A conjugate vaccine using enantiopure hapten imparts superior nicotine-binding capacity

Article abstract of DOI:10.1021/jm501625j

A leading nicotine conjugate vaccine was only efficacious for one-third of clinical trial participants, likely due in part to its use of racemic nicotine hapten, (±)-3′-AmNic. Immunization of male Wistar rats with (+)-, (-)-, or (±)-3′-AmNicSucTT and subsequent antibody immunoassays suggest that a vaccine using enantiopure (-)-3′-AmNic hapten imparts superior capacity to bind (-)-nicotine. Future nicotine vaccine clinical candidates must incorporate this design consideration (i.e., hapten enantiopurity) in order to maximize efficacy.

Full text of DOI:10.1021/jm501625j

Brief Article
pubs.acs.org/jmc
A Conjugate Vaccine Using Enantiopure Hapten Imparts Superior
Nicotine-Binding Capacity
Jonathan W. Lockner,† Jenny M. Lively,† Karen C. Collins,† Janaína C. M. Vendruscolo,‡ Marc R. Azar,‡
and Kim D. Janda*,†
†Departments of Chemistry and Immunology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California
92037, United States
‡Behavioral Pharma Inc., 505 Coast Boulevard South, La Jolla, California 92037, United States
S
* Supporting Information
ABSTRACT: A leading nicotine conjugate vaccine was only efficacious for one-third of
clinical trial participants, likely due in part to its use of racemic nicotine hapten, ( )-3′-
AmNic. Immunization of male Wistar rats with (+)-, (−)-, or ( )-3′-AmNicSucTT and
subsequent antibody immunoassays suggest that a vaccine using enantiopure (−)-3′-
AmNic hapten imparts superior capacity to bind (−)-nicotine. Future nicotine vaccine
clinical candidates must incorporate this design consideration (i.e., hapten
enantiopurity) in order to maximize efficacy.
nicotine vaccine to date, having progressed all the way through
phase III.8−11 It was safe and well tolerated, but effective for
only a fraction of clinical trial participants.12,13 Nevertheless,
given the huge promise of a clinically approved nicotine
vaccine, research continues unmitigated.14 Many design and
formulation aspects have been scrutinized in recent years to
furnish something better than ( )-3′-AmNic-rEPA. Efforts
include boosting immunogenicity through the use of newer
adjuvants,15−18 improving practicality through alternative
routes of administration,19 and adopting multivalent strat-
egies20−23 to increase antinicotine antibody binding capacity.
For a vaccine aimed at conferring protective immunity
against a specific small molecule such as nicotine, it is important
that the vaccine displays adequate chemical epitope homoge-
neity.24−26 Other vaccines may be engineered to simultaneously
target multiple prevailing epitopes, as in the case of diphtheria−
tetanus−acellular pertussis (DTaP), measles−mumps−rubella
(MMR), and 23-valent pneumococcal combination vac-
cines.27,28 Here, however, the aim is to specifically and
selectively target only (−)-nicotine; thus, a vaccine should
efficiently elicit antibodies capable of sequestering only
(−)-nicotine. The notion that antibodies can enantiodifferen-
tiate was first appreciated by Landsteiner nearly a century
ago29,30 and continues to be exploited to this day. Such work
includes enantioselective catalytic antibodies31−33 and stereo-
specific mAb to nicotine34 and cocaine.35−37 In the case of the
nicotine mAb study, hybridomas were selected using (S)-
(−)-[3H]nicotine, thereby optimizing for antibodies specific for
INTRODUCTION
■
According to the World Health Organization, there are over 1
billion smokers worldwide, and smoking is responsible for
nearly 6 million deaths annually.1 The economic impact is also
sobering: in the United States alone, smoking costs nearly $300
billion in medical expenses and lost productivity each year.2
The epidemiological link between chronic tobacco use and
myriad diseases is well understood, and while many smokers
wish to quit, currently available cessation aids do not help
much. Synthetic small molecule agonists or antagonists target
brain receptors involved in nicotine dependence.3−5 Acting
centrally, these medicines have a variety of side effects.6
Meanwhile, we have been pursuing a pharmacokinetic
(antibody-based) instead of a pharmacodynamic (drug-based)
strategy to aiding smokers’ efforts to quit.7 Nicotine plays a
central role in precipitating addiction to smoking tobacco. A
nicotine vaccine stimulates the immune system to identify
nicotine as a foreign antigen, eliciting antibodies that alter
nicotine pharmacokinetics. Antinicotine antibodies reduce the
concentration of free nicotine in the blood and prevent it from
entering the central nervous system. Blocking the activation of
brain reward systems can facilitate extinction of the addictive
behavior, leading to better smoking cessation outcomes. A
clinically approved nicotine vaccine would be a complementary
addition to available therapies, which, when leveraged
appropriately, could afford significantly better rates of sustained
smoking abstinence.
A leading nicotine conjugate vaccine, consisting of racemic
hapten 3′-aminomethylnicotine conjugated to Pseudomonas
aeruginosa exoprotein A (( )-3′-AmNic-rEPA) and formulated
with alum adjuvant, represents the most clinically advanced
Received: April 7, 2014
© XXXX American Chemical Society
A
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a
Scheme 1. Preparation of Nicotine Vaccine Conjugates Using Enantiopure Nicotine Haptens
a
Reagents and conditions: (a) NH4HCO3, pyridine, Boc2O, CH3CN, H2O, rt, 4 h, 40 °C, 30 min, 84%; (b) Red-Al, toluene, rt, 18 h, 19%; (c) chiral
SFC, ∼80% recovery of each enantiomer; (d) EDC, (−)-2 or (+)-2, pH 5.80 MES buffer, final dialysis against pH 7.4 PBS, hapten densities 41−44;
(e) succinic anhydride, pH 8.65 Tris buffer, linker density 53.
the naturally occurring isomer.34 The present study demon-
strates that the capacity of antibodies to enantiodifferentiate
should not be ignored in development of vaccines for drugs of
abuse.
derived male rats (n = 10−12 per group, 250−300 g) were
assigned randomly to (−)- or (+)-groups (first study), or (−)-,
(+)-, and ( )-groups (second study). Rats were given free
access to food and water during the immunization schedule
(Figure 1), which consisted of a series of three (200 μL)
Many of the nicotine vaccines that have undergone clinical
evaluation began with manipulation of racemic trans-cotinine
carboxylic acid (( )-1, Scheme 1). To the best of our
knowledge, no chiral separation step (nor asymmetric synthesis
step) was included in the production of the hapten−protein
conjugate that would become ( )-3′-AmNic-rEPA. Hence, we
set out to examine the notion that a nonracemic, fully
(−)-nicotine vaccine conjugate would be a superior immu-
nogen, owing to the exquisite ability of antibodies to
stereodifferentiate. For the present study, we chose to focus
our attention on 3′-AmNic, the hapten used in ( )-3′-AmNic-
rEPA.
Figure 1. Rat immunization and bleed schedule.
intramuscular injections on days 0, 21, and 42. On days 28 and
49, plasma samples were obtained by tail vein bleed. On day 63,
rats were anesthetized, bled by cardiac puncture, and
euthanized.
RESULTS
■
First, racemic trans-3′-aminomethylnicotine (3′-AmNic, ( )-2)
was prepared from commercially available racemic trans-
cotininecarboxylic acid (( )-1). Next, using chiral supercritical
fluid chromatography (SFC), ∼600 mg of ( )-2 was separated
into ∼250 mg of each enantiomer (Scheme 1). Given our prior
experience38−40 coupling carboxylate-containing nicotine hapt-
ens to carrier proteins, we tried to do the same in the present
context. Thus, each enantiomer of 2 was acylated with succinic
anhydride. However, activation of succinylated haptens and
mixing with TT gave conjugates with low hapten densities.
Consequently, we employed an alternative choreography
(Scheme 1, inset), in which the carrier protein (rather than the
hapten) is first succinylated.18,41,42 First, TT was treated with
succinic anhydride in pH 8.65 Tris buffer. Then, SucTT was
treated with EDC and either (−)- or ( )-2 in pH 5.80 MES
buffer, with final dialysis against pH 7.4 PBS. At this point, we
had separate batches of (−)- and (+)-3′-AmNicSucTT (hapten
densities ∼44 and ∼41 by MALDI-TOF MS), ready for
formulation with adjuvants. For the second rat study, these
procedures were repeated in order to furnish (−)-, (+)-, and
( )-3′-AmNicSucTT (hapten densities ∼56, ∼55, and ∼67).
Each hapten−protein conjugate was formulated with
phosphorothioated CpG ODN 182643−45 (Eurofins) and
Alhydrogel 2% (InvivoGen). Vaccines prepared in this manner
contained 100 μg of conjugate, 100 μg of CpG, and 100 μL of
Alhydrogel per 200 μL of complete formulation.46 Wistar-
Enzyme-linked immunosorbent assays (ELISAs) were carried
out using either (−)- or (+)-3′-AmNicSucBSA, prepared in a
manner analogous to the TT conjugates above. For self-reactive
ELISA, plasma samples were run against their respective
haptens: rat plasma from the (−)-3′-AmNicSucTT group was
assayed on (−)-3′-AmNicSucBSA plates, and rat plasma from
the (+)-3′-AmNicSucTT group was assayed on (+)-3′-
AmNicSucBSA plates. For cross-reactive ELISA, plasma
samples were run against their antipodes: rat plasma from the
(−)-3′-AmNicSucTT group was assayed on (+)-3′-AmNic-
SucBSA plates, and rat plasma from the (+)-3′-AmNicSucTT
group was assayed on (−)-3′-AmNicSucBSA plates. The
( )-group was assayed on both (−)- and (+)-plates.
(−)-Nicotine-specific plasma antibody binding affinities and
antibody concentrations were determined by competitive
radioimmunoassay (RIA).47 First, the plasma dilution that
3
binds ∼50% of H-labeled (−)-nicotine is determined. Then,
the affinity constant is calculated by competition with unlabeled
nicotine. Because the plasma samples were pooled for each
vaccine group, the measured affinity constants are average
affinities (Kd,avg) for each group.
DISCUSSION AND CONCLUSIONS
■
The results of ELISA (first rat study) are summarized in Figure
2. By bleed 2 (day 49), titers were approximately 100000.
Furthermore, cross-reactive ELISA results demonstrate an
B
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Figure 2. Average ELISA titers for plasma from (−)-, or (+)-3′-AmNicSucTT groups (n = 12; first rat study). Each animal was assayed in duplicate;
error bars are SEM. Left panel: Rat plasma on (−)-3′-AmNicSucBSA coated plates. Two-way repeated measures ANOVA (treatment: F1,22 = 17.29,
***P < 0.001). Right panel: Rat plasma on (+)-3′-AmNicSucBSA coated plates. Two-way repeated measures ANOVA (treatment: F1,22 = 15.29,
***P < 0.001).
Table 1. Anti-(−)-nicotine Antibody Binding Affinities and Concentrations from Radioimmunoassay (RIA)
bleed 2
[Ab]avg (μg/mL)
47.5 6.6
bleed 3
a
b
b
c
b
b
c
vaccine
Kd,avg (nM)
X (g/μmol)
Kd,avg (nM)
[Ab]avg (μg/mL)
X (g/μmol)
(−)-3′-AmNicSucTT
(+)-3′-AmNicSucTT
23.9 3.2
23.8 3.2
1.99
0.50
250 22
118 2.7
30.4 2.6
0.47
0.27
12.0 1.2
111
9
a
b
Formulated with CpG ODN 1826 and Alhydrogel. Values are average (with SEM) Kd and [Ab] determined by RIA using (−)-[N-methyl-3H]-
c
nicotine and pooled rat plasma samples (n = 12; first rat study) assayed in triplicate. X is the quotient of [Ab]avg divided by Kd,avg. Units for X (μg/
mL/nM) have been reduced to g/μmol.
approximately 3−5-fold difference in titers, suggesting that
plasma antibodies produced by these two groups of rats possess
a measurable level of enantiodifferentiation. Importantly,
plasma from the (−)-3′-AmNicSucTT group has superior
capacity to bind to natural (−)-nicotine displayed by (−)-3′-
AmNicSucBSA. Implications of this phenomenon will be
discussed below.
Radioimmunoassay provides a means for determining the
average binding affinity and average antibody concentration for
a soluble ligand. Because the ligand is soluble and free to
associate/dissociate in the analysis milieu, it offers significant
advantage over ELISA, in which the ligand is immobilized on
the plate surface, not to mention conjugated to a carrier protein
(e.g., BSA). Thus, the equilibrium environment simulated in a
RIA experiment much more closely mimics that of free nicotine
that would distribute in the blood and brain during tobacco use.
It behooves researchers in the field to routinely incorporate
RIA to evaluate the immunogenic efficacy of drug of abuse
vaccine formulations.
From each binding curve generated (see Supporting
Information), average binding affinity (Kd,avg) and average
antinicotine antibody concentration ([Ab]avg) were obtained
(Table 1). By bleed 2, an approximately 4-fold difference in
[Ab]avg was observed between the (−)-3′-AmNicSucTT group
and the (+)-3′-AmNicSucTT group. The approximately 4-fold
difference in antibody concentration observed in bleed 2 was
maintained in bleed 3. It is interesting to note that ELISA
results showed 3−5-fold difference in titers; the results of these
two immunoassays (ELISA and RIA) correlate with one other.
However, we were surprised to see higher affinities for
(−)-nicotine in the (+)-group. Scrutiny of the ELISA results
and the RIA results might suggest conflicting interpretations. In
particular, it seems surprising that the binding affinity for
nicotine is superior (lower Kd,avg at bleed 3) for the rats that
received the (+)-vaccine. The (+)-group’s plasma antibodies
(Kd,avg 111 nM) have 2-fold higher binding affinity for
(−)-nicotine than the (−)-group’s plasma antibodies (Kd,avg
250 nM). This feature is congruent with the findings of
others,48 who reported that a human antinicotine mAb bound
(+)-nicotine with slightly higher affinity than (−)-nicotine.
Incidentally, this mAb (Nic12) was derived from another
clinically evaluated nicotine vaccine that fell short in phase II.49
The seemingly counterintuitive result for the measured Kd,avg
values may be rationalized by bearing in mind that nicotine
(unlike cocaine and heroin) possesses greater conformational
flexibility because it consists of two heterocyclic rings joined via
a single carbon−carbon bond. Either enantiomer of nicotine
can adopt an appropriate conformation suitable for making
critical binding interactions with an antibody’s binding site.
These include the pyridyl nitrogen serving as a hydrogen-bond
acceptor and the pyrrolidinium nitrogen engaging in charge−
charge and/or cation−π interaction(s).
Linker attachment can also play a role in directing antihapten
antibody quantity and quality. For instance, if morphine is
coupled through its 3-position to a carrier protein, codeine (3-
methylmorphine) is a more effective inhibitor (than morphine)
of the resultant morphine antiserum.50 In the present study,
nicotine is linked to protein via the 3′-position on its
pyrrolidine ring. This 3′-linkage may impose constraints on
antibody elicitation such that the measured antinicotine
antibodies, while being of lower quantity, nevertheless exhibit
slightly higher affinity for free (−)-nicotine than antibodies
elicited by the (−)-3′-AmNic conjugate.
As a means for reconciling this seeming discrepancy between
average antibody affinity and average antibody concentration,
we propose the use of a composite parameter, X, defined as the
ratio of [Ab]avg over Kd,avg for a given pool of antisera.
Supposing that two ways for improving vaccine performance
are to elicit higher [Ab]avg (antibody abundance) and lower
Kd,avg (antibody utility) values, then as improvements are made
in either/or/both of these terms, the ratio (X) will become
larger. Thus, for bleed 3, (−)-group’s X = 0.47, while
(+)-group’s X = 0.27. Conceptually speaking, the aim is to
optimize protein design for a given ligand target (“antibody
efficiency”); the inverse is widely applied in medicinal
chemistry: optimizing a ligand design for a given protein target
C
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(e.g., “ligand efficiency”). That said, the ratio (X) is a means for
assessing antibody efficiency and hence vaccine efficacy.
Comparing X values for bleeds 2 and 3, we see the negative
impact of decreasing Kd,avg over the time course of
immunization. While RIA affinity measurements to (−)-nic-
otine were never undertaken for the clinical vaccine, it is
tempting to speculate that, besides antibody titer, untoward
antibody affinity decrease may have also contributed to the
vaccine’s inability to achieve the desired clinical outcomes.
To validate our estimation (by algebraic extrapolation51) that
racemic ( )-3′-AmNicSucTT would only be ∼63% as effective
as enantiopure (−)-3′-AmNicSucTT, we conducted a second
rat immunization study in which groups (n = 10) were
administered (−)-, (+)-, or ( )-3′-AmNicSucTT in formula-
tion with phosphorothioated CpG ODN 1826 and Alhydrogel.
Average bleed 1 ELISA titers for the (−)-3′-AmNicSucTT
group were similar (first study, ∼66000; second study,
∼60000). Importantly, however, the average bleed 1 ELISA
titers (Figure 3) for the ( )-3′-AmNicSucTT group were only
Chart 1. Structure of Nicotine Hapten (−)-CNI
Recently, others have suggested that coadministration of two
(“bivalent”) or three (“trivalent”) structurally distinct nicotine
immunogens may be a viable means to compensate for
suboptimal nicotine vaccine performance.20−23 The individual
nicotine immunogens elicited antibodies with nonoverlapping
specificities. If one accepts that (−)-3′-AmNic is structurally
distinct from (+)-3′-AmNic, then a mixture of the two (racemic
( )-3′-AmNic) would itself constitute a “bivalent” nicotine
vaccine.
The data herein demonstrate that these two immunogens are
not equally capable of eliciting anti-(−)-nicotine antibodies, and
a mixture of the two would be inferior to pure (−)-3′-AmNic.
Empirically, we find that the (+)-vaccine is only one-fourth as
effective as the (−)-vaccine (4-fold lower [Ab]avg, Table 1) and
the ( )-vaccine is only ∼60% as effective as the (−)-vaccine
(Figure 3). Because structurally distinct immunogens activate
distinct populations of antibody-producing B cells, we reckon
the racemic hapten approach (as in ( )-3′-AmNic-rEPA) to be
a readily remediable limitation. Instead, by using a vaccine
consisting of a single optimized immunogen, bearing solely
(−)-nicotine hapten, we target (with laser-like precision) a
relatively tighter distribution of antibody-producing B cells.
Serological analyses of groups of rats vaccinated with (−)-,
(+)-, or ( )-3′-AmNic conjugates suggest that a vaccine using
an enantiopure (−)-hapten may elicit plasma antibodies with
superior capacity to bind natural (−)-nicotine. This will
undoubtedly be a critical vaccine design consideration that is
heeded in nicotine vaccines following ( )-3′-AmNic-rEPA.
Future research in the nicotine vaccine field should incorporate
this significant aspect in order to produce the most efficacious
formulations capable of eliciting protective immunity against
(−)-nicotine. A logical next step will be to correlate these
serological data with in vivo efficacy data (for example, by
measuring brain/plasma nicotine concentrations following
acute nicotine administration).
By taking full advantage of the capacity of antibodies to
enantiodifferentiate, in combination with hapten conforma-
tional rigidity (e.g., via (−)-CNI), we anticipate a maximal
impact on the ability of such a vaccine to elicit antibodies with
even greater capacity to sequester (−)-nicotine. Additionally,
for situations in which [Ab]avg and Kd,avg do not neatly correlate,
use of the composite parameter, X, may be of benefit for
ranking the effectiveness of vaccine formulations being tested.
Put simply, one should seek to maximize X, by increasing
[Ab]avg, decreasing Kd,avg, or both. The results of investigations
along these lines will be reported in due course.
Figure 3. Average ELISA titers for plasma from (−)-, (+)-, or ( )-3′-
AmNicSucTT groups (n = 10; second rat study; bleed (1) on (−)- or
(+)-3′-AmNicSucBSA coated plates. Each animal was assayed in
duplicate; error bars are SEM. One-way ANOVA (F5,54 = 22.47, P <
0.001); Sidak test (***P < 0.001, ##P < 0.05).
∼61% as high as titers for the (−)-3′-AmNicSucTT group. All
else with regard to vaccine composition and dose being equal,
there is significant gain achieved by using enantiopure hapten.
The present results validate the notion that one must factor
chirality into vaccine design, taking care to mimic the natural
stereochemistry of the small molecule, be it (−)-cocaine,
(−)-heroin, (−)-nicotine, or any other intended target. The
situation is more nuanced in the case of nicotine, which
presumably has much to do with the conformational flexibility
of nicotine itself, as well as linker placement. As a ligand,
nicotine can bind in a variety of orientations within the binding
site of an antinicotine antibody. By contrast, the additional
structural constraints in cocaine or heroin impose greater
conformational rigidity, and one observes unsurprising results
for relative binding affinities of antibodies for natural versus
unnatural enantiomers of these illicit drugs.
With regard to nicotine, we have previously made the case
that one may gain advantage by “building in” further
conformational rigidity in hapten design, by recourse to ring
fusion, as in CNI.39 We contend that an enantiopure version of
CNI (Chart 1) would combine the benefits of conformational
rigidity and optical purity, with synergistic effect for eliciting
optimal antibody defense against (−)-nicotine in the blood-
stream.
EXPERIMENTAL SECTION
■
Chemistry. All reactions were carried out under an argon
atmosphere with dry solvents using anhydrous conditions unless
otherwise stated. Most chemicals were purchased from Sigma-Aldrich
(St. Louis, MO) and used as received. L-(−)-[N-Methyl-3H]-nicotine
with specific activity of 81.7 Ci/mmol (product no. NET827250UC)
was purchased from PerkinElmer (Boston, MA). BSA (product no.
D
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77110) and EDC (product no. 22980) were purchased from Thermo
Scientific Pierce Biotechnology (Rockford, IL). Tetanus toxoid (TT)
was purchased from Statens Serum Institut (Copenhagen, Denmark).
Yields refer to chromatographically (HPLC) and spectroscopically (1H
NMR) homogeneous (≥95%) materials. Reactions were monitored by
thin layer chromatography (TLC) carried out on 0.25 mm E. Merck
silica gel plates (60F-254) using UV light as the visualizing agent. Flash
column chromatography was performed using E. Merck silica gel (60,
particle size 0.040−0.063 mm). Organic solvents were concentrated
on a rotary evaporator under reduced pressure, followed by further
evacuation using a dual stage mechanical pump. NMR spectra were
recorded on a Bruker Avance III HD with DCH CryoProbe (600
MHz) instrument or a Bruker BioSpin DRX (500 MHz) instrument
and calibrated using residual undeuterated solvent as an internal
reference (CD3OD @ δ 4.87 ppm 1H NMR, δ 49.00 ppm 13C NMR).
The following abbreviations (or combinations thereof) are used to
explain 1H NMR multiplicities: s = singlet, d = doublet, t = triplet, m =
multiplet. High-resolution mass spectra (HRMS) were recorded on an
Agilent LC/MSD TOF mass spectrometer by electrospray ionization
time-of-flight reflectron experiments. IR spectra were recorded on a
Thermo Scientific Nicolet 380 FTIR spectrometer.
column. Each cycle was 5 min. Peak 1 was collected by UV detection
during 2.96−3.35 min, and peak 2 was collected during 3.78−4.30
min. All peak 1 and peak 2 collections were combined separately and
rotary evaporated to dryness at 35 °C (5−10 mmHg). Peak 1: (−)-2,
2.712 min (2.875 min); 241.3 mg, >99% ee; [α]D22, −31.2 (c 0.25,
MeOH). Peak 2: (+)-2, 3.664 min (3.860 min); 272.5 mg, >99% ee;
22
[α]D +30.6 (c 0.15, MeOH).
Hapten−Protein Conjugations. A solution of BSA (∼4 mg/mL)
or TT (∼2 mg/mL) in pH 8.65 Tris buffer was treated with succinic
anhydride (1.0 M in DMSO, 750−3000 equiv) at room temperature
for 1 h. The bulk of the material was dialyzed against pH 5.80 MES
buffer for the subsequent step (below), while a small portion of the
material was dialyzed against PBS pH 7.4 for BCA assay and MALDI-
TOF analysis. Succinyl group densities of 47 per BSA or 53 per TT
were obtained. A solution of SucBSA (∼4 mg/mL) or SucTT (∼2
mg/mL) in pH 5.80 MES buffer was treated with (−)-2, (+)-2, or
( )-2 (0.2 M in H2O, 100−800 equiv) and EDC (2.0 M in H2O, 1000
equiv) at room temperature for 20−24 h. Final dialyses against PBS
pH 7.4 removed excess reagents. Hapten densities of 67−68 per
SucBSA, 41−44 per SucTT (first study), and 55−67 per SucTT
(second study) were obtained. Concentrations of proteins and
hapten−protein conjugates were determined using a BCA Protein
Assay Kit (Thermo Scientific, product no. 23225) according to
manufacturer’s directions.
Biology. Each hapten−protein conjugate was mixed with
phosphorothioated CpG ODN 1826 (Eurofins MWG Operon) and
diluted to 1.0 mg/mL in pH 7.4 PBS. Then, an equal volume of
Alhydrogel 2% (vac-alu-50, InvivoGen) was added dropwise, followed
by 10 min of gentle inversion. Vaccines prepared in this manner
contained 100 μg of conjugate, 100 μg of CpG, and 100 μL of
Alhydrogel per 200 μL of complete formulation. All animal care and
use was performed according to NIH guidelines and in compliance
with protocols approved by the Institutional Animal Care and Use
Committee at Behavioral Pharma, Inc. Wistar-derived male rats (n =
10−12 per group, 250−300 g) were purchased from Harlan (Indiana,
USA) and assigned randomly to (−)-, (+)-, or ( )-groups. Rats were
given free access to food and water during the immunization schedule,
which consisted of three (200 μL) intramuscular injections on days 0,
21, and 42. On days 28 and 49, plasma samples were obtained by tail
vein bleed. On day 63, animals were anesthetized, bled by cardiac
puncture, and euthanized.
ELISA. Plates (96-well Costar 3690) were coated with either
(−)-3′-AmNicSucBSA or (+)-3′-AmNicSucBSA diluted in PBS pH 7.2
(5 μg/mL, 25 μL/well) and incubated at 37 °C overnight. Then, plates
were fixed with MeOH (50 μL/well) at room temperature for 5 min,
blocked with 5% nonfat powdered milk in PBS pH 7.2 (50 μL/well)
37 °C for 30 min, and charged with 2% BSA in PBS pH 7.2 (25 μL/
well). Plasma samples (diluted 1:100 in 1% BSA in PBS pH 7.2) were
added to the first column and serially diluted across the plate. Plates
were incubated at 37 °C for 90 min, washed with H2O, and treated
with secondary antibody (goat-antirat Ig(H+L)-HRP, Southern
Biotech, catalogue no. 3010-05, diluted 1:5000 in 2% BSA in PBS
pH 7.2, 25 μL/well). Plates were incubated at 37 °C for 30 min,
washed with H2O, treated with developing reagent (TMB Pierce
Substrate Kit, 50 μL/well), and incubated at room temperature in the
dark for 10−20 min. Color development was halted by addition of 2 M
H2SO4 (50 μL/well), and plates were read on a SpectraMax M2e
(Molecular Devices, Sunnyvale, CA) at 450 nm. Midpoint titers were
obtained using Microsoft Excel and GraphPad Prism.
( )-trans-3′-Aminomethylnicotine (( )-2). A suspension of
( )-trans-4-cotininecarboxylic acid ( )-1 (5.18 g, 23.5 mmol) and
ammonium bicarbonate (2.60 g, 32.9 mmol) in CH3CN (104 mL) was
treated with a solution of pyridine (0.95 mL, 11.8 mmol) in CH3CN
(5 mL). The mixture was stirred at room temperature for 10 min, then
treated with a solution of di-tert-butyl dicarbonate (7.57 mL, 32.9
mmol) in CH3CN (21 mL). The mixture was stirred at room
temperature for 15 min, then treated with H2O (2.6 mL). The mixture
was stirred at room temperature for 4 h, then at 40 °C for 30 min. The
mixture was concentrated in vacuo. Purification by silica gel
chromatography (10:1 CH2Cl2/MeOH) afforded ( )-S1 (4.33 g,
84%) as a white solid. Rf = 0.20 (silica gel, 10:1 CH2Cl2/MeOH). IR
1
(neat) νmax 3304, 1651, 1393, 1299, 1128, 713 cm−1. H NMR (600
MHz, CD3OD) δ 8.57 (dd, J = 4.9, 1.6 Hz, 1 H), 8.53 (d, J = 2.0 Hz, 1
H), 7.82 (dt, J = 7.9, 1.9 Hz, 1 H), 7.54 (dd, J = 8.1, 4.7 Hz, 1 H),
4.87−4.86 (m, 3 H), 3.08 (ddd, J = 9.6, 7.9, 6.5 Hz, 1 H), 2.86 (dd, J =
17.0, 9.5 Hz, 1 H), 2.70 (dd, J = 17.1, 8.0 Hz, 1 H), 2.66 (s, 3 H). 13C
NMR (150 MHz, CD3OD) δ 176.1, 175.8, 150.5, 149.4, 137.2, 136.9,
125.9, 66.5, 47.9, 35.5, 28.7. HRMS (ESI-TOF) calcd for
C11H13N3O2H+ [M + H+], 220.1080; found, 220.1082. A solution of
( )-S1 (4.13 g, 18.8 mmol) in toluene (261 mL) was treated with a
solution of Red-Al (65 wt % in toluene, 25.4 mL, 84.8 mmol). The
mixture was stirred at room temperature for 18 h, then poured into a
suspension of Celite (4.13 g) and Darco G60 charcoal (2.07 g) in H2O
(41 mL). The quenched reaction mixture was filtered, and the filter
cake was washed with H2O (2 × 8 mL). The filtrate was transferred to
a separatory funnel, and layers were separated. The aqueous layer was
concentrated in vacuo. Purification by silica gel chromatography
(85:15:1 CH2Cl2/MeOH/NH4OH) afforded ( )-2 (0.68 g, 19%) as a
pale-yellow syrup. Rf = 0.13 (silica gel, 85:15:1 CH2Cl2/MeOH/
NH4OH). IR (neat) νmax 3282, 1646, 1578, 1432, 1320, 1025, 713
cm−1. 1H NMR (500 MHz, CD3OD) δ 8.56 (d, J = 1.9 Hz, 1 H), 8.50
(dd, J = 4.9, 1.5 Hz, 1 H), 7.92 (dt, J = 8.0, 1.8 Hz, 1 H), 7.48 (dd, J =
7.8, 4.9 Hz, 1 H), 4.87 (s, 2 H), 3.29−3.25 (m, 1 H), 2.88 (d, J = 8.0
Hz, 1 H), 2.69 (dd, J = 12.7, 4.5 Hz, 1 H), 2.60 (dd, J = 12.7, 8.1 Hz, 1
H), 2.45 (q, J = 8.9 Hz, 1 H), 2.26−2.21 (m, 2 H), 2.16 (s, 3 H),
1.78−1.72 (m, 1 H). 13C NMR (125 MHz, CD3OD) δ 150.3, 149.5,
139.3, 137.9, 125.5, 74.2, 56.8, 51.8, 45.2, 40.6, 28.4. HRMS (ESI-
TOF) calcd for C11H17N3H+ [M + H+], 192.1495; found, 192.1496.
Chiral SFC Separation of (−)-2 and (+)-2. Column, Chiralpak
AD-H, 21 mm × 250 mm; mobile phase, 85:15 CO2/MeOH (0.05%
Et2NH); pressure, 120 bar; flow rate, 60.0 mL/min; injection amount,
0.8 mL (∼24 mg); temperature, 36 °C; wavelength, 260 nm; sample
preparation, ∼600 mg of ( )-2 was dissolved in ∼20 mL MeOH;
operational procedure, a preparative Berger MultiGram II SFC
(Waters Inc., Milford, MA) instrument was equilibrated at conditions
above. Dual Varian SD-1 pumps delivered CO2 (liquid) and MeOH
(containing 0.05% Et2NH). Injections of 0.8 mL (∼24 mg compound,
total 28 injections) onto a Chiralpak AD-H 21 mm ID × 250 mm
RIA. Competitive RIA was carried out in a 5 kDa MWCO
Equilibrium Dialyzer-96 (Harvard Apparatus, Holliston, MA) to allow
easy separation of bound and free L-[N-methyl-3H]-nicotine tracer;
specific activity = 81.7 Ci/mmol (PerkinElmer, Boston, MA). Pooled
rat plasma were diluted in 2% BSA to a concentration that bound
3
∼50% of ∼30000 dpm of H-nicotine tracer. Each sample chamber
was loaded with 75 μL of diluted plasma and 75 μL of radiolabeled
tracer (∼30000 dpm), and each buffer chamber was loaded with 150
μL of unlabeled (−)-nicotine at varying concentrations in 1% BSA.
Chamber contents were equilibrated at room temperature for at least
22 h. A 75 μL aliquot was removed from each sample/buffer chamber
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dx.doi.org/10.1021/jm501625j | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Brief Article
and added to 5 mL of scintillation fluid (Ecolite(+), MP Biomedicals,
Santa Ana, CA). Radioactivity (dpm) was measured in a Beckman LS
6500 scintillation counter. Kd,avg and [Ab]avg values were obtained
using Microsoft Excel and GraphPad Prism.
(9) Wagena, E. J.; de Vos, A.; Horwith, G.; van Schayck, C. P. The
immunogenicity and safety of a nicotine vaccine in smokers and
nonsmokers: results of a randomized, placebo-controlled phase 1/2
trial. Nicotine Tob. Res. 2008, 10, 213−218.
(10) Hatsukami, D. K.; Jorenby, D. E.; Gonzales, D.; Rigotti, N. A.;
Glover, E. D.; Oncken, C. A.; Tashkin, D. P.; Reus, V. I.; Akhavain, R.
C.; Fahim, R. E.; Kessler, P. D.; Niknian, M.; Kalnik, M. W.; Rennard,
S. I. Immunogenicity and smoking-cessation outcomes for a novel
nicotine immunotherapeutic. Clin. Pharmacol. Ther. 2011, 89, 392−
399.
(11) Hoogsteder, P.; Kotz, D.; Viechtbauer, W.; Brauer, R.; Kessler,
P.; Kalnik, M.; Fahim, R.; Spiegel, P. V.; Schayck, O. V. The efficacy
and safety of a nicotine conjugate vaccine (NicVAX) or placebo co-
administered with varenicline (Champix) for smoking cessation: study
protocol of a phase IIb, double blind, randomized, placebo controlled
trial. BMC Public Health 2012, 12, 1052.
(12) Nabi Biopharmaceuticals Announces Results of First NicVAX Phase
III Clinical Trial: Smoking Cessation Immunotherapy Failed to Meet
Primary Endpoint; Nabi Biopharmaceuticals: Rockville, MD, July 18,
2011.
(13) Nabi Biopharmaceuticals Announces Results of Second NicVAX
Phase III Clinical Trial: Smoking Cessation Immunotherapy Failed to
Meet Primary Endpoint; Nabi Biopharmaceuticals: Rockville, MD,
November 7, 2011.
ASSOCIATED CONTENT
* Supporting Information
Tabular summary of nicotine vaccine clinical candidates, assay
results, NMR spectra, HPLC chromatograms, and MALDI-
TOF spectra. This material is available free of charge via the
Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
*Phone: (858)-785-2516. Fax: (858) 784-2595. E-mail:
kdjanda@scripps.edu.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Jeff Elleraas at Pfizer La Jolla for separating (−)-2
and (+)-2 using chiral SFC, Mark Hixon at Takeda California
for useful discussion regarding composite parameter X, and
Nirvan Rouzbeh for technical assistance with rat injections and
bleeds. This work was supported by Tobacco-Related Disease
Research Program (TRDRP) grant no. 20XT-0156 (to K.D.J.).
This is manuscript no. 27033 from The Scripps Research
Institute.
(14) A complete listing of nicotine vaccine clinical candidates is
provided in the Supporting Information.
(15) Selecta Biosciences Initiates Phase 1 Clinical Study of SEL-068, a
First-in-Class Synthetic Nicotine Vaccine for Smoking Cessation and
Relapse Prevention; Selecta Biosciences: Watertown, MA, November
21, 2011.
(16) Pittet, L.; Altreuter, D.; Ilyinskii, P.; Fraser, C.; Gao, Y.; Baldwin,
S.; Keegan, M.; Johnston, L.; Kishimoto, T. Development and
preclinical evaluation of SEL-068, a novel targeted Synthetic Vaccine
Particle (tSVP) for smoking cessation and relapse prevention that
generates high titers of antibodies against nicotine. J. Immunol. 2012,
188.
(17) Lockner, J. W.; Ho, S. O.; McCague, K. C.; Chiang, S. M.; Do,
T. Q.; Fujii, G.; Janda, K. D. Enhancing nicotine vaccine
immunogenicity with liposomes. Bioorg. Med. Chem. Lett. 2013, 23,
975−978.
(18) McCluskie, M. J.; Pryde, D. C.; Gervais, D. P.; Stead, D. R.;
Zhang, N.; Benoit, M.; Robertson, K.; Kim, I. J.; Tharmanathan, T.;
Merson, J. R.; Davis, H. L. Enhancing immunogenicity of a
3′aminomethylnicotine−DT-conjugate anti-nicotine vaccine with
CpG adjuvant in mice and non-human primates. Int. Immunopharma-
col. 2013, 16, 50−56.
ABBREVIATIONS USED
■
3′-AmNic, 3′-aminomethylnicotine; BSA, bovine serum albu-
min; CpG ODN, cytosine-phosphorothioate-guanine oligo-
deoxynucleotide; EDC, 1-ethyl-3-[3-(dimethylamino)propyl]-
carbodiimide hydrochloride; ELISA, enzyme-linked immuno-
sorbent assay; mAb, monoclonal antibody; MES, 2-(N-
morpholino)ethane-sulfonic acid; Suc, succinyl; TT, tetanus
toxoid
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