Probing active cocaine vaccination performance through catalytic and noncatalytic hapten design

Article abstract of DOI:10.1021/jm400228w

Presently, there are no FDA-approved medications to treat cocaine addiction. Active vaccination has emerged as one approach to intervene through the rapid sequestering of the circulating drug, thus terminating both psychoactive effects and drug toxicity. Herein, we report our efforts examining two complementary, but mechanistically distinct active vaccines, i.e., noncatalytic and catalytic, for cocaine treatment. A cocaine-like hapten GNE and a cocaine transition-state analogue GNT were used to generate the active vaccines, respectively. GNE-KLH (keyhole limpet hemocyannin) was found to elicit persistent high-titer, cocaine-specific antibodies and blunt cocaine-induced locomotor behaviors. Catalytic antibodies induced by GNT-KLH were also shown to produce potent titers and suppress locomotor response in mice; however, upon repeated cocaine challenges, the vaccine's protecting effects waned. In depth kinetic analysis suggested that loss of catalytic activity was due to antibody modification by cocaine. The work provides new insights for the development of active vaccines for the treatment of cocaine abuse.

Full text of DOI:10.1021/jm400228w

Article
pubs.acs.org/jmc
Probing Active Cocaine Vaccination Performance through Catalytic
and Noncatalytic Hapten Design
Xiaoqing Cai,† Timothy Whitfield,‡ Mark S. Hixon,§ Yanabel Grant,‡ George F. Koob,‡
and Kim D. Janda*,†
†Departments of Chemistry and Immunology, The Skaggs Institute for Chemical Biology, The Worm Institute of Research and
Medicine, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, United States
‡Committee on the Neurobiology of Addictive Disorders, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla,
California 92037, United States
§Department of Discovery Biology, Takeda California, Inc., 10410 Science Center Drive, San Diego, California 92121, United States
S
* Supporting Information
ABSTRACT: Presently, there are no FDA-approved medications to treat cocaine addiction. Active vaccination has emerged as
one approach to intervene through the rapid sequestering of the circulating drug, thus terminating both psychoactive effects and
drug toxicity. Herein, we report our efforts examining two complementary, but mechanistically distinct active vaccines, i.e.,
noncatalytic and catalytic, for cocaine treatment. A cocaine-like hapten GNE and a cocaine transition-state analogue GNT were
used to generate the active vaccines, respectively. GNE−KLH (keyhole limpet hemocyannin) was found to elicit persistent high-
titer, cocaine-specific antibodies and blunt cocaine-induced locomotor behaviors. Catalytic antibodies induced by GNT−KLH
were also shown to produce potent titers and suppress locomotor response in mice; however, upon repeated cocaine challenges,
the vaccine’s protecting effects waned. In depth kinetic analysis suggested that loss of catalytic activity was due to antibody
modification by cocaine. The work provides new insights for the development of active vaccines for the treatment of cocaine
abuse.
passage of cocaine across the blood−brain barrier (BBB) into
the central nervous system (CNS), where the drug exerts its
addictive effects.5 Two vaccine strategies have been targeted:
the use of simple binding antibodies or catalytic antibodies,
both of which invoke the tenet of preventing cocaine from
crossing the blood−brain barrier, thus terminating the drug-
induced efforts.
A number of anticocaine vaccines based upon simply
sequestering the drug have been disclosed. Their origins
include the linking of various cocaine-like haptens to carrier
proteins, such as bovine serum albumin (BSA), keyhole limpet
hemocyannin (KLH), and cholera toxin, indeed, one termed
TA-CD has reached phase II clinical trials.6 Our group reported
a first-generation hapten termed GNC appended to KLH,
INTRODUCTION
■
Cocaine is a powerful psychoactive substance whose abuse
remains a prevalent health and societal crisis.1 Furthermore,
numerous medical complications including cardiovascular
toxicity, brain damage, and death often accompany cocaine
abuse. In addition, the association of the drug with the spread
of AIDS is also of great concern.2 Presently, there is no proven
medications to treat cocaine addiction, and while a number of
direct and indirect agonists or antagonists have been examined
for the treatment of cocaine dependence, overall, these have
met limited success.3 Over the past two decades, immuno-
pharmacotherapy has been explored sporadically as an
alternative strategy for the treatment of cocaine abuse. Cocaine
vaccines either active or passive have been created to induce the
production of anticocaine antibodies (Abs).4 Immune stim-
ulation and antibody production is engaged to bind and/or
modify peripherally circulating cocaine, thus blocking the
Received: February 18, 2013
Published: April 29, 2013
© 2013 American Chemical Society
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Figure 1. The structure of (−)-cocaine and cocaine-like haptens GNC, GND, GNE, SNC, and GNT.
Scheme 1. Synthesis of Cocaine Hapten GNE
which elicited potent titers of anticocaine antibodies with high
affinity and specificity for cocaine (Figure 1). The induced
antibodies significantly sequestered cocaine in the periphery of
rodents and suppressed cocaine-induced psychomotor stim-
ulation.7 A second-generation hapten termed GND was
developed to increase hapten stability by replacing the relatively
labile C2 and C3 ester bonds with amides (Figure 1).
Accordingly, GND−KLH conjugates provided greater and
longer-lasting protection against cocaine.8 However, from a
synthetic perspective, GND presents a unique challenge,
therefore GND is impractical for a viable clinical vaccine. To
prioritize access to a stable antigen, we synthesized a third-
generation cocaine hapten termed GNE (Figure 1 and Scheme
1) via a C2 ester-amide interchange. We hypothesized that an
ester-amide interchange at this position would prevent loss of
the cocaine scaffold from the carrier protein in the same way as
seen for GND. By this approach, we have easier synthetic
access to a structure that would still maximize anticocaine
immune response. Indeed, an anticocaine vaccine based on
coupling GNE (3, Scheme 1) to the highly immunogenic
adenovirus capsid protein has been reported to evoke
persistent, high titer anticocaine antibodies.9
In addition to active vaccine sequestering antibodies, catalytic
antibodies have also emerged as a powerful tool with the
potential to treat cocaine addiction. The unique feature of a
catalytic antibody is that after it hydrolyzes its substrate and
releases its metabolites, the antibody becomes free for further
binding, hydrolysis, and thus turnover. Seminal research by
Landry and co-workers demonstrated how a transition-state-
analogue strategy for hapten design elicited several cocaine-
hydrolyzing mAbs.10 Here, the transition-state analogue used
was a phenylphosphonate, which granted antibodies that
hydrolyzed the benzoyl ester of cocaine, resulting in the
inactive psychometabolite ecgonine methyl ester. A second
approach reported by Basmadjian and co-workers used again
transition-state analogue haptens, but now polyclonal sera was
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Scheme 2. Synthesis of Cocaine Hapten GNT
examined for antibody catalysis.11 Here, although cocaine
hydrolysis was observed, no kinetic data was reported and no
correlation was seen between titers and catalysis. Preparations
of efficient catalytic antibodies that readily catalyze cocaine’s
degradation, to a great extent, rely on the design of the
transition state moiety embedded within the hapten used for
immunization.12 Yet, previous studies have also shown that the
linker, including length, site of attachment, and heteroatom
choice can all be very critical for producing efficient catalytic
antibodies.10b,13 On the basis of the chemical scaffold of the
only hapten that has reached clinical trials, succinyl norcocaine
(SNC),6d,e we sought a new transition-state hapten, termed
GNT (10, Scheme 2) in which a succinyl linker is attached at
the tropane nitrogen. In contemplating this hapten design, we
were aware of the lability of the C-2 methyl ester mediated by
the adjacent tropane nitrogen, which is predominately
protonated at physiologic pH.14 We anticipated this lability
would be nullified in GNT through the bridge-head amide of
the installed linker. Furthermore, the distal linker site in
relationship to the anticipated benzoyl ester dominant epitope
might better expose the unique transition-state epitope for
added immune recognition.
Cocaine vaccines have been examined from many different
perspectives for treating addiction and overdose, but to our
knowledge, there has been no investigation examining active
vaccination with a transition-state analogue hapten in a
behavioral model of cocaine’s psychostimulant actions. In this
study, we prepared two distinct haptens, GNE and GNT, and
examined their active vaccines in an animal behavioral model.
We compared their corresponding immunological properties
and efficacy as judged from reduction of the psychomotor
activation produced by acute cocaine administration. We
demonstrated that both GNE−KLH and GNT−KLH evoked
potent immune response with rapid generation of robust
polyclonal antibody titers. The sequestering antibodies from
GNE vaccination were shown to possess high cocaine binding
specificity and confer significant protection against cocaine-
induced spontaneous locomotion. Intriguingly, although the
GNT active vaccine suppressed locomotor behavioral responses
to cocaine initially, it slowly lost its protection with repeated
cocaine injections. To investigate the loss of efficacy in the
locomotor behavioral model, GNT polyclonal antibodies were
examined pre- and post-cocaine challenge through kinetic
analysis, which indicated that the catalytic antibody component
was modified in vivo by cocaine.
RESULTS
■
Synthesis of Cocaine Haptens and Hapten−Protein
Immunoconjugates. The synthesis of cocaine hapten GNE is
illustrated in Scheme 1. The synthesis commenced with
(−)-ecgonine, which was coupled with amine 4 in the presence
of EDC and 4-methylmorpholine in DCM to afford amide 1 in
45% yield. Compound 4 was prepared by the protection of the
commercially available Boc-6-aminohexanoic acid with benzyl
alcohol followed by the removal of the Boc-protecting group.
Benzoylation of the hydroxyl group of compound 1 was
achieved in 40% yield by the use of benzoyl chloride, Et3N, and
DMAP in DCM. The benzoylated compound 2 was subjected
to hydrogenolysis using 1 atm of H2 and 10% Pd−C in MeOH
to generate the desired compound 3 (GNE). The new cocaine
transition-state analogue GNT was designed and synthesized as
shown in Scheme 2. The synthesis commenced with ecgonine
methyl ester 5, which was prepared from (−)-cocaine
hydrochloride in two steps.12,15 Ecgonine methyl ester 5 was
treated with lithium diisopropylamide in THF, followed by the
addition of compound 612 at 0 °C to provide the required
phosphonate diester 7 in 60% yield. Demethylation of 7 was
achieved by forming a carbamate intermediate before treatment
with zinc dust, providing norcocaine derivative 8 in 41% yield
over two steps. Amide 9 was prepared by N-acylation of amine
8 with succinic anhydride in the presence of Et3N in DMF.
Finally, the desired phenylphosphonate 10 (GNT) was
obtained in 85% yield by catalytic hydrogenolysis.
Following by the synthesis of cocaine haptens GNE and
GNT, the immunoconjugates GNE−KLH and GNT−KLH
were prepared for vaccination. Cocaine haptens were first
activated by the use of EDC/sulfo-NHS in DMF, followed by
the addition of carrier protein KLH in PBS solution. The
resulting mixtures were allowed to stand at 4 °C overnight and
then subjected to dialysis to remove excess reagents. Prior to
administration, each protein conjugate as well as the control
vehicle, KLH, were formulated with SAS (Sigma Adjuvant
System), a stable oil-in-water emulsion derived from bacterial
and mycobacterial cell walls. In addition, both GNE and GNT
were also coupled to bovine serum albumin (BSA) for ELISA
microtiter plate coating as well as to monitor the coupling
efficiency using MALDI-TOF mass spectrometry. The
molecular haptenation ratios (number of moles of hapten per
mole of carrier protein BSA) were found to be about 20 for
both GNE and GNT.
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Titers and Affinity of Cocaine-Specific Antibodies
Generated by Active Immunization in Mice. The efficacy
of GNE−KLH and GNT−KLH immunoconjugates was
assessed by vaccination into groups of eight Swiss Webster
mice. We have chosen Swiss Webster mice because they have
been reported to show locomotor habituation in activity
monitors and significant cocaine-induced hyperactivity once
habituated.16 Test groups included KLH negative control,
GNE−KLH, and GNT−KLH. Vaccine was administered on
days 0, 21, and 42, with bleeds taken one week post-
vaccination. This vaccination schedule has previously been
reported by our laboratory.7,8 Titer levels were assessed by
ELISA. Titer is defined as the inverse of the serum dilution
needed to generate a half-maximal response when binding to
the antigen. All bleeds from the KLH control group showed no
significant titer either to GNE or GNT. For GNE−KLH group
and GNT−KLH group, a significant titer response to the
corresponding hapten was observed after the third injection (t
= 42 days) compared to that after the second injection (t = 21
days). As shown in Table 1, immunization of mice with either
GNE−KLH or GNT−KLH evoked a high-titer response to
cocaine with both titer levels in the range of ∼1:30000.
Psychomotor Activation Effects of Cocaine in Mice
Immunized with GNT and GNE Vaccines. Three groups of
vaccinated mice (KLH negative control, GNE−KLH, and
GNT−KLH) were assessed for the psychomotor stimulant
effects of cocaine seven days after the second immunization
boost. Following three days of habituation and intraperitoneal
injections of saline, the mice were challenged with injections of
15 mg/kg cocaine and evaluated for drug-induced locomotor
hyperactivity each day for an additional three days (Supporting
Information Figures S1−S4). In response to cocaine challenge,
mice vaccinated with KLH showed significant increases in the
number of beam breaks (Figure 3A, session effect F (3,6) =
5.344, p < 0.01) and crossovers (Figure 3B, session effect F
(3,6) = 4.522, p < 0.05) compared to saline injection. Beam
breaks indicate when either one of the two photocell beams
across the long access to the cage are broken; crossovers reflect
beam 1 being broken after beam 2 and vice versa, showing
actual movement across the length of the cage. The anticocaine
antibodies elicited by GNE−KLH vaccination completely
prevented the cocaine-induced locomotor hyperactivity in
response for the duration of the testing period. In contrast,
the antibodies generated by the GNT−KLH vaccinated mice
had significant protection against cocaine during the first and
second days. However, the antibodies lost some of their
protecting effects against cocaine administration in the third
day of cocaine challenge, where the number of beam breaks
(Figure 3A, session effect F (3,7) = 4.174, p < 0.01), but not
crossovers, was significantly increased compared to saline
injection.
Kinetic Analysis of GNT Polyclonal Antibody Sera. A
group of 20 Swiss Webster mice was vaccinated on days 0, 21,
and 42 with a suspension of GNT−KLH in PBS in formulation
with SAS adjuvant. The first set of 10 mice were sacrificed on
day 50, and the sera were collected and purified by protein G
affinity chromatography to obtain the polyclonal antibody
before cocaine challenge (pretreated GNT pAbs). Following
two days of habituation, the second set of 10 mice were
challenged with injections of 15 mg/kg cocaine on days 50, 51,
and 52 and sacrificed. The corresponding sera were collected at
the time of sacrifice and further purified by protein G affinity
chromatography to obtain polyclonal antibodies, which had
been subjected to cocaine challenges (post-treated GNT pAbs).
The pretreated GNT pAbs and post-treated GNT pAbs were
tested for their ability to hydrolyze cocaine at a concentration
of 1 μM of pAbs and varying concentrations of cocaine. Their
catalytic activity was examined through LC/MS detection of
benzoic acid. Michaelis−Menten saturation curves were
observed (Supporting Information Figure S5). Comparison of
the Michaelis−Menten kinetics of the pretreated and post-
treated GNT pAbs (Table 2) shows that treatment of cocaine
in vivo resulted in a lower catalytic rate and higher apparent Km.
The catalytic rate of the post-treated pAbs was 40% below that
of pretreated pAbs, while apparent Km increased 2-fold. The
catalytic efficiency kcat/Km of pretreated GNT pAbs was about
4.18 times higher than that of the post-treated GNT pAbs
(84.69 M−1 s−1 versus 16.36 M−1 s−1).
Table 1. Average ELISA Titer and Average Relative Affinity
of Antisera from Immunized Mice against Cocaine As
a
Determined by Equilibrium Dialysis
immunoconjugate
titer
Kd (nM)
[Ab] (μg/mL)
GNE−KLH
GNT−KLH
31200
29600
6.36 1.56
85.12 17.34
b
b
ND
b
ND
a
All assays were performed in triplicate. ND, not detectable.
Another important parameter in determining the antibody
efficacy is the ability of the polyclonal antibody to bind cocaine.
Accordingly, cocaine affinity of the antibodies was determined
by a soluble radioimmunoassay (RIA) using 3H-cocaine (Table
1).17 Antibodies induced by GNE-KLH in the second bleed
provided a Kd of 6.36 1.56 nM (Figure 2). IgG was assumed
Figure 2. Affinity of GNE polyclonal antibody sera.
to have a molecular weight of 150 kDa and two cocaine-binding
sites per molecule. This cocaine specific IgG concentration in
serum corresponds to a cocaine binding capacity in serum of
GNT Vaccine’s Reduction in Catalysis upon Cocaine
Exposure. 3H-cocaine was incubated with pools of GNT
antibodies that had been previously untreated or treated with
cocaine under physiological conditions for 16 and 64 h. After
the removal of free H-cocaine by dialysis, a portion of each
sample was separated by SDS/PAGE. The band at 150 kDa,
corresponding to IgG, was excised and the protein was
85.12
17.34 μg/mL. Strikingly, the antibodies elicited by
GNT−KLH showed no binding affinity for cocaine. We
suspected that this could be due to the catalytic antibodies
elicited from the GNT vaccine and the time course required for
the RIA assay. Indeed, LC/MS analysis of the solution
presented only cocaine metabolite ecgonine methyl ester (see
LC/MS data, Supporting Information).
3
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Figure 3. Catalytic vaccine and noncatalytic vaccines prevent the psychomotor stimulating effects of repeated cocaine treatment in mice. * p < 0.05
and ** p < 0.01.
affinity, and specificity. Most elaborations within cocaine
vaccine preparations can be seen in hapten design. Alternation
to scaffolding of the cocaine hapten and the positioning of the
linker, including linker length and its chemical composition,
have made significant impact on vaccine efficacy. To expand
upon the current arsenal of haptens for cocaine vaccine
development, we were mindful of the spontaneous hydrolysis of
the methyl and benzoyl esters embedded within this tropane
alkaloid framework.19 Thus, our second-generation GND
hapten obviates hydrolytic lability issues, however, its synthetic
sequence accessibility diminishes the robust efficacy seen within
animal behavioral studies. To simplify the synthesis of the
former reported hapten GND (Figure 1),8 we designed the new
hapten GNE in which we replaced the C2-ester with a C2-
amide while maintaining the C3-benzoyl ester (Scheme 1). In
collaboration with the Crystal laboratory, we have demon-
strated that GNE-based cocaine vaccine dAd5GNE, in which
GNE was attached to the disrupted serotype 5 adenovirus (Ad)
gene transfer vector, evoked persistent high-titer and high
affinity IgG anticocaine antibodies.9 To further probe the value
of our GNE hapten design, we simply linked GNE to the
immunogenic carrier protein KLH using SAS as the adjuvant.
Our immunological assays presented evidence that the GNE−
KLH vaccine was able to evoke potent-titer and highly specific
cocaine antibodies (Figure 2, Table 1). In addition, GNE’s
performance as an immunotherapeutic vaccine was examined in
a mouse locomotor test and the antibodies elicited were found
capable of completely blunting cocaine-induced locomotor
hyperactivity over the entire testing period (Figure 3). These
results demonstrate that replacement of the C2-ester with the
corresponding C2-amide affords a stable hapten with
sustainable and robust immunogenicity, thus hapten GNE
represents a promising hapten candidate for anticocaine
vaccination development.
Table 2. Kinetic Data for GNT pAbs (Pretreated pAbs) and
Cocaine-Treated GNT pAbs (Post-treated pAbs)
kcat
Km
(μM)
kcat/Km
a
b
pAbs
(min−1
)
(M−1 s−1
)
kcat/k′uncat
pretreated pAbs
0.3143
0.1989
61.85
84.69
16.36
7.67 × 104
4.85 × 104
post-treated pAbs
121.60
a
b
Reactions were carried out in PBS (pH 7.4) at 23 °C. k′uncat = 4.1 ×
10−6 min−1.18
extracted and assayed by liquid scintillation counting. As
presented in Figure 4, active counts, and thus cocaine
Figure 4. Radioactivity (counts/min) of GNT pAbs (pretreated pAbs)
and cocaine-treated GNT pAbs (post-treated pAbs) after incubation
3
with H-cocaine for 16 and 64 h, respectively.
incorporation, increased over time, wherein the radioactivity
of the pretreated GNT pAbs was more intense than that of the
post-treated GNT pAbs after either 16 or 64 h.
To examine the potential of an active catalytic antibody
vaccine, we synthesized a new cocaine transition-state analogue,
GNT (Scheme 2). Although hapten GNT appeared equally
immunogenic as GNE using titer as the metric, the antibodies
elicited by GNT showed no appreciable affinity to cocaine as
measured by RIA (Table 1). This finding while seemingly
DISCUSSION
■
Significant effort has been devoted to the preparation of
anticocaine vaccines in the hopes of increasing antibody titer,
Figure 5. Proposed mechanism of antibody acylation by cocaine.
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unexpected is readily interpreted. Most likely, cocaine was
hydrolyzed during the time course required for the equilibrium
dialysis assay (22 h), and indeed LC/MS analysis of the
solution showed only cocaine metabolized products (LC/MS
data in Supporting Information). In the animal behavioral test,
catalytic antibodies produced by GNT−KLH provided
significant protection against cocaine’s induced hyperlocomotor
activity in the first two days of testing but slowly lost their
protection with repeated cocaine challenges (Figure 3). Landry
et al. reported how cocaine was able to covalently modify a
protein through an acylation reaction of the lysine ε-amino
groups (Figure 5).20 We posit that the loss of vaccine potency
upon repeated exposure to cocaine could be due to
modification of the antibody catalysts produced in response
to GNT. Using LC/MS analysis, we determined the initial rates
of hydrolysis in the presence of the pretreated and post-treated
GNT pAbs as a function of substrate concentration (Table 2).
Initial rates of antibody-based hydrolysis displayed saturation or
Michaelis−Menten kinetics with respect to cocaine concen-
tration. To rationalize the GNT vaccine’s loss of efficacy in our
locomotor behavioral model, we performed a kinetic study
comparing the catalytic activity of pretreated and post-treated
pAbs. Although, we did not observe a high enzymatic efficiency
for either of these two pAbs, it is evident that repeated cocaine
administration compromised the catalysis observed for the
GNT vaccine (Table 2). On the basis of radiolabeling data
(Figure 4), cocaine-protein labeling appears plausible for the
time dependent loss of catalytic activity. Interestingly, post-
treated cocaine sera antibodies were less affected versus the
pretreated cohort, which aligns with protein-cocaine mod-
ification in that available modification sites have already been
partially blocked. It is tempting to speculate that the catalytic
sites of the GNT vaccine are being modified because simple
sequestering antibodies evoked by GNE appeared not to be
duly affected as viewed by RIA analysis (Table 1) or locomotor
behavior (Figure 3). Future analysis of GNT sera where
catalysts are physically separated from simple binding antibod-
ies could shed light on this hypothesis.
EXPERIMENTAL SECTION
■
All reactions involving air- or moisture-sensitive reagents or
intermediates were performed under an argon atmosphere. Chemicals
and solvents were of reagent grade and were used without further
purification. The (−)-cocaine hydrochloride was supplied by the
National Institute on Drug Abuse (NIDA). Flash chromatography was
performed on silica gel 60 (230−400 mesh), and analytical TLC was
carried out on glass plates coated with a 0.25 mm layer of silica gel 60
F-254. HPLC separations were performed on a Vydac 218TP C18
reversed phase preparative (10−15 μm) HPLC column using a
gradient of acetonitrile and water. The LC/MS analysis was performed
using an Agilent G-1956D single quadrupole mass spectrometer
equipped with an 1100 series LC system from Agilent Technologies.
1H and 13C NMR spectra were obtained using a Bruker 500 or 600
MHz instrument. Chemical shifts were reported in parts per million
(ppm, δ) referenced to the residual 1H resonance of the solvent
(CDCl3, 7.26 ppm) or (CD3OD, 3.31 ppm). 13C spectra were
referenced to the residual 13C resonance of the solvent (CDCl3, 77.0
ppm) or (CD3OD, 49.0 ppm). Splitting patterns were designated as
follows: s, singlet; br, broad; d, doublet; dd, doublet of doublets; t,
triplet; q, quartet; m, multiplet. High resolution mass spectra were
obtained in the Scripps Center for Mass Spectrometry. Hapten protein
conjugates were analyzed using MALDI-TOF MS. The purity of all
synthetic compounds was higher than 95% as determined by HPLC
and/or LC/MS.
Benzyl 6-((1R,2R,3S,5S)-3-Hydroxy-8-methyl-8-azabicyclo-
[3.2.1]octane-2-carboxamido)hexanoate (1). To a solution of
44.3 mg (0.20 mmol) of (−)-ecgonine in 4 mL of DCM was added
22.0 μL (0.50 mmol) of N-methylmorpholine and 49.7 mg (0.26
mmol) of EDC, followed by 48.6 mg (0.22 mmol) of 4 in 1 mL of
DCM and 7.3 mg (0.06 mmol) of DMAP at 0 °C. The reaction
mixture was maintained at 0 °C and stirred for another 3 h. The
solvent was removed under diminished pressure, and the crude
products were purified on a VYDAC C18 reversed phase semi-
preparative (250 mm × 22 mm, 10−15 μm) HPLC column using
water and 0.1% TFA in CH3CN mobile phases. A linear gradient was
employed (90:10 H2O/0.1%TFA in CH3CN → 10:90 H2O/0.1%TFA
in CH3CN) over a period of 40 min at a flow rate of 10 mL/min.
Fractions containing the desired product were collected, frozen, and
1
lyophilized to give 1 as TFA salt: yield 37.4 mg (45%). H NMR
(CDCl3) δ 1.32−1.35 (m, 2H), 1.52 (quin, 2H, J = 6.0 Hz), 1.62
(quin, 2H, J = 6.0 Hz), 2.06−2.17 (m, 4H), 2.27−2.35 (m, 4H), 2.74
(s, 3H), 3.15−3.28 (m, 3H), 3.81−3.87 (m, 2H), 4.32 (quin, 1H, J =
6.0 Hz), 5.08 (s, 2H), 7.25−7.36 (m, 5H) and 8.40−8.45 (br, 2H). 13C
NMR (CDCl3) δ 24.42, 25.15, 26.93, 29.13, 34.86, 36.44, 39.02, 40.43,
48.39, 61.11, 63.93, 67.00, 128.87, 128.89, 129.01, 129.39, 136.87,
173.75, 174.56 and 174.58; mass spectrum (ESI), m/z 389.2446 (M +
H)+ (C22H33N2O4 requires m/z 389.2435).
(1R,2R,3S,5S)-2-((6-(Benzyloxy)-6-oxohexyl)carbamoyl)-8-
methyl-8-azabicyclo[3.2.1]octan-3-yl Benzoate (2). To a sol-
ution of 37.4 mg (0.096 mmol) of 1 in 2 mL of DCM was added 29.5
μL (0.21 mmol) of Et3N followed by 12.3 μL (0.11 mmol) of benzoyl
chloride and 1.2 mg (0.06 mmol) of DMAP at 0 °C. The reaction
mixture was slowly warmed to room temperature and stirred for
another 3 h. The solvent was removed under diminished pressure, and
the crude products were purified on a VYDAC C18 reversed phase
semipreparative (250 mm × 22 mm, 10−15 μm) HPLC column using
water and 0.1% TFA in CH3CN mobile phases. A linear gradient was
employed (90:10 H2O/0.1%TFA in CH3CN → 10:90 H2O/0.1%TFA
in CH3CN) over a period of 40 min at a flow rate of 10 mL/min.
Fractions containing the desired product were collected, frozen, and
CONCLUSIONS
■
In summary, we have synthesized a cocaine-like hapten GNE
and a cocaine transition-state analogue GNT as well as their
KLH immunoconjugates. Both GNE−KLH and GNT−KLH
cocaine vaccines were found to elicit high-titer antibodies.
While the GNE vaccine granted complete protection from the
cocaine stimulatory effects in a locomotor model, GNT−KLH
vaccine lost protection after repeated cocaine challenges in vivo.
We hypothesize that the reduction of catalytic activity of GNT
pAbs could be due to the acylation of the active site upon
repeated cocaine challenges. We note that the catalytic
efficiency of catalytic antibodies generated by hapten GNT is
modest as judged by enzyme standards, yet this response was
competent enough to provide protection in a mouse locomotor
model, albeit it does wane upon repeated cocaine challenges.
With these findings, it is warranted to examine other catalytic
antibody hapten design principles to see if this loss of catalysis
can be tempered or if it is a general liability of an active catalytic
antibody vaccine. Finally, inspired by a recent report on the
combination treatments of cocaine hydrolase and anticocaine
antibodies,21 we speculate that a combination of sequestering
and catalytic antibody active vaccines may also provide a potent
dual vaccine approach for examining cocaine-induced behavior
in animal models.
1
lyophilized to give 2 as TFA salt: yield 19.0 mg (40%). H NMR
(CD3OD) δ 1.15−1.25 (m, 2H), 1.27−1.36 (m, 3H), 1.46−1.50 (m,
2H), 2.02−2.09 (m, 2H), 2.21−2.23 (m, 3H), 2.34−2.42 (m, 3H),
2.67 (s, 3H), 3.06−3.17 (m, 2H), 3.21−3.25 (m, 1H), 3.81−3.91 (m,
2H), 5.09 (s, 2H), 5.46 (quin, 1H, J = 6.0 Hz), 7.33−7.35 (m, 5H),
7.39−7.44 (m, 2H) and 7.93−7.95 (m, 3H). 13C NMR (CD3OD) δ
24.09, 24.98, 26.82, 29.44, 34.21, 38.67, 39.60, 65.38, 66.57, 95.73,
100.43, 128.21, 128.66, 128.99, 129.09, 129.74, 130.09, 130.27, 130.99,
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134.15, 137.17, 137.67, 166.15, 172.50 and 174.35; mass spectrum
(ESI), m/z 493.2697 (M + H)+ (C29H37N2O5 requires m/z 493.2697).
6-((1R,2R,3S,5S)-3-(Benzoyloxy)-8-methyl-8-azabicyclo-
[3.2.1]octane-2-carboxamido)hexanoic Acid (3). A mixture of
23.0 mg (0.05 mmol) of 2 and 15 mg of 10% Pd/C in 1 mL of ethanol
was stirred overnight under a H2 atm at room temperature. The
catalyst was removed by filtration, and the crude products were
purified on a VYDAC C18 reversed phase semipreparative (250 mm ×
22 mm, 10−15 μm) HPLC column using water and 0.1% TFA in
CH3CN mobile phases. A linear gradient was employed (90:10 H2O/
0.1%TFA in CH3CN → 10:90 H2O/0.1%TFA in CH3CN) over a
period of 40 min at a flow rate of 10 mL/min. Fractions containing the
desired product were collected, frozen, and lyophilized to give 3 as
132.12, 132.21, 132.29, 132.77, 132.84, 136.65, 136.70, 170.71 and
170.91; mass spectrum (MALDI-TOF), m/z 430.5 (M + H)+
(theoretical 430.2).
(1R,2R,3S,5S)-Methyl 3-(((benzyloxy)(phenyl)phosphoryl)-
oxy)-8-azabicyclo[3.2.1]octane-2-carboxylate (8). To a solution
of 46.6 mg (0.11 mmol) of amide 7 in 1 mL of benzene was added
134.4 μL (0.98 mmol) of Troc-Cl and 4.5 mg (0.033 mmol) of
K2CO3. After being refluxed at 80 °C overnight, the reaction mixture
was quenched with saturated NH4Cl aqueous solution and then
extracted with EtOAc. The combined organic layer was washed with
brine, dried (MgSO4), and concentrated under diminished pressure.
The filtrate was concentrated under diminished pressure. The
remaining residue was placed under high vacuum for 2 h, followed
by the addition of 2 mL of DMF, 11.1 mg (0.17 mmol) of Zn dust,
and 2.1 μL (0.056 mmol) of formic acid. The resulted mixture was
stirred at room temperature overnight. The reaction mixture was
quenched with saturated NH4Cl aqueous solution and then extracted
with EtOAc. The combined organic layer was washed with brine, dried
(MgSO4), and concentrated under diminished pressure to give the
product 8, which was used for the next step promptly without any
purification or characterization.
TFA salt: yield 11.2 mg (60%); [α]2D5 −21.08° (c 0.65, MeOH). H
1
NMR (CD3OD) δ 1.12−1.21 (m, 2H), 1.27−1.36 (m, 3H), 1.37−1.46
(m, 2H), 2.12 (t, 2H, J = 6.0 Hz), 2.15−2.24 (m, 2H), 2.33−2.37 (m,
1H), 2.33−2.37 (m, 1H), 2.47−2.52 (m, 2H), 2.58 (td, 1H, J = 12.0,
6.0 Hz), 2.84 (s, 3H), 3.04−3.10 (m, 1H), 3.21−3.26 (m, 2H), 4.00−
4.01 (m, 1H), 4.15 (d, 1H, J = 6.0 Hz), 5.52−5.56 (m, 1H), 7.49 (t,
2H, J = 6.0 Hz), 7.63 (t, 1H, J = 6.0 Hz), 7.98 (d, 2H, J = 6.0 Hz) and
8.43 (br, 1H). 13C NMR (CD3OD) δ 23.71, 24.26, 24.94, 26.80, 29.37,
33.68, 34.02, 38.14, 39.91, 46.64, 63.70, 65.00, 65.87, 129.20, 130.02,
130.12, 134.36, 166.02, 172.39 and 176.73; mass spectrum (ESI), m/z
403.2222 (M + H)+ (C22H31N2O5 requires m/z 403.2227).
4-((1R,2R,3S,5S)-3-(((Benzyloxy)(phenyl)phosphoryl)oxy)-2-
(methoxycarbonyl)-8-azabicyclo[3.2.1]octan-8-yl)-4-oxobuta-
noic Acid (9). To a solution of 50.9 mg (0.12 mmol) of amine 8 in 2
mL of DMF was added 34.2 μL (0.25 mmol) of Et3N and 24.5 mg
(0.25 mmol) of succinic anhydride. The reaction mixture was then
heated to 45 °C and stirred for 16 h. The reaction mixture diluted with
5 mL of H2O and extracted with three 10 mL portions of EtOAc. The
combined organic layer was washed with brine, dried (MgSO4), and
concentrated under diminished pressure. The crude products were
purified on a VYDAC C18 reversed phase semipreparative (250 mm ×
22 mm, 10−15 μm) HPLC column using water and 0.1% TFA in
CH3CN mobile phases. A linear gradient was employed (90:10 H2O/
0.1%TFA in CH3CN → 10:90 H2O/0.1%TFA in CH3CN) over a
period of 40 min at a flow rate of 10 mL/min. Fractions containing the
desired product were collected, frozen, and lyophilized to give 9 as
Benzyl 6-Aminohexanoate (4). To a solution of 0.5 g (2.16
mmol) of Boc-6-aminohexanoic acid in 15 mL of DCM was added 497
mg (2.29 mmol) of EDC followed by 269 μL (2.59 mmol) of benzyl
alcohol and 26.4 mg (0.22 mmol) of DMAP at 0 °C. The reaction
mixture was slowly warmed to room temperature and stirred for
another 16 h. The reaction mixture was quenched by the addition of
10 mL of satd aq NH4Cl. The mixture was extracted with EtOAc. The
combined organic layer was washed with brine, dried (MgSO4), and
concentrated under diminished pressure. The residue was purified by
flash chromatography on a silica gel column (25 cm × 3.2 cm). Elution
with 10:1 hexanes/ethyl acetate gave the product as a yellow oil: yield
1
0.64 g (92%); silica gel TLC Rf 0.25 (8:2 hexanes/ethyl acetate). H
1
TFA salt: yield 39.8 mg (63%). H NMR (CD3OD) δ 1.55−1.74 (m,
NMR (CDCl3) δ 1.45−1.49 (m, 2H), 1.58 (s, 9H), 1.60−1.64 (m,
1H), 1.76−1.84 (m, 3H), 2.50 (t, 2H, J = 8.0 Hz), 3.21−3.25 (m, 2H),
4.63 (br, 1H), 5.25 (s, 2H) and 7.44−7.53 (m, 5H). To 0.64 g (1.99
mmol) of the obtained benzylated product in 10 mL of DCM at 0 °C
was added 5 mL of TFA. The reaction was stirred at 0 °C for 2 h
before the solvent was removed under diminished pressure to give 4 as
light-yellow oil: yield 408 mg (85% over two steps). 1H NMR
(CDCl3) δ 1.49−1.52 (m, 2H), 1.75−1.82 (m, 4H), 2.49 (t, 2H, J =
7.2 Hz), 3.06−3.11 (m, 2H), 5.24 (s, 2H), 7.41−7.90 (m, 5H) and
7.92 (br, 2H). 13C NMR (CDCl3) δ 24.34, 25.88, 27.28, 34.08, 40.15,
66.79, 128.46, 128.66, 128.94, 136.12 and 174.15; mass spectrum
(ESI), m/z 222.1492 (M + H)+ (C13H19NO2 requires m/z 222.1488).
(1R,2R,3S,5S)-Methyl 3-(((benzyloxy)(phenyl)phosphoryl)-
oxy)-8-methyl-8-azabicyclo[3.2.1]octane-2-carboxylate (7). To
a solution of 39.0 mg (0.20 mmol) of 5 in 1 mL of THF was added
120 μL of LDA (0.22 mmol, 1.8 M in cyclohexane) of LDA followed
by 52.2 mg (0.23 mmol) of 6 at 0 °C. The reaction mixture was slowly
warmed to room temperature and stirred for another 16 h. The
reaction mixture was concentrated under diminished pressure and
purified by flash chromatography on a silica gel column (25 cm × 3.2
cm). Elution with 40:1 chloroform/MeOH gave the product 7 as a
yellow oil: yield 51.5 mg (60%); silica gel TLC Rf 0.5 (9:1 chloroform/
2H), 1.77−2.05 (m, 2H), 2.11−2.38 (m, 1H), 2.45−2.75 (m, 4H),
2.95−3.12 (m, 1H), 3.49 (s, 1H), 3.53 (s, 0.5H), 3.57 (2, 1H), 3.64 (s,
0.5H), 4.41−4.45 (m, 0.6H), 4.55−4.60 (m, 0.4H), 4.65−4.70 (m,
0.4H), 4.86−4.86 (m, 2H), 4.97−5.13 (m, 2.6H), 7.32−7.37 (m, 5H),
7.49−7.52 (m, 2H), 7.60−7.63 (m, 1H), and 7.68−7.75 (m, 2H). The
product was shown to exist as a mixture of amide rotamers of benzyl
ester diastereomers, and the ratio of two rotamers was estimated to be
approximately 2:3. 13C NMR (CD3OD) δ 26.52, 26.94, 26.95, 28.38,
28.53, 28.60, 28.62, 28.81, 51.47, 51.51, 51.75, 51.84, 51.88, 51.91,
53.86, 53.88, 54.27, 54.32, 56.32, 68.74, 68.76, 68.87, 70.26, 70.30,
70.33, 70.37, 70.30, 128.66, 128.70, 129.20, 129.29, 129.39, 131.96,
132.03, 132.04, 132.07, 132.12, 133.76, 136.86, 169.71, 170.79, 170.89,
170.94, 171.01, 175.65 and 175.70; mass spectrum (ESI), m/z
516.1773 (M + H)+ (C26H31NO8P requires 516.1782).
4-((1R,2R,3S,5S)-3-((Hydroxy(phenyl)phosphoryl)oxy)-2-
(methoxycarbonyl)-8-azabicyclo[3.2.1]octan-8-yl)-4-oxobuta-
noic Acid (10). A mixture of 11.2 mg (0.02 mmol) of 9 and 5 mg of
10% Pd/C in 1 mL of ethanol was stirred overnight under a H2 atm at
room temperature. The catalyst was removed by filtration, and the
crude products were purified on a VYDAC C18 reversed phase
semipreparative (250 mm × 22 mm, 10−15 μm) HPLC column using
water and 0.1% TFA in CH3CN mobile phases. A linear gradient was
employed (90:10 H2O/0.1%TFA in CH3CN → 10:90 H2O/0.1%TFA
in CH3CN) over a period of 40 min at a flow rate of 10 mL/min.
Fractions containing the desired product were collected, frozen, and
lyophilized to give 10 as TFA salt: yield 7.8 mg (85%); [α]2D5 −12.78°
1
MeOH). H NMR (CD3OD) δ 1.48−1.54 (m, 2H), 1.65−1.69 (m,
0.4H), 1.82−1.86 (m, 0.6H), 1.98−2.05 (m, 0.6H), 2.15 (s, 3H), 2.36
(td, J = 12, 6 Hz, 0.4H), 2.49 (td, J = 12, 6 Hz, 0.6H), 2.77−2.79 (t, J =
6 Hz, 0.6H), 2.95−2.97 (t, J = 6 Hz, 0.4H), 3.14−3.16 (m, 0.4H),
2.96−3.06 (m, 1H), 3.19−3.21 (m, 0.6H), 3.38−3.40 (m, 0.6H),
3.44−3.64 (m, 0.4H), 3.58 (s, 1.8H), 3.67 (s, 1.2H), 4.61−4.69 (m,
1H), 4.92−5.10 (m, 2H), 7.28−7.35 (m, 5H), 7.39−7.45 (m, 2H),
7.51−7.54 (m, 1H) and 7.74−7.84 (m, 2H). The product was shown
to exist as a mixture of benzyl ester diastereomers. 13C NMR
(CD3OD) δ 25.45, 25.74, 30.12, 37.84, 41.41, 51.71, 51.79, 50.02,
52.05, 52.28, 61.87, 61.97, 65.17, 65.17, 65.21, 67.78, 67.82, 70.00,
70.04, 128.21, 128.65, 128.69, 128.73, 128.81, 128.85, 128.89, 132.04,
1
(c 1.15, MeOH). H NMR (CD3OD) δ 1.66−2.34 (m, 6H), 2.45−
2.76 (m, 4H), 2.96−3.06 (m, 1H), 3.34 (s, 1.2H), 3.56 (s, 1.8H),
3.63−3.65 (m, 1H), 4.44−4.46 (m, 0.6H), 4.57−4.59 (m, 0.4H),
4.67−4.69 (m, 0.4H), 4.80−4.81 (m, 1.6H), 7.47−7.50 (m, 2H),
7.56−7.59 (m, 1H) and 7.72−7.76 (m, 2H). The product was shown
to exist as a mixture of amide rotamers of benzyl ester diastereomers,
and the ratio of two rotamers was estimated to be approximately 2:3.
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13C NMR (CD3OD) δ 26.59, 27.03, 28.46, 28.54, 28.61, 28.86, 29.32,
29.62, 35.61, 35.64, 36.59, 36.61, 51.20, 51.22, 51.40, 51.58, 51.68,
51.75, 53.87, 53.89, 54.40, 56.32, 68.93, 68.97, 128.97, 129.07, 131.71,
131.78, 132.80, 132.82, 169.69, 169.75, 171.06, 171.16, 175.66 and
175.72; mass spectrum (ESI), m/z 426.1310 (M + H)+ (C19H25NO8P
requires 426.1312).
Hapten−Protein Immunoconjugates. Haptens GNT and GNE
were activated, respectively, at room temperature for 6 h using
standard EDC/sulfo-NHS (2 equiv each) coupling procedure in DMF.
After DMF removal under reduced pressure, to the residue was slowly
added the corresponding amount of KLH protein (1 mg of hapten
versus 1 mg of protein) in 1 mL of 100 mM PBS, pH 7.2. The
resultant solution was allowed to stand for 12 h at 4 °C. Coupling
efficiencies were monitored using MALDI-TOF MS, save for KLH,
which cannot be directly analyzed.
Enzyme-Linked Immunosorbent Assay (ELISA). Production of
cocaine-specific IgG was monitored by ELISA using a GNE−BSA or
GNT−BSA conjugate as the coating antigen. Titers were calculated
from the plot of absorbance versus log dilution, as the dilution
corresponding to an absorbance reading 50% of the maximal value.
BSA conjugate was added 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 serum was
serially diluted in a 1% BSA solution across the plate and allowed to
incubate for 1−2 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 2-step kit (Pierce; Rockford, IL).
50:50 mixture of 5 mM ammonium acetate buffer (pH 4.5) and
methanol at the flow rate of 0.2 mL/min. Mass calibration curves of
BzOH and BzOH-d5 were obtained at six different concentration ratios
with 10 μM of BzOH-d5. The kinetic analysis was conducted at
ambient temperature in PBS (pH 7.4) with 1.0 μM polyclonal
antibody and cocaine at four concentrations from 5 to 200 μM. The
total volume of all reactions was 100 μL. Experiments were performed
by adding the appropriate volume of a 1.0 mM cocaine solution to a
solution of polyclonal antibody in PBS. The background assays are
conducted at ambient temperature in PBS with the same
concentration of cocaine. Then 10 μL of sample was removed and
mixed with 10 μL of 20 μM BzOH-d5 for analysis every 30 min. The
concentration of benzoic acid was determined by interpolation of peak
height and area relative to standard curves. Initial rates were calculated
by linear regression analysis of plots of [benzoic acid] versus time. The
kinetic parameters kcat and Km were calculated by fitting plots of initial
rate versus cocaine concentration to the Michaelis−Menten equation
using Prism Version 5.0b software (GraphPad Software, USA).
Scintillation Method. 3H-cocaine (∼0.3 mM, 20−50 Ci/mmol, 2
μL) was incubated with GNT antibodies treated or untreated with
cocaine in 200 μL of 10 mM PBS, pH 7.4, at 37 °C. After 16 and 64 h,
100 μL of the mixture was taken and subjected to dialysis against 1 L
of 10 mM PBS (2 exchanges). Then 10 μL of samples were separated
by SDS/PAGE. The band at 150 kDa was excised and placed in a clean
microcentrifuge tube. To the tube was added 1 mL of elution buffer
(50 mM Tris-HCl, 150 mM NaCl, and 0.1 mM EDTA; pH 7.5). The
gel pieces were crushed using a clean pestle and incubated in a rotary
shaker at 37 °C overnight and then centrifuged at 10000g for 10 min.
The supernatant was pipetted into a scintillation tube, mixed with 5
mL of scintillation cocktail, and assayed by liquid scintillation
counting.
Radioimmunoassay (Equilibrium Dialysis). Refined values of
antibody affinity and cocaine binding capacity were determined via a
soluble radioimmunoassay (RIA). A modified version of Muller’s
̈
method19 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 L-[benzoyl1−3,4-3H(N)]-cocaine tracer; specific activity = 26
Ci/mmol (PerkinElmer, Boston, MA). Mice serum was diluted in RIA
buffer (sterile filtered 2% BSA in 1× PBS, pH 7.4) to a concentration
ASSOCIATED CONTENT
* Supporting Information
■
S
Additional biological assays and compound characterization
spectra (1H NMR and 13C NMR). This material is available
free of charge via the Internet at http://pubs.acs.org.
3
that would bind 50% of ∼30000 decays/min of H-cocaine tracer. A
AUTHOR INFORMATION
Corresponding Author
■
75 μL aliquot of serum was combined with 75 μL of radiolabeled
tracer (∼30000 decays/min); 150 μL of unlabeled cocaine at varying
concentrations in RIA buffer (sterile filtered 1% BSA in 1× 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 75 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 assay.
*Phone: (858) 784-2515. Fax: (858) 784-2595. E-mail:
kdjanda@scripps.edu.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We acknowledge the Skaggs Institute for Chemical Biology and
the National Institute on Drug Abuse (DA08590 to K.D.J.) for
financial support.
Locomotor Testing. Sixteen identical hanging wire cages (26 cm
wide × 35 cm long × 20 cm high) were used to monitor locomotor
activity following daily intraperitoneal injections of saline (1 mg/mL)
or cocaine (15 mg/kg) in the testing context (n = 7−8 per group).
Each cage was equipped with two pairs of infrared emitter−detector
photocells that were positioned along the long axis 1 cm from the floor
and 8 cm from the front and back of the cage. Photocell interruptions
served as a measure of locomotor activity. Beam breaks and cage
crossovers were recorded by a PC in 10 min bins (Supporting
Information Figures S1−S4). Area under the curve (AUC) was
calculated using the 30 min preinjection as the threshold, then
determining the total peak area in the 60 min post-injection. AUCs
were compared for each vaccine group using a repeated-measure one-
way ANOVA, and a Dunnett’s test was used for post hoc comparisons.
LC/MS Analysis. The LC/MS analysis was performed using an
Agilent G-1956D single quadrupole mass spectrometer equipped with
an 1100 Series LC system from Agilent Technologies. Initial rates of
antibody-catalyzed degradation of cocaine were determined by
monitoring the formation of benzoic acid (BzOH) on a Waters
Symmetry C18 column (2.1 mm × 150 mm, 3.5 μm particle size) with
a guard column. Analytical separation was performed using an isocratic
ABBREVIATIONS USED
■
Abs, antibodies; BzOH, benzoic acid; DMAP, dimethylamine-
pyridine; EDC, 1-ethyl-3-(3-dimethylaminopropyl)-
carbodiimide; kcat, turnover number; Kd, dissociation constant;
KLH, keyhole limpet hemocyannin; pAbs, polyclonal antibod-
ies; SAS, Sigma Adjuvant System; SNC, succinyl norcocaine;
sulfo-NHS, N-hydroxysulfosuccinimide sodium salt; Troc-Cl,
2,2,2-trichloroethyl chloroformate
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