loading efficacy via
a noncompeting bioconjugation technique.
Synthetic Preparation of Haptens. All haptens were prepared
from commercially available starting materials using standard
reaction conditions. Full synthetic details and characterization of
all haptens are given in the Supporting Information. We note that
whenever possible we obtained enantiomerically pure materials to
match the most active stereoisomer of methamphetamine. How-
ever, MH1, MH3, and MH5 were synthesized in racemic form and
were not further resolved. Additionally, MH3 yielded two regio-
isomersthatwerenot separated further, asthey onlyvaryatthe site
of linker attachment within the aromatic ring (positions 6 and 7,
Supporting Information). Molecular modeling of both regioi-
somers deemed this not to be critical for immune presentation.
A brief description of each synthesis is shown, vide infra.
First, we hypothesized that targeting of the lowest energy
conformations could be achieved by either a constrained or
unconstrained approach. Unconstrained structures would effec-
tively mimic the target by theoretically converging onto the same
energy conformation of the parent structure. However, depend-
ing on the energy expenditure required to go from one potential
energy sink to another, a series of structures could exist through
time. Previous reports on development of small molecule
haptens for vaccines against nicotine abuse have provided
compelling evidence that application of conformational con-
straints can be used to reduce the hapten’s internal rotational
degree of freedom, thus minimizing the entropic loss upon
antibody binding.6a,22 Methamphetamine, like nicotine, is a small
molecule with ample degrees of freedom provided by rotation
along its sigma bonds. Thus, we hypothesized that rational
introduction of a strategic molecular constraint would be able
to “guide” the immune response toward the most stable and,
thus, most prevalent conformation of the parent structure. On
the basis of our modeling experiments, vide supra, we proposed
two specific ways to introduce molecular constraint in order to
target two distinct low energy conformations for methamphet-
amine. This allowed us to categorize our haptens based on the
identity of their core structures. MH1 and MH2 present a
constrained piperazine nucleus, MH3 and MH5 are constrained
based on a tetrahydroisoquinoline scaffold, and finally MH6 and
MH7 are linker-functionalized versions of the free rotating
methamphetamine molecule.
MH1 and MH2(R), constrained anti-anti METH mimetics, were
synthesized by functionalization of the commercially available
piperazine cores with the appropriate linker, 6-(tritylthio)hexan-1-
amine for MH1 and 6-(tritylthio)hexanoic acid for MH2. Carbo-
diimide activation chemistry was used for linker coupling; this was
followed by acid deprotection of the trityl group to yield the desired
haptens (Scheme 1). In short, we took advantage of the fact that the
piperazine core needed to prepare MH2 was commercially available
as either enantiomer, thus allowing easy access to structures that
targeted both the (þ) and (ꢀ) enantiomers of methamphetamine.
This was significant, as it would allow us to gauge the importance of
retaining the stereochemical requirements of our target, (þ)-
methamphtamine, the most active isomer, during hapten design.
In contrast for the MH1 hapten, starting material was only available
for the opposite enantiomer; thus, to access both isomers, racemiza-
tion was accomplished using 1,8-diazabicycloundec-7-ene (DBU)
and heat (see Supporting Information). The two isomers of MH1
were not further resolved, as we have previously shown how the
immune system can readily generate antibodies able to selectively
recognize either antipode from a racemic synthetic hapten.25
The syntheses of tetrahydroisoquinoline (THIQ) haptens
MH3 and MH5 are shown within Scheme 2. The synthesis of
MH3 was initiated with indium-mediated reduction of 3-methy-
lisoquinoline to the corresponding 1,2,3,4-tetrahydroisoquionline,
1; this was followed by aromatic nitration, which resulted in 2 as a
mixture of regioisomers at positions 6 and 7, as stated, vide supra,
that were not further resolved. Boc-protection of the reactive
secondary amine of 2 followed by reduction of the nitro moiety
granted 3 and allowed for facile coupling of the 6-(tritylthio)-
hexanoic acid linker. Global acidic deprotection provided MH3.
The synthesis of MH5 was initiated with the reduction and
reoxidation of the carboxylic acid from commercially available
(R)-N-Boc-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid
Second, it is well established in the literature that (þ)-metham-
phetamine is about a five times more potent stimulant drug than its
(ꢀ)-methamphetamine enantiomer, thus increasing its liability for
abuse.23,24 During our hapten design, we hypothesized that paying
special attention to the stereochemical requirements of the more
potent enantiomer of methamphetamine would allow for superior
immune tuning. Thus, the design of our haptens focused on
targeting the appropriate (þ)-configuration.
Finally, we hypothesized that maximizing hapten load onto the
carrier proteins could provide more potent immune stimulation.
Previous work focused on the use of carbodiimide activation of
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Scheme 1. Synthesis of Constrained Anti-Anti METH Mimetic Haptens, MH1/MH2
Scheme 2. Synthetic Routes toward (ꢀ) Gauche-Anti METH Mimetic Haptens, MH3/MH5
(Fluka), providing 5. Aldehyde 5 was subjected to reductive
amination using sodium cyanoborohydride and 6-(tritylthio)-
hexan-1-amine; this was followed by global acidic deprotection to
yield MH5.
cross-reactivity, i.e., MH3 preferentially identified MH7, while MH5
preferentially identified MH6. This bias was removed by use of
equilibrium dialysis, a solution-based assay we feel more closely
resembles the in vivo interaction. All binding constants as well as
antibody concentrations reported were calculated from a solution-
based radioimmunoassay (RIA) and normalized, thus allowing for
direct comparison of values between test groups.
The synthesis of the unconstrained hapten cores proceeded in
the following manner. MH6 was accessed by simple N-alkylation
of (þ)-amphetamine with 6-(tritylthio)hexyl methanesulfonate
followed by acid deprotection. The synthesis of MH7 was more
involved and began from (þ)-methamphetamine that was found
to be quite volatile as its free base, thus requiring protection of the
secondary amine with trifluoroacetic anhydride in order to allow
for easier handling. Thus, aromatic nitration gave rise to para-
substituted trifluoroacetamide methamphetamine, 6, as the
major product, which was separated using silica chromatography.
Reduction of the nitro functionality yielded 7, which was
followed by linker coupling using 6-(tritylthio)hexanoic acid,
providing 8. Base/acid deprotection of the trifluoroacetamide
and trityl moieties gave the final product MH7.
Characterization of anti-METH Antibodies. As stated, success
of active vaccination is contingent upon both the magnitude of the
response as well as the affinity and specificity of the antibodies (Abs)
generated. The magnitude of the immune response was initially
assessed by ELISA on microtiter plates coated with either MH6ꢀ or
MH7ꢀBSA conjugates. It was immediately apparent that the site of
linker attachment played an important role in determining relative
We hypothesized that mimicking of the target structure via either
introduction of strategic molecular constraint or effective functio-
nalization would maximize immune recognition of the target
structure as demonstrated by higher antibody affinity and specificity.
Gratifyingly, the highest affinity anti-METH Abs were observed
with constrained hapten MH2(R), an anti-anti METH mimetic as
determined by molecular modeling, followed by unconstrained
haptens MH6 and MH7. By the end of the study, polyclonal
responses had an affinity well within the range of previous anti-
methamphetamine mAbs tested (10ꢀ250 nM).26 Nonetheless,
unconstrained haptens MH6 and MH7 produced antibody con-
centrations in the 150ꢀ220 μg/mL range which corresponds to 3ꢁ
and 2ꢁ larger responses than that of constrained hapten MH2(R).
To put these values into perspective, a nicotine vaccine that has
advanced to clinical trials generated antibodies in rats within the
same range (184 μg/mL).27 Furthermore, these values are about 2
orders of magnitude higher than those reported by Duryee et al.13
wherein vaccination showed moderate effects in the rates of
-
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Scheme 3. Synthetic Routes toward Unconstrained METH Haptens, MH6/7
Table 1. Average Relative Affinities of Antisera from Immunized Mice against Amphetamine and Methamphetamine As
Determined by Equilibrium Dialysis
immunized antigen
MH2(R)
[abs] ( SE μg/mL
(þ)-METH KD(μM)
(þ)-amph KD(μM)
first bleed
70.83 ( 4.05
44.53 ( 2.54
220.13 ( 19.69
107.83 ( 7.22
152.53 ( 9.08
89.85 ( 11.89
0.218 ( 0.055
0.082 ( 0.018
0.266 ( 0.034
0.130 ( 0.019
0.152 ( 0.011
0.169 ( 0.023
1.267 ( 0.309
0.356 ( 0.093
1.12 ( 0.171
0.724 ( 0.082
23.73 ( 3.52
22.54 ( 4.70
second bleed
first bleed
MH6
MH7
second bleed
first bleed
second bleed
methamphetamine intravenous self-administration. On the other
hand, vaccination with MH1 as well as the tetrahydroisoquinoline
(THIQ) haptens MH3 and MH5 produced antibodies with mini-
mal affinity for METH in solution (>50 μM) and thus were
discarded from future testing.
We note that in contrast to what was expected, bleeds following a
third injection showed reduced antibody concentrations. Additional
booster injections are usually expected to increase the magnitude of
the response as well as focus the antibody population toward the
preferred target. In our case, antibody affinity was greatly increased
for MH2(R) and MH6 yet remained the same for MH7 across both
bleeds. We interpret the decrease in antibody concentration in one
of two ways. It is possible that the immune response prior to the last
injection was still in the top part of the bell shape curve, and thus
upon antigen presentation, part of the current stock of circulating
antibodies went to “neutralize the infection”, thus reducing the
overall antibody concentration. Alternatively, it is possible that
overall antibody count was lowered in favor of increasing antibody
affinity. We suspect an optimized vaccination schedule wherein
animals are allowed a longer “rest” period between injections would
be able to rule in favor of one hypothesis.
Amphetamine (amph) is a closely related drug of abuse as well as
a METH metabolite; thus, the binding affinity of the antisera for this
drug is of interest and was also assayed. Ideally, a clinically viable
METH vaccine would produce a response able to provide good
recognition for both related structures, thus maximizing its protec-
tive effects. Obtaining anti-METH antibodies that cross-react with
amphetamine has historically been a challenge with the traditional
hapten design, i.e., linker attachment at the aromatic moiety.28
Gratifyingly, the antisera of MH2(R) (anti-anti constrained METH
mimetic) and MH6 (unconstrained hapten) vaccinated animals had
moderate affinity for amphetamine which improved upon boosting.
MH7, consistent with what has been typically observed with this
hapten design,28 had overall poor affinity (Table 1). The differences
These findings suggest that targeting the (ꢀ) gauche/anti
conformation of methamphetamine does not appear to be as
effective at raising an immune response against the global mini-
mum anti/anti conformation. Among the possible reasons for the
apparent inability of the tetrahydroisoquinoline (THIQ) hapten
(MH3 and MH5)-based vaccines to raise sufficient antibody titers,
the lower pKa of the requisite secondary amino group appears to
offer one plausible explanation. Furthermore, it seems reasonable
to conclude that positioning of the aromatic ring is also a critical
determinant of efficacy. In the case of MH1, an anti-anti METH
mimetic similar to MH2, the steric repulsion between the aromatic
group and the carbonyl group of the MH1 2-piperazinone
template resulted in a misalignment of the aromatic ring as
compared to the global minimum structure of METH, and in
contrast toMH2, whichshowed a near-perfectfit (Figure5). Thus,
the better fit of the MH2 hapten structure with the global
minimum conformation of methamphetamine seems to correlate
with the higher titers produced with this hapten. This rationale is
further supported by the fact that (1) both of the THIQ haptens
which showed poor efficacy also see a misalignment of their
aromatic moiety with that of the target structure, and (2) both
unconstrained haptens, whose minima structures showed a better
fit with that of METH, showed a measurable immune response
(Figures 6 and 7).
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Table 2. Average Antibody Affinity against Both Isomers of
Methamphetamine As Determined by Equilibrium Dialysis
development of a clinically useful vaccine against metham-
phetamine abuse.
’ MATERIALS AND METHODS
Synthetic Generation of Methamphetamine Haptens.
Synthetic details for the three haptens of interest are detailed below.
Full synthetic detail and characterization of all haptens is given in the
Supporting Information.
Synthesis of MH2(R). (R)-1-Boc-2-Benzylpiperazine was purchased
from Synthonix and used without further purification. A 0.153 mmol
amount of 6-(tritylthio)hexanoic acid was mixed with 0.2 mmol of EDC
and 0.046 mmol of DMAP in 0.7 mL of DCM. A 0.184 mmol amount of
(R)-1-Boc-2-benzylpiperazine and 0.3 mmol of 4-methylmorpholine were
added, and the reaction mixture was stirred under argon at room
temperature for 3 h. The mixture was diluted with ethyl acetate, and the
organic layer was washed 3ꢁ with saturated sodium bicarbonate, 3ꢁ with
10% citric acid, and 1ꢁ with water. The organic layer was then dried over
sodium sulfate and rotoevaporated. The residue was then passed through a
short plug of silica using 80% ethyl acetate/hexane as eluent. The crude
product was used without further purification. Global deprotection was
achieved by addition of trifluoroacetic acid in a 1:1 dilution with DCM.
Drops of triisopropylsilane were added to scavenge the trityl groups. After 2
h, the mixture was rotoevaporated and purified by preparative HPLC.
Method = 0ꢀ5 min 30%B, to 33%B over 2 min, to 40% B over 27 min, to
95%B over 5 min, hold for 10 min, requilibrate. Product retention time = 13
min. Experimental yield over two steps = 56%. Observed amide rotamers
with NMR: 1H NMR (600 MHz, MeOD) δ 7.44ꢀ7.24 (m, 13H), 4.58 (s,
1H), 4.44 (s, 1H), 4.08 (d, J = 14.4 Hz, 1H), 3.95 (d, J = 14.5 Hz, 1H), 3.54
(dd, J = 15.6, 12.2 Hz, 1H), 3.50ꢀ3.34 (m, 5H), 3.16 (ddd, J = 29.6, 24.5,
15.1 Hz, 2H), 3.05ꢀ2.85 (m, 9H), 2.51ꢀ2.36 (m, 8H), 2.23 (d, J = 4.2 Hz,
2H), 1.64ꢀ1.54 (m, 6H), 1.53ꢀ1.46 (m, 3H), 1.43 (d, J = 7.1 Hz, 4H),
1.31 (d, J = 6.9 Hz, 2H). 13C NMR (600 MHz, MeOD) δ = 174.15, 135.89,
130.46, 130.30, 129.03, 128.84, 57.72, 57.41, 49.67, 48.14, 44.70, 43.75,
43.37, 39.37, 37.65, 37.37, 34.93, 34.78, 33.61, 28.99, 25.67, 25.62, 24.87.
LRMS (M þ H)þ: calcd for C17H26N2OS = 307.18, found 307.1.
Synthesis of MH6. A 0.126 mmol amount of D-amphetamine
sulfate was dissolved in 0.9 mL of ethanol. A 0.164 mmol amount of
6-(tritylthio)hexyl methanesulfonate and 0.38 mmol of potassium
carbonate were added, and the solution was heated to reflux overnight.
After 14 h, ethanol was removed under vacuum and the residue dissolved
in ethyl acetate. The organic layer was washed 2ꢁ with water, dried over
sodium sulfate, and rotoevaporated. The residue was then dissolved in
5 mL of 10% TFA/DCM, and drops of TIS were added as a trityl
scavenger. The solution was stirred for 1 h, rotoevaporated, and purified
by prep HPLC. Method = 0ꢀ5 min 35%B, to 40%B over 2 min, to 44% B
over 27 min, to 95%B over 5 min, hold for 10 min, requilibrate. Product
retention time = 17 min. Experimental yield over two steps = 50%. 1H
NMR (600 MHz, MeOD) δ 7.31 (m, J = 7.5 Hz, 2H), 7.23 (m, 3H),
3.48ꢀ3.41 (m, 1H), 3.16 (dd, J = 13.2, 4.4 Hz, 1H), 3.07ꢀ2.97 (m, 2H),
2.66 (dd, J = 9.3, 3.9 Hz, 1H), 2.48 (t, J = 7.6 Hz, 1H), 1.73ꢀ1.55 (m,
4H), 1.47ꢀ1.35 (m, 4H), 1.17 (d, J = 6.6 Hz, 3H). 13C NMR (600 MHz,
MeOD) δ = 137.32, 130.41, 130.03, 128.44, 56.95, 49.61, 46.31, 40.35,
39.22, 34.85, 29.88, 28.92, 28.82, 27.47, 27.26, 27.11, 24.79, 16.07.
LRMS (M þ H)þ: calcd for C15H25NS = 252.17, found 252.2.
Synthesis of MH7 (four steps). Synthesis of (S)-2,2,2-Trifluoro-N-
(þ)-
(ꢀ)-METH
KD
immunized
antigen
METH
KD (μM)
(μM)
MH2 (S)
MH6
second bleed
first bleed
1.720
0.266
0.130
0.276
3.050
2.010
second bleed
for amphetamine cross-reactivity between MH6 and MH7, both
unconstrained cores, is not obvious based on our modeling and is
likely the result of an unintended linker effect.
In order to test the importance of retaining the stereochemical
requirements of the more potent enantiomer of methampheta-
mine during hapten design, we synthesized the opposite (S)-
enantiomer for the best constrained hapten MH2 (a piperazine
core, anti-anti METH mimetic). The response elicited from
immunization with MH2(S) was assayed for relative affinity to
both isomers of METH. The first bleed contained Abs with little
(þ)-METH affinity, thus making it difficult to quantify. Gratify-
ingly, the second bleed antisera of MH2(S) immunized mice had
six times better affinity for the (ꢀ)-METH isomer as predicted
by molecular modeling (Table 2). Furthermore, to prove if
stereochemical requirements were also critical for unconstrained
METH mimetics, the affinity of MH6 (modeled after (þ)-
METH) antisera for (ꢀ)-METH was measured. MH6 was
chosen over MH7 because of its higher antibody concentrations
as well as lower binding constants for both METH and AMPH.
Competitions with (ꢀ)-METH show that MH6 preferentially
bound (þ)-METH by at least 10-fold (Table 2).
’ CONCLUSIONS
In summary, the current paper details the rational design of six
METH haptens, five with unique structural characteristics, and their
ability for precise generation of anti-METH immune responses.
Three haptenic compounds, MH2(R), MH6, and MH7, show
particular promise in generation of a potentially clinically relevant
METH vaccine based on both an elevated antibody titer as well as
nanomolar range (þ)-METH affinity. Introduction of strategic
molecular constraint and stereochemical requirements in MH2(R)
allowed for generation of a polyclonal response well within the range
of previous monoclonal antibodies tested. Additionally, MH2(R)
generated antibodies with moderate affinity for amphetamine, a
related drug and methamphetamine metabolite. However, magnitude
of the response remained highest in the two unconstrained structures
MH6 and MH7. MH6 is of particular interest, as not only did it
present a surprisingly high antibody concentration but it also showed
good specificity toward METH and discrete affinity toward amphet-
amine. Further studies including improvements onto the adjuvant
activity of the formulations as well as behavioral studies with our
prime hapten leads will provide a practical approach toward
methyl-N-(1-(4-nitrophenyl)propan-2-yl)acetamide.
A 0.269 mmol
amount of D-methamphetamine hydrochloride was dissolved in 1.8 mL
DCM. A 0.538 mmol amount of triethylamine was added, and the mixture
was cooled to 0 °C. A 0.323 mmol amount of trifluoroacetic anhydride was
added dropwise, and the mixture was stirred at room temperature for 2.5 h.
The solvent was removed under vacuum and the residue dissolved in ethyl
ether which resulted in the formation of a precipitate. The solution was
passed through a short plug of silica topped with basic alumina. The filtrates
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were combined and rotoevaporated to yield a single spot by TLC which was
carried forward without further purification. The residue was dissolved in
0.51 mL of DCM and added dropwise to a chilled solution consisting of 3.26
mmol of potassium nitrate and 3.09 mmol of concentrated sulfuric acid
previously dissolved in 1.63 mL of DCM. The slurry was stirred overnight at
room temperature. An aqueous solution of sodium sulfate was used to
quench the reaction. The two layers were separated, and the organic layer
was washed twice more with aqueous sodium sulfate. Organics were
combined, dried with anhydrous sodium sulfate, and rotoevaporated. The
product was purified using silica chromatography with 10% ethyl acetate/
hexane as the eluent. The major product of reaction was para-substituted as
evidenced by NMR. Experimental yield = 54% (para). TLC conditions 15%
EtOAc/hex Rf = 0.16. Observed rotamers 1:0.5 by NMR; shifts and
integration given for main rotamer: 1H NMR (600 MHz, CDCl3) δ 8.17
(d, J = 8.7 Hz, 2H), 7.36 (d, J = 8.6 Hz, 2H), 4.87 (dq, J = 13.8, 6.9 Hz, 1H),
3.00ꢀ2.90 (m, 5H), 1.29ꢀ1.25 (m, 3H). 13C NMR (600 MHz, CDCl3)
δ = 176.84, 147.35, 147.20, 145.28, 144.79, 130.04, 129.90, 124.24, 124.05,
54.10, 52.40, 40.92, 39.50, 29.92, 29.37, 28.23, 18.49, 17.08. LRMS (M þ
H)þ: calcd for C12H13F3N2O3= 291.09, found 291.4.
sodium sulfate, and rotoevaporated. The residue was dissolved in 6 mL of
10% TFA/DCM, and drops of TIS were added to scavenge the trityl
groups. The solution was stirred for 1 h, rotoevaporated, and purified by
prep HPLC. Method = 0ꢀ5 min 25%B, to 30%B over 2 min, to 40% B
over 27 min, to 95%B over 5 min, hold for 10 min, requilibrate. Product
retention time = 19 min. Experimental yield over two steps = 40%. 1H
NMR (500 MHz, MeOD) δ 7.54 (dd, J = 8.6, 2.4 Hz, 2H), 7.21 (d, J = 8.5
Hz, 2H), 3.48ꢀ3.40 (m, 1H), 3.34 (s, 1H), 3.07 (dd, J = 13.7, 5.3 Hz, 1H),
2.71 (s, 3H), 2.51 (t, J = 7.6 Hz, 2H), 2.37 (t, J = 7.5 Hz, 2H), 1.74ꢀ1.61
(m, 4H), 1.52ꢀ1.45 (m, 2H), 1.23 (d, J = 6.6 Hz, 3H). 13C NMR (500
MHz, MeOD) δ= 130.81, 121.84, 57.81, 39.71, 37.81, 34.88, 30.95, 28.95,
26.35, 24.83, 15.80. LRMS (M þ H)þ: calcd for C16H26N2OS = 295.18,
found 295.2.
HaptenꢀProtein Immunoconjugates. Immunoconjugates
were prepared by reaction of the thiol presenting haptens with mal-
eimide-activated protein, either KLH or BSA. Briefly, protein activation
was accomplished by reacting 1 mg of protein with 1 mg of S-GMBS
(N-[γ-maleimidobutyryloxy]sulfosuccinimide ester, Pierce) at a con-
centration of 5.4 mg protein/mL of EDC conjugation buffer. The
solution was shaken at room temperature for 3 h and dialyzed
thoroughly in order to remove unreacted material. The concentration
of the activated solution was determined via the BCA assay. Preweighed
haptens were then dissolved directly into the protein solution at a ratio of
0.5 mg of hapten:1 mg of protein. The mixture was shaken for 30 min at
room temperature followed by overnight shaking at 4 °C. The solution
was once again dialyzed and characterized. Coupling efficiencies were
monitored using MALDI-TOF MS for all BSA conjugates. Because of
the size of KLH, conjugates to this protein could not be directly
analyzed. For BSA, all haptens showed similar coupling efficiencies of
about 24ꢀ29 copies per BSA protein molecule.
Synthesis of (S)-N-(1-(4-Aminophenyl)propan-2-yl)-2,2,2-trifluoro-N-
methylacetamide. A 0.174 mmol amount of (S)-2,2,2-trifluoro-N-methyl-
N-(1-(4-nitrophenyl)propan-2-yl)acetamide was dissolved in MeOH, and
12 mg of 10% activated palladium on carbon was added under a hydrogen
balloon. The reaction was stirred for 2 h before being filtered on a Celite
plug. The plug was washed with methanol, filtrates were combined, and
solvent was removed under vacuum. The residue was purified using silica
chromatography using 20% ethyl acetate/hexane as eluent. Experimental
yield = 97%. TLC conditions 30% EtOAc/hex Rf = 0.3. Observed rotamers
1
by NMR 1:0.7; shifts and integration given for main rotamer: H NMR
(500 MHz, CDCl3) δ 6.95 (d, J = 8.2 Hz, 2H), 6.66ꢀ6.59 (m, 2H),
4.82ꢀ4.70 (m, 1H), 3.60 (broad s, 2H), 2.92 (s, 3H), 2.83ꢀ2.62 (m, 2H),
1.29ꢀ1.17 (m, 3H). 13C NMR (500 MHz, CDCl3) δ = 183.74, 145.33,
143.76, 130.13, 130.00, 127.62, 115.69, 115.63, 54.92, 53.03, 40.58, 38.97,
28.31, 18.17, 16.90. LRMS (M þ H)þ: calcd for C12H15F3N2O = 261.11,
found 261.1.
Vaccination Protocols for Mice Studies. Groups of n = 4 129GIx
mice (6ꢀ8 weeks, 23ꢀ28 g) were immunized i.p. on days 0, 14, and 35
with a suspension of each haptenꢀKLH conjugate (100 μg) in formula-
tion with Sigma Adjuvant System (SAS, Sigma) according to the manu-
facturer’s instructions. SAS is a stable oil-in-water emulsion that may be
used as an alternative to the classical Freund0s water-in-oil emulsions. This
adjuvant is derived from bacterial and mycobacterial cell wall components
such as detoxified monophosphoryl lipid A derived from Salmonella
minnesota and synthetic trehalose dicorynomycolate that provide a potent
stimulus to the immune system. Following vaccine administration, serum
(0.1 mL) was collected on days 21 and 42 via tail-bleed. All biological
samples were stored at ꢀ80 °C until use to preserve integrity.
Synthesis of (S)-N-(4-(2-(2,2,2-trifluoro-N-methylacetamido)propyl)-
phenyl)-6-(tritylthio)hexanamide. A 0.172 mmol amount of (S)-
N-(1-(4-aminophenyl)propan-2-yl)-2,2,2-trifluoro-N-methylacetamide
was dissolved in 0.368 mL of DCM. A 0.157 mmol amount of 6-
(tritylthio)hexanoic acid, 0.204 mmol of EDC, and 0.047 mmol of
DMAP were added, and the mixture was stirred. A 0.314 mmol amount
of 4-methylmorpholine was added, and the reaction was stirred for 4 h. A
3 mL volume of ethyl acetate was added and washed 2ꢁ with saturated
sodium bicarbonate, 4ꢁ with 10% citric acid, and 1ꢁ with water. The
organic layer was dried over sodium sulfate and rotoevaporated to a
yellow oil which corresponded to single spot on TLC. Experimental
yield = 93%. TLC conditions 20% EtOAc/hex Rf = 0.13. Observed
amide rotamers at a ratio of 1:0.63. 1H NMR (600 MHz, CDCl3) δ 7.41
(dd, J = 12.7, 8.7 Hz, 13H), 7.27 (dd, J = 10.5, 4.3 Hz, 12H), 7.20 (t, J =
7.6 Hz, 4H), 7.16ꢀ7.04 (m, 5H), 4.85ꢀ4.73 (m, 1H), 2.91 (s, J = 25.0
Hz, 5H), 2.87ꢀ2.70 (m, 3H), 2.25 (dd, J = 12.1, 7.2 Hz, 3H), 2.16 (t, J =
6.9 Hz, 3H), 1.64ꢀ1.53 (m, 4H), 1.45ꢀ1.38 (m, 3H), 1.37ꢀ1.27 (m,
4H), 1.21 (t, J = 7.0 Hz, 7H). 13C NMR (600 MHz, CDCl3) δ = 171.16,
145.14, 136.95, 136.73, 129.76, 129.67, 129.53, 128.02, 126.74, 120.19,
120.03, 66.65, 54.55, 52.67, 40.61, 39.01, 37.64, 31.92, 28.66, 28.63,
28.49, 28.18, 25.22, 18.08, 16.81. LRMS (M þ Na)þ: calcd for
C37H39F3N2O2S = 655.25, found 655.3.
Immunoassays: ELISA. Production of methamphetamine-specific
IgG was initially monitored by ELISA using MH6ꢀ and MH7ꢀBSA
conjugates as the coating antigen. Titers were calculated from the plot of
absorbance versus log dilution and were defined as the dilution
corresponding to an absorbance reading 50% of the maximal value.
MH6ꢀBSA, MH7ꢀBSA, and protein only controls were added indivi-
dually to COSTAR 3690 microtiter plates and allowed to dry at 37 °C
overnight. Following methanol fixation, nonspecific binding was
blocked with a solution of 5% nonfat powdered milk in PBS for 0.5 h
at 37 °C. Next, mouse sera was serially diluted in a 1% BSA solution
across the plate and allowed to incubate for 1ꢀ2 h at 37 °C in a moist
chamber. Plates were then washed with DI H2O and treated with goat
antimouse-HRP antibody for 0.5 h at 37 °C. Following another wash
cycle, plates were developed with the TMB two-step kit (Pierce;
Rockford, IL).
Synthesis of MH7. A 0.145 mmol amount of (S)-N-(4-(2-(2,2,2-
trifluoro-N-methylacetamido)propyl)phenyl)-6-(tritylthio)hexanamide
was dissolved in 1 mL of methanol and drops of water. A 0.436 mmol
amount of potassiumcarbonate was added and themixture stirred atroom
temperature for 50 h. The methanol was removed under vacuum, and the
residue was dropped in water. The aqueous layer was basified and
extracted 3ꢁ with DCM. The organic layers were combined, dried over
Equilibrium Dialysis. Refined values for antibody affinity, speci-
ficity, and concentration were determined using a solution-based radio-
immunoassay (RIA). A modified version of Muller’s method29 was
followed, as it allows for determination of both affinity constant and
concentration of specific antibody in serum. The RIA was carried out in a
96-well equilibrium dialyzer MWCO 5000 Da (Harvard Apparatus,
Holliston, MA) to allow easy separation of bound and free (þ)-[20,60-
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ARTICLE
3H(n)] methamphetamine tracer; specific activity = 39 Ci/mmol
(obtained from the National Institute on Drug Abuse (Bethesda,
MD) and synthesized at Research Triangle Institute (Research Triangle
Park, NC). Briefly, mouse sera for each bleed for each hapten were
pooled together and diluted in RIA buffer (sterile filtered 2% BSA in 1ꢁ
PBS pH = 7.4) to a concentration that would bind 11ꢀ30% of ∼24 000
decays/min of 3H-methamphetamine tracer. A 100 μL aliquot of sera
was combined with 50 μL of radiolabeled tracer (∼24 000 decays/min)
and 150 μL of unlabeled competitor [(þ)-methamphetamine or (þ)-
amphetamine] at varying concentrations in PBS pH = 7.4 was added to
the solvent chamber, and the samples were allowed to reach equilibrium
on a plate rotator (Harvard Apparatus, Holliston, MA) at room
temperature for at least 22 h. A 100 μL aliquot from each sample/
solvent chamber was slowly aspirated and suspended in 5 mL of
scintillation fluid (Ecolite, ICN, Irvine, CA), and the radioactivity of
each sample was determined by liquid scintillation spectrometry.
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’ AUTHOR INFORMATION
(21) Shelley, J.; Cholleti, A.; Frye, L.; Greenwood, J.; Timlin, M.;
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Corresponding Author
kdjanda@scripps.edu
’ ACKNOWLEDGMENT
The authors appreciate the financial support provided by
NIDA (DA024705) and The Skaggs Institute for Chemical
Biology. (þ)-[20,60-3H(n)] methamphetamine for soluble radio-
immunoassays was obtained from the National Institute on Drug
Abuse (Bethesda, MD) and synthesized at Research Triangle
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