Reduced antinociception of opioids in rats and mice by vaccination with immunogens containing oxycodone and hydrocodone haptens

Article abstract of DOI:10.1021/jm3013745

Prescription opioids abuse and associated deaths are an emerging concern in the USA. Vaccination against prescription opioids may provide an alternative to pharmacotherapy. An oxycodone hapten containing a tetraglycine linker at the C6 position (6OXY(Gly)₄OH) conjugated to keyhole limpet hemocyanin (KLH) has shown early proof-of-efficacy in rodents as a candidate immunogen (6OXY(Gly)₄-KLH) for the treatment of oxycodone abuse. In this study, oxycodone-based and hydrocodone-based haptens were conjugated to KLH to generate immunogens that would recognize both oxycodone and hydrocodone. Vaccination with 6OXY(Gly)₄-KLH increased drug binding in serum, reduced drug distribution to brain, and blunted analgesia for both oxycodone and hydrocodone. An analogous C6-linked hydrocodone vaccine blocked hydrocodone effects but less so than 6OXY(Gly)₄-KLH. C8-Linked hydrocodone immunogens had only limited efficacy. Amide conjugation showed higher haptenation ratios and greater efficacy than thioether conjugation to maleimide activated KLH (mKLH). The 6OXY(Gly)₄-KLH vaccine may be used for treatment of prescription opioid abuse.

Full text of DOI:10.1021/jm3013745

Article
pubs.acs.org/jmc
Reduced Antinociception of Opioids in Rats and Mice by Vaccination
with Immunogens Containing Oxycodone and Hydrocodone
Haptens
Marco Pravetoni,*,†,‡,§ Morgan Le Naour,∥ Ashli M. Tucker,† Theresa M. Harmon,† Tara M. Hawley,†
Philip S. Portoghese,§,∥ and Paul R. Pentel†,‡,§
†Minneapolis Medical Research Foundation, 701 Park Avenue, Minneapolis, Minnesota 55404, United States
‡University of Minnesota, School of Medicine, Department of Medicine, 420 Delaware Street SE, MMC 194, Suite 14-110,
Phillips−Wangensteen Building, Minneapolis, Minnesota 55455, United States
§University of Minnesota, School of Medicine, Department of Pharmacology, 6-120 Jackson Hall, 321 Church Street SE, Minneapolis
Minnesota 55455, United States
∥University of Minnesota, College of Pharmacy, Department of Medicinal Chemistry, 8-101 Weaver Densford Hall, 308 Harvard
Street SE, Minneapolis, Minnesota 55455, United States
S
* Supporting Information
ABSTRACT: Prescription opioids abuse and associated deaths are an
emerging concern in the USA. Vaccination against prescription opioids may
provide an alternative to pharmacotherapy. An oxycodone hapten containing a
tetraglycine linker at the C6 position (6OXY(Gly)4OH) conjugated to
keyhole limpet hemocyanin (KLH) has shown early proof-of-efficacy in
rodents as a candidate immunogen (6OXY(Gly)4−KLH) for the treatment of
oxycodone abuse. In this study, oxycodone-based and hydrocodone-based
haptens were conjugated to KLH to generate immunogens that would
recognize both oxycodone and hydrocodone. Vaccination with 6OXY(Gly)4−KLH increased drug binding in serum, reduced
drug distribution to brain, and blunted analgesia for both oxycodone and hydrocodone. An analogous C6-linked hydrocodone
vaccine blocked hydrocodone effects but less so than 6OXY(Gly)4−KLH. C8-Linked hydrocodone immunogens had only
limited efficacy. Amide conjugation showed higher haptenation ratios and greater efficacy than thioether conjugation to
maleimide activated KLH (mKLH). The 6OXY(Gly)4−KLH vaccine may be used for treatment of prescription opioid abuse.
prescription opioids such as oxycodone, oxymorphone, or
hydrocodone.5d,6
INTRODUCTION
■
Prescription opioid abuse is increasing and is now more
common than heroin abuse in the USA.1 Oxycodone,
oxymorphone, and hydrocodone are the most commonly
abused prescription opioids.1a,b,2 Treatment of prescription
opioid abuse is complex because addicts may switch or
transition between different prescription opioids to overcome
lack of availability.3 The prevalence of prescription opioid abuse
highlights the need for effective therapies.
Vaccines to treat addiction are a promising alternative to
current pharmacotherapies. Addiction vaccines act by stimulat-
ing production of drug-specific antibodies that bind and retain
the target drug in serum. Vaccination reduces the distribution
of drug to the brain and its ability to have central effects, thus
attenuating behavioral effects. Proof-of-principle of clinical
efficacy has been shown with nicotine and cocaine vaccines.4
Several groups have developed immunogens based upon the
morphine structure to block the effects of heroin and its active
metabolites 6-monoacetylmorphine and morphine.5 Heroin/
morphine vaccine efficacy has been established in animal
models but has not been evaluated in humans. Fewer studies
have focused on development of immunogens to target
Evaluation of a recently described oxycodone immunogen,
designated as 6OXY(Gly)4−KLH (6, Figure 1), showed that
modifying oxycodone at the C6 position and conjugating this
hapten to a carrier protein through a tetraglycine linker
provided an immunogen that generates a strong immune
response in mice and rats.5d,6b The tetraglycine linker was more
effective than a shorter succinic acid based linker.6b Vaccination
with 6 elicited antibodies that recognize oxycodone and its
active metabolite, oxymorphone, and reduced oxycodone
distribution to brain and blocked its analgesia.6b
In the current study, we adopted an analogous approach and
employed the C6 position for attachment of the tetraglycine
linker to generate a candidate hydrocodone immunogen,
designated as 6HYDROC(Gly)4−KLH (7) and the C8 position
to generate 8HYDROC(Gly)4−KLH (9). Derivatization at the
C8 position was thought to mask differences between
oxycodone and hydrocodone, thus providing an immunogen
Received: September 21, 2012
Published: December 18, 2012
© 2012 American Chemical Society
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Figure 1. Oxycodone, hydrocodone, and derivatized haptens.
Scheme 1. Synthesis of Haptens Derivatized at the C6 Position
Reactants. (a) O-(carboxymethyl) hydroxylamine hemichloride, pyridine, MeOH, 80 °C, 4 h; (b) (i) Gly4OtBu, DCC/HOBt, DMAP, anh. DMF, rt,
24 h, (ii) TFA/DCM, rt, overnight; (c) (i) S-tritylcysteamine, DCC/HOBt, DMAP, anhyd DMF, rt, 24 h, (ii) TFA, AcOH, DCM, rt, overnight.
Synthesis yields are reported as % of starting material for both haptens.
that would recognize both drugs. At both positions, the
thioether−maleimide conjugation method was compared to
amide linkage for coupling haptens [[[17-methyl-4,5α -epoxy-
14-hydroxy-3-methoxy-morphinan-6-[[((ylidene)-amino)-oxy]-
N-(2-mercaptoethyl)-acetamido]morphinan (3) and 17-meth-
yl-4,5α-epoxy-6-one-3-methoxy-8-[thio-N-(2-mercaptoethyl)-
acetamido]morphinan (5) to maleimide-activated KLH
(mKLH). The resulting immunogens, 6OXY(S)−mKLH (8)
and 8HYDROC(S)−mKLH (10) were compared to the
previously characterized 6. The effect of vaccination on the
distribution of oxycodone and hydrocodone to the brain was
determined in rats. Vaccine efficacy in blunting oxycodone and
hydrocodone behavioral effects was determined in a test of
thermal nociception in mice and rats. This study represents an
early screening of oxycodone- and hydrocodone-based
immunogens to identify candidate vaccines for further
development. Results show that the previously described
oxycodone immunogen 6 had efficacy against hydrocodone as
well as oxycodone and that the oxycodone-based haptens 1 and
3 were more effective than the hydrocodone-based haptens 17-
methyl-4,5α-epoxy-3-methoxy-6-[[((ylidene)-amino)-oxy]-N-
(glycylglycylglycylglycine)-acetamido]morphinan (2), 17-meth-
yl-4,5α-epoxy-6-one-3-methoxy-8-[thio-N-(glycylglycylglycyl-
glycine)-acetamido]morphinan (4), and 5.
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Scheme 2. Synthesis of Haptens Derivatized at the C8 Position
Reactants. (a) thioglycolic acid, THF, rt, 8 h, 87%; (b) (i) Gly4OtBu, DCC/HOBt, DMAP, anhyd DMF, rt, 24 h, (ii) TFA/DCM, rt, overnight; (c)
(i) S-tritylcysteamine, DCC/HOBt, DMAP, anhyd DMF, rt, 24 h, (ii) TFA, AcOH, DCM, rt, overnight. Synthesis yields are reported as % of starting
material for both haptens.
a
Table 1. Characterization of Serum Antibody Titers (× 103) from Vaccinated Rats
immunogen used for vaccination
ELISA coating immunogen
6OXY(Gly)4−KLH 6
8HYDROC(Gly)4−KLH 9
6OXY(S)−mKLH 8
82
23 31
18
20 21
8HYDROC(S)−mKLH10
6OXY(Gly)4−BSA
8HYDROC(Gly)4−BSA
6OXY(S)−mBSA
186 71
77 92
76 48
32 11
6
1.0 1.4
3
1
5
4
144 72
15 11
5
1.0 0.4
1.5
8HYDROC(S)−mBSA
2
3
1
a
Data are expressed as mean SD and represent serum antibody titers obtained by ELISA. Serum antibody titers from rats vaccinated with each
individual immunogen (6, 9, 8, and 10) were measured against each respective hapten bound to bovine serum albumin (BSA) or maleimide activated
BSA (mBSA) as coating immunogen in the ELISA assay. Sera were measured against other conjugates to determine cross-reactivity across haptens.
1
the H NMR of the mixture of α- and β-epimers was made
possible as H1 and H5 had a slightly different chemical shift
between the two isomers. Integration of the two peaks showed
a ratio of 75/25 in favor of the 8β-isomer as reported in similar
studies.10 The identification of the β-adduct as the major
isomer is consistent with the less hindered 8β position of the
morphinan scaffold. As the isomers could not be separated by
conventional chromatographic methods, the isomeric mixture
was used for conjugation to proteins.
Conjugation of Haptens to Carrier Proteins. Haptena-
tion ratios for the KLH conjugates could not be measured due
to the large size of KLH, so conditions were standardized using
bovine serum albumin (BSA) as a model carrier protein.
Haptenation ratios for 1 and 4 to BSA were comparable and
ranged from 17 to 21 moles of hapten per mole of carrier
protein as determined by MALDI-TOF. The hapten 2
exhibited molar haptenation ratios of 12−14 bound to BSA.
Activation of BSA resulted in 15−17 maleimide per mole of
BSA (mBSA), as determined by MALDI-TOF, similar to
activated proteins from commercial sources and in the same
range as previous reports by our and other groups.11
Conjugation ratios for hapten 3 bound to mBSA were ∼5−
10 and similar to previously described haptens conjugated
through maleimide,11a although higher haptenation ratios have
been reported with a morphine hapten.5e The haptenation ratio
was lower than ∼5 for hapten 5 conjugated to mBSA. To
minimize concerns of maleimide activation efficacy, hapten 3
conjugated to commercially available mKLH was compared to
immunogen 8 containing the in-house mKLH by evaluation of
the effect of vaccination on analgesia in mice but was not found
to be more effective.
RESULTS
■
Hapten Design and Synthesis. Critical parameters for the
generation of hapten−protein conjugate vaccines are the
derivatization site on the hapten to which the linker is attached,
linker length, the conjugation method used, and the
immunogenicity of the carrier protein. In prior studies, a 12
atom tetraglycine linker was found superior to a shorter linker
for an oxycodone hapten.6b For these reasons, this linker was
used to generate tetraglycine-containing hydrocodone haptens.
In general, the immunogenicity of hapten−protein conjugate
vaccines is improved with a higher ratio of haptenation, so this
study investigated if thiol-based maleimide conjugation would
improve haptenation ratios compared to the carbodiimide
method of conjugation. The synthesis of haptens 2 and 3 is
shown in Scheme 1.
To generate a hydrocodone vaccine, a close analogue of our
lead oxycodone hapten 1 was synthesized using hydrocodone
as starting material. Conditions similar to those previously
described6b led to intermediate 17-methyl-4,5α-epoxy-3-
methoxy-6-[[((ylidene)-amino)-oxy]acetic acid]morphinan
(11).7 Carboxylic acids 11 and 12 were then coupled to
linkers Gly4OtBu or S-tritylcysteamine8 using the dicyclohex-
ylcarbodiimide/hydroxybenzotriazole (DCC/HOBt) proce-
dure. Hydrolysis of the tertbutyl ester protecting group with
trifluoroacetic acid (TFA) in dichloromethylene (DCM)
produced hapten 2 in good yield (68% overall). The precursor
of the sulfhydryl-containing hapten 12 was detritylated in situ
by treatment with acetic acid (AcOH) and trifluoroacetic acid
in dichloromethane to give the free thiol hapten 3 in a similar
yield (77%). To investigate the role of the 6-keto group on the
opioid scaffold, these linkers were also attached at the C8
position. Thus, the Michael addition of thioglycolic acid to
codeinone9 (Scheme 2) allowed modification at the C8
position with retention of the 6-keto-group of the correspond-
ing 8-substituted dihydrocodeinone (4 and 5).
Biological Studies. Immunogenicity and Effect of
Vaccination on Oxycodone Distribution after Intravenous
Drug Dosing. The immunogenicity of 6, 8, 9, and 10 was first
tested in rats. Vaccination elicited higher oxycodone-specific
serum antibody titers generated by 6 compared to 9 (Table 1).
Oxycodone-specific serum antibody titer generated from the
Under these conditions, the reaction afforded a mixture of
8α- and 8β- isomers as determined by 1H NMR. Assignment of
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immunogens 8 and 10 were generally lower due to apparent
interference in the ELISA with use of the SH linkage. However,
serum antibodies directed against immunogen 8 recognized
hapten 1 and serum antibodies against immunogen 6
recognized hapten 3 (Table 1). These data suggest that
immunogens 6 and 8, despite their different linkers, generate
antibodies with similar specificity. Serum antibodies against
immunogens 6 and 8 showed higher affinity (lower IC50) for
oxycodone than antibodies directed against immunogen 9
(Supporting Information Table 1) and were the most efficient
in blocking oxycodone distribution (Figure 2).
blocking oxycodone antinociception and distribution to brain
(Figure 3) and also markedly increased oxycodone protein
binding in serum from 10
5 to100
3% (Supporting
Information Table 2). In these rats, no titers are available
because trunk blood was collected by euthanasia after drug
administration; the presence of oxycodone interfered with
measurement of serum antibody titers by ELISA (data not
shown).
In this experiment, immunogen 6 elicited serum antibody
titers of 142000
103000 (mean
SD), which were
significantly higher than those elicited by 7 (50000 48000,
p < 0.05). The immunogen 6 generated a stronger blockage of
hydrocodone analgesia than immunogen 7 (p < 0.05; Figure
3C). This finding is consistent with the greater effect of 6 on
hydrocodone distribution and protein binding (Figure 3B and
Supporting Information Table 2). Rats immunized with 6
showed a stronger reduction in brain oxycodone (74%, p < 0.05
compared to KLH control, Figure 3) than brain hydrocodone
(54%, p < 0.05 compared to KLH control, Figure 3).
Vaccination with 6 comparably attenuated both oxycodone
and hydrocodone analgesia in rats, but fentanyl analgesia was
preserved (data not shown) at 82 18 (mean SEM) MPE%.
Effect of Vaccination on Oxycodone Antinociception after
Subcutaneous Drug Dosing in Mice. To investigate generality
across species and using a different adjuvant and route of
administration (alum, sc), the effect of vaccination with the 6,
8, or 9 immunogens was compared using the hot plate assay in
BALB/c mice. Similar to rats, mice immunized with 6 and 8
showed a significant decrease in oxycodone behavioral effects
compared to KLH controls (p < 0.05, Figure 4). Conjugation
conditions for 8 were optimized through changes of hapten to
protein ratios and evaluated for their ability to block oxycodone
analgesia in mice (Supporting Information Figure 1).
Conjugation conditions used a range of hapten to carrier
protein ratios previously shown to be effective with a nicotine
hapten conjugated through maleimide chemistry.11b The effect
of vaccination on blockage of oxycodone analgesia suggested
optimal ratios between hapten 3 and mKLH, and the
commercially available mKLH was not found superior to the
in-house activated mKLH.
Figure 2. Effect of immunization on oxycodone distribution after
intravenous administration in rats. Vaccine effects on (A) serum and
(B) brain oxycodone after administration of 0.1 mg/kg oxycodone iv.
The % values above columns indicate decreases relative to the KLH
control group. Data are the mean SD; * p < 0.05 compared to KLH
control; # p < 0.05 compared to other vaccines.
DISCUSSION
■
The main finding of this study was that haptens based on
modifications of oxycodone at the C6 position produced more
effective immunogens than hydrocodone haptens modified at
the C6 or C8 positions for eliciting antibodies against both
oxycodone and hydrocodone and in blocking their distribution
to brain or their behavioral effects. Use of a (Gly)4OH linker
and amide linkage to KLH resulted in a greater efficacy
probably due to higher protein haptenation ratios than
thioether linkage to mKLH. These data suggest that the
previously characterized immunogen 6 may be useful in treating
both oxycodone and hydrocodone abuse.
In rats immunized with either 6 or 8 immunogens, the
retention of oxycodone in serum was increased compared to
the unconjugated KLH control group at 5 min after the
oxycodone dose (p < 0.05, Figure 2, upper panel). The
immunogen 6 was more effective than 8 (p < 0.05, see Figure 2,
upper panel). The immunogens 9 and 10 did not increase the
serum oxycodone concentration. Both immunogens 6 and 8
significantly reduced the distribution of oxycodone to brain (p
< 0.05, Figure 2, lower panel). The effect of vaccination on
oxycodone distribution was further confirmed by the
dramatically increased protein binding of oxycodone in serum
from 14 10 to 100 2% (Supporting Information Table 2);
the unbound drug concentrations were not different across
groups, but there was considerable variability in serum
concentrations and a trend toward lower unbound concen-
tration in the group vaccinated with 6.
Linker position and composition of hapten−protein
conjugate vaccines are important determinants of their
immunogenicity, but the number of haptens attached to each
molecule of protein also profoundly affects immunogenicity.
Haptenation ratios are not always measured when investigating
or comparing conjugate vaccines, so it can be difficult to know
whether an immunogen with greater immunogenicity is
intrinsically more stimulating to the immune system or simply
more efficiently linked to its carrier protein so that it provides a
higher density of hapten epitopes. KLH is a commonly used
Effect of Vaccination on Oxycodone and Hydrocodone
Distribution and Antinociception after Subcutaneous Drug
Dosing in Rats. Immunogen 6 was more efficient than 9 in
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Figure 3. Effect of immunization on oxycodone or hydrocodone distribution and antinociception after subcutaneous drug administration in rats.
Vaccine effects on (A) serum concentrations, (B) brain concentrations, and (C) antinociception after administration of 2.25 mg/kg oxycodone sc or
6.75 mg/kg hydrocodone sc. The % values above columns indicate decreases relative to the KLH control group. Distribution data are the mean
SD, while behavioral data are mean SEM; * p < 0.05 compared to KLH control; # p < 0.05 compared to other vaccines.
carrier protein for small molecule haptens because of its efficacy
and acceptability for human use. As KLH haptenation ratios
cannot be measured by mass spectrometry because of its large
size, conjugation reactions in the current study were first carried
out using BSA as a carrier protein because it allows such
measurement as well as optimization of conjugation conditions.
We have found previously that conjugation procedures that
increase the haptenation and immunogenicity of nicotine−BSA
conjugates also increase the immunogenicity of the correspond-
ing nicotine−KLH conjugates.11b Haptenation ratios to BSA
were therefore used as a surrogate for a direct measure of KLH
haptenation.
having a protein haptenation ratio equal to that of the more
effective immunogen 6. This is somewhat surprising because
the C8 substitution was expected to generate antibodies that
would recognize both oxycodone and hydrocodone by masking
their C14 position. Because this immunogen was derived from
an unresolvable mixture of 8α- and 8β- epimers, 25/75,
respectively, the density of the active isomer on KLH may have
been lower. However, it has been shown that specific antibodies
can be selectively generated for each isomer of a racemic hapten
mixture.12 The immunogen 9 was also less effective than both
C6-linked OXY haptens even though 8 had a substantially
lower haptenation ratio than 9. This suggests that the 6OXY
hapten structure is intrinsically more immunogenic than the
8HYDROC hapten structure and that the absence of
The 8-substituted immunogen 9 had only limited efficacy in
blocking oxycodone distribution and antinociception despite
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codone and effective in blocking its effects. The immunogen 7
blocked hydrocodone distribution and antinociception but,
surprisingly, less so than 6. The cross-reactivity of antibodies
generated by 6 suggests that the presence of substitution at the
14-position, the only structural difference between oxycodone
and hydrocodone, is not recognized by the antibodies
generated by this immunogen. The lesser immunogenicity of
the hydrocodone-based immunogen may have been due to the
possibility that hapten 2 is less efficient for haptenation of KLH
than hapten 1. However, this seems less likely given that the
haptenation ratio was only 10% lower.
The 12 atom (Gly)4OH linker was used because it has been
found to be suitable and superior to shorter linkers containing
0−2 glycines for an analogous morphine vaccine (not shown).
The linker used for the SH immunogens was shorter than
(Gly)4OH, but its effective length is similar given that, like the
carbodiimide conjugates, it attaches to mKLH through the ε-
amino group of lysine residues. In a previous study, 6 was
compared with an oxycodone immunogen containing a shorter
hemisuccinate-like linker at the C6 position, and the
tetraglycine linker was more effective in evoking serum
antibody titers.6b
Figure 4. Comparison of immunogens on oxycodone antinociception
in mice. Vaccine effects on oxycodone analgesia, after administration of
2.25 mg/kg oxycodone sc in BALB/c mice immunized with alum
adjuvant sc. The % values above columns indicate decreases relative to
the KLH control group. Data are mean SEM; * p < 0.05 compared
to KLH control.
Because the purpose of this study was to identify an effective
hydrocodone immunogen, not all immunogens were compared
in each assay. However all immunogens were compared to the
lead compound 6 and by all measures this immunogen was the
most effective. Prescription opioid abuse is a challenging
treatment target because abusers may use different prescription
opioids at different times. A vaccine that blocks both oxycodone
and hydrocodone, two of the most commonly abused
prescription opioids, might be of value. These data suggest
that the immunogen 6 is a reasonable candidate for generating
polyspecific antibodies against both drugs and for further
investigation of the merit of this therapeutic approach.
The structure of fentanyl differs substantially from that of
oxycodone and hydrocodone. As expected, rats vaccinated with
the conjugate 6 did not show a significant reduction in fentanyl
antinociception, indicating that the efficacy of this immunogen
does not extend to all opioids. This may be therapeutically
useful because it would allow individuals vaccinated with this
immunogen to still obtain full effect from fentanyl or other
opioids if required for their medical needs. On the other hand,
if fentanyl abuse should emerge as a common problem, it may
be necessary to combine 6 with an immunogen specifically
targeting this drug.
substitution at C8 is important for preserving this immunoge-
nicity. In fact, serum antibodies generated from the 6 and 8
immunogens recognized both haptens 1 and 3 despite the
different linker composition and linker chemistry. Serum
antibodies generated from the immunogens 6 and 8 did not
recognize the hydrocodone-based haptens 4 and 5, which
contrasts with the in vivo data showing that the immunogen 6
blocked the distribution to brain and nociception of both
oxycodone and hydrocodone. This result was unexpected, since
we have previously shown that vaccination with 6 elicits serum
antibody displaying high affinity for oxycodone and its
metabolite oxymorphone but lower affinity for hydrocodone.6b
It is possible that derivatization at the C8 position masked
critical bindings sites for antibody recognition of hydrocodone
or that characterization of vaccine-generated serum antibodies
by the ELISA method may not fully predict the effect of
vaccination against opioids in vivo. Similar discrepancies
between ELISA data and in vivo efficacy have been recently
reported for a morphine conjugate vaccine.13 Recognition of
linker by antibodies is not a likely contributor to antibody
efficacy because similar efficacy was found with immunogens
utilizing either tetraglycine or the thiol-maleimide linkers. Also,
we have previously shown that antibodies raised by immunogen
6 do not recognize the (Gly4)OH linker.6b
Maleimide activation of BSA resulted in a number of
maleimide groups similar to that of mBSA obtained from
commercial sources and in the same range as previous reports
by our and other groups.11a,14 The hapten 3 conjugated to
mBSA showed a haptenization ratio similar to previously
described haptens conjugated through maleimide,11a although
higher haptenization ratios have been reported with a
maleimide-containing morphine conjugate.5e Despite a rela-
tively low haptenation ratio, the immunogen 8 elicited an
efficient immune response that blocked the distribution and the
behavioral effects of oxycodone. These data suggest that if the
haptenation were further improved, immunogen 8 could be an
alternative to 6.
CONCLUSIONS
■
A previously described oxycodone-based immunogen, 6, was
found to generate polyspecific antibodies with activity against
hydrocodone as well as oxycodone, and was more effective in
doing so than hydrocodone-based immunogens. The greater
efficacy of this immunogen was likely due to both its intrinsic
immunogenicity and a high protein haptenation ratio. The
ability of 6 to attenuate the distribution of both oxycodone and
hydrocodone to brain, as well as their analgesic activity,
identifies it as a useful tool for studying the potential use of
vaccination to treat prescription opioid abuse.
EXPERIMENTAL SECTION
■
Drugs and Reagents. Opioids were obtained through the NIDA
Drug Supply Program, Sigma (St. Louis, MO), and Mallinckrodt (St.
Louis, MO). All drug doses and concentrations are expressed as the
weight of the base.
Because the previously studied 6 was highly effective in
blocking oxycodone effects in rats, the analogous conjugate 7
was anticipated to be similarly immunogenic against hydro-
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Synthesis of Oxycodone and Hydrocodone Haptens.
General Information. NMR spectra were recorded on a Bruker
Avance 400 MHz spectrometer (Bruker Daltonics, Billerica, MA).
Chemical shifts are in parts per million (ppm). The assignments were
Hz), 6.73 (d, 1H, JH1−H2 = 8.1 Hz), 6.82 (d, 1H, JH2−H1 = 8.1 Hz). 13
C
NMR (DMSO) 17.82, 21.34, 28.45, 28.51, 31.64, 39.78, 41.34, 42.13,
42.33, 42.56, 42.63, 44.65, 49.37, 59.72, 70.20, 85.74, 116.74, 119.54,
126.70, 136.57, 142.40, 144.46, 156.68, 168.23, 171.0, 171.30, 171.32,
174.61; mp 176 °C (decomposition). Anal. Calcd for C28H36N6O9: C,
55.99; H, 6.04; N, 13.99. Found: C, 55.83; H, 5.97; N, 13.87. ESI-TOF
MS calculated for C28H36N6O9, m/z 600.620, found 601.548 (MH)+.
[[[17-Methyl-4,5α -epoxy-14-hydroxy-3-methoxy-morphinan-6-
[[((ylidene)-amino)-oxy]-N-(2-mercaptoethyl)-acetamido]-
morphinan [6OXY(SH)] (3). Reacting S-tritylcysteamine (121 mg, 0.38
mmol), acid 12 (122 mg, 0.31 mmol), DCC (128 mg, 0.62 mmol),
HOBt (84 mg, 0.62 mmol), and DMAP (4 mg, 0.03 mmol), followed
by treatment with TFA and AcOH as reported above, gave the target
compound 3, which was purified by flash chromatography on silica gel;
white solid; yield 77%. 1H NMR (DMSO) δ: 1.28 (m, 1H), 1.61−1.67
(m, 2H), 2.34−3.14 (m, 14H with s, 3H, NCH3 at 2.67), 3.71 (m,
1H), 3.86 (s, 3H), 4.51 (s, 2H), 4.99 (s, 1H), 6.73 (d, 1H, JH1−H2 = 8.1
Hz), 6.82 (d, 1H, JH2−H1 = 8.1 Hz). 13C NMR (DMSO) 17.96, 23.25,
26.84, 27.34, 28.17, 28.42, 41.13, 45.26, 46.28, 56.32, 65.14, 69.47,
70.34, 85.27, 115.30, 119.68, 123.56, 128.84, 142.26, 144.46, 155.83,
170.66; mp 184 °C. ESI-TOF MS calculated for C22H29N3O5S, m/z
447.548, found 448.370 (MH)+.
17-Methyl-4,5α-epoxy-6-one-3-methoxy-8-[thio-N-(glycylglycyl-
glycylglycine)-acetamido]morphinan [8HYDROC(Gly)4OH] (4). Re-
acting amine GlyO4tBu (112 mg, 0.37 mmol), acid 13 (120 mg, 0.31
mmol), DCC (128 mg, 0.62 mmol), HOBt (84 mg, 0.62 mmol), and
DMAP (4 mg, 0.03 mmol), followed by treatment with TFA as
reported above, gave the target compound 4, which was purified by
reverse-phase HPLC; product retention time, 12 min; slightly
brownish solid; yield 56%. 1H NMR (DMSO) δ (major isomer):
1.53 (m, 1H), 2.05 (m, 2H), 2.32 (s, 3H, NCH3), 2.39−2.47 (m, 5H),
2.75 (m, 1H), 2.91 (m, 2H), 3.31−3.35 (m, 10H), 3.79 (s, 3H,
OCH3), 4.95 (s, 1H), 6.62 (d, 1H, JH1−H2 = 8.1 Hz), 6.72 (d, 1H,
JH2−H1 = 8.1 Hz). ESI-TOF MS calculated for C28H35N5O9S, m/z
617.671, found 618.723 (MH)+.
1
made using one-dimensional H and 13C spectra. ESI mass spectra
were recorded on a BrukerBioTof II system. Preparative high-
performance liquid chromatography (HPLC) was performed using a
VERSAPrep system with a Haisil column (100 C-18, 5 mm, 10 mm ×
250 mm). A gradient starting from 90% CH3CN/10% H20/0.1%
trifluoroacetic acid (TFA) reaching 70% CH3CN/30% H2O/0.1%
TFA was used. Flash chromatography on silica gel were performed on
EM Science silica gel 60 (230−400 mesh). A gradient starting from 2%
methanol/97% dichloromethane (DCM)/1% NH4OH reaching 10%
methanol/89% DCM/1% NH4OH was used. Purity (%) was
determined by reverse phase HPLC, using UV detection (254 nm),
and all compounds showed purity greater than 95%. Melting points
were determined on a Thomas−Hoover melting point apparatus and
are uncorrected. All commercial reagents and solvents were used
without further purification.
17-Methyl-4,5α-epoxy-3-methoxy-6-[[((ylidene)-amino)-oxy]-
acetic acid]morphinan (11). To a solution of hydrocodone base (300
mg, 1 mmol) in methanol (5 mL) were added pyridine (196 μL, 2
mmol) and carboxymethoxylamine hemichloride (262 mg, 1.2 mmol).
The reaction was stirred at 80 °C for 4 h. After the solvent was
removed in vacuo, the crude residue was taken up and triturated in an
1
acetone/diethyl ether mixture to afford 2 (97%) as a white solid. H
NMR (DMSO) δ: 1.13 (m, 1H), 1.85−1,88 (m, 2H), 2.14 (m, 1H),
2.41−2.49 (m, 2H), 2.55 (s, 3H), 2.99−3.01 (m, 2H), 3.22−3.25 (m,
2H), 3.87 (s, 3H, OCH3), 4.47 (s, 2H), 4.94 (s, 1H), 6.64 (d, 1H,
JH1−H2 = 8.1 Hz), 6.72 (d, 1H, JH2−H1 = 8.1 Hz). ESI-TOF MS
calculated for C20H24N2O3, m/z 372.415, found 373.367 (MH)+.
17-Methyl-4,5α-epoxy-6-one-3-methoxy-8-[thioacetic acid]-
morphinan (13). A solution of codeinone (9 250 mg, 8.4 mmol)
and thioglycolic acid (65 μL, 0.92 mmol) in anhydrous tetrahydrofur-
ane (THF, 5 mL) was stirred at room temperature for 20 h. A solid
began to form almost immediately. The suspension was filtered, the
resulting white solid washed with water and diethyl ether, and dried to
17-Methyl-4,5α-epoxy-6-one-3-methoxy-8-[thio-N-(2-mercap-
toethyl)-acetamido]morphinan [8HYDROC(SH)] (5). Reacting S-
tritylcysteamine (83 mg, 0.34 mmol), acid 13 (110 mg, 0.28 mmol),
DCC (115 mg, 0.56 mmol), HOBt (76 mg, 0.56 mmol), and DMAP
(4 mg, 0.03 mmol), followed by treatment with TFA and AcOH as
reported above, gave the target compound 5, which was purified by
reverse-phase HPLC; product retention time, 10 min; slightly
1
afford 13 (87%) as a 25/75 mixture of isomers. H NMR (DMSO) δ
major isomer): 1.52 (m, 1H), 2.05 (m, 2H), 2.37 (s, 3H, NCH3),
2.41−2.59 (m, 5H), 2.77 (m, 1H), 2.94 (m, 2H), 3.30 (dd, 2H, J = 6.4
Hz, J = 15.4 Hz), 3.77 (s, 3H, OCH3), 4.93 (s, 1H), 6.65 (d, 1H,
JH1−H2 = 8.1 Hz), 6.73 (d, 1H, JH2−H1 = 8.2 Hz). 1H NMR (DMSO) δ
(minor isomer): 1.52 (m, 1H), 2.05 (m, 2H), 2.37 (s, 3H, NCH3),
2.41−2.59 (m, 5H), 2.77 (m, 1H), 2.94 (m, 2H), 3.30 (m, 2H), 3.77
(s, 3H, OCH3), 4.86 (s, 1H), 6.62 (d, 1H, JH1−H2 = 8.3 Hz), 6.73 (d,
1H, JH2−H1 = 8.2 Hz). ESI-TOF MS calculated for C20H23NO5S, m/z
389.461, found 390.258 (MH)+.
General Procedure for Coupling and Deprotection of
Haptens 2−5. To a solution of carboxylic acid (1 mol equiv) in
N,N-dimethylformamide (DMF), amine (1.2 mol equiv), dicyclohex-
ylcarbodiimide (DCC, 2.0 mol equiv), hydroxybenzotriazole (HOBt, 2
mol equiv), and catalytic N,N-dimethylaminopyridine (DMAP, 0.1
mol equiv) were sequentially added. After stirring at room temperature
for 24 h, the reaction was evaporated and the residue was dissolved in
DCM and then filtered to remove dicyclohexylurea. The resulting
solution was treated with TFA (10 mol equiv) and glacial acetic acid
(AcOH 10 mol equiv) when a trityl protecting group was used. After
stirring at room temperature for 16 h, the solvent was removed by
evaporation and the residue was purified by column chromatography
on silica gel or on a reverse-phase HPLC column.
17-Methyl-4,5α-epoxy-3-methoxy-6-[[((ylidene)-amino)-oxy]-N-
(glycylglycylglycylglycine)-acetamido]morphinan [6HYDROC-
(Gly)4OH] (2). Reacting amine GlyOtBu (180 mg, 0.6 mmol), acid
11 (185 mg, 0.5 mmol), DCC (205 mg, 1.0 mmol), HOBt (134 mg,
1.0 mmol), and DMAP (6 mg, 0.05 mmol), followed by treatment
with TFA as reported above, gave the target compound 2, which was
purified by reverse-phase HPLC; product retention time, 9 min; white
solid; yield 68%. 1H NMR (CD3OD) δ: 1.28 (m, 1H), 1.61−1,67 (m,
2H), 2.34−3.14 (m, 14H with s, 3H, NCH3 at 2.67), 3.71 (m, 1H),
3.86 (s, 3H, OCH3), 4.50 (s, 2H), 5.00 (s, 1H), 6.28 (t, 1H, NH, J = 8.
1
brownish solid; yield 56%. H NMR (CD3OD) δ (major isomer):
1.53 (m, 1H), 2.07 (m, 2H), 2.35 (s, 3H, NCH3), 2.40−2.62 (m, 7H),
2.72−2.81 (m, 3H), 2.95 (m, 2H), 3.78 (s, 3H, OCH3), 4.94 (s, 1H),
6.67 (d, 1H, JH1−H2 = 8.1 Hz), 6.71 (d, 1H, JH2−H1 = 8.1 Hz). ESI-TOF
MS calculated for C22H28N2O4S2, m/z 448.599, found 449.641
(MH)+.
Conjugation of 6, 7, and 9 Immunogens. Glycine linker
containing haptens for use in the vaccines were conjugated to KLH
using carbodiimide conjugation chemistry as described previously.5d,6b
For use as coating antigen in ELISA assays, haptens were conjugated
to bovine serum albumin (BSA). Molar hapten:protein conjugation
ratios (moles of hapten conjugated per mole of protein) for the BSA
conjugates were quantitated by mass spectrometry (Reflex III, Bruker),
as described previously.6b
Conjugation of 8 and 10 Immunogens. Sulfhydryl-containing
haptens 3 and 5 were conjugated to maleimide activated BSA (mBSA)
and KLH (mKLH) as described before with minor modifications.11b
BSA and KLH (Thermo Scientific, Rockford, IL) were activated with
sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate
(maleimide moles 200×:BSA moles) (sulfo-SMCC, Thermo Scien-
tific) in degassed 50 mM phosphate buffered saline (PBS) containing 1
mM ethylenediaminetetraacetic acid (EDTA, Sigma) for 1 h at room
temperature under nitrogen (N2) purge to avoid oxidation. Maleimide-
activated proteins were purified by dialysis in degassed 0.05 mM PBS
under N2 for 4 h and stored at +4 °C. The haptens 3 and 5 were
dissolved in 1 mL of degassed 50 mM PBS, 1 mM EDTA containing
15 mM TCEP (Sigma), and added to 2 mg of mBSA or mKLH in a
final 2 mL reaction volume. For 6OXY(S)−mBSA, the following
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Journal of Medicinal Chemistry
Article
hapten to protein molar ratios were used to determine the optimal
reaction conditions: 1000×, 825×, 496×, 331×, and 165×. For 8, the
following hapten to protein molar ratios were used to determine the
optimal reaction conditions: 33500×, 11200×, 5600×, 2800×, 700×.
Reactions were stirred for 2 h at room temperature in glass vials sealed
with N2, followed by dialysis in degassed 0.05 mM PBS under N2 for 6
h and stored at +4 °C. All 8 conjugation conditions were replicated
using commercially available mKLH (Thermo Scientific) to verify the
immunogenicity of in-house mKLH. Conjugation of 10 to BSA and
KLH was based upon optimal conditions employed for the preparation
of 8.
Vaccination. Male Holtzman rats and BALB/c mice (Harlan
Laboratories, Madison, WI) were housed with a 12/12 h standard
light/dark cycle. In rats, conjugates or unconjugated KLH control were
injected ip in a final volume of 0.4 mL in complete Freund’s adjuvant
for the first injection and incomplete Freund’s adjuvant for two
subsequent booster injections at 3 and 6 weeks as described.6b Mice
were vaccinated on days 0, 14, and 28 with 25 μg of immunogen or
unconjugated protein control; vaccine was administered sc in a final
volume of 0.2 mL containing alum adjuvant as described.5d Blood was
obtained on day 35 for serum antibody titer measurement by facial
vein sampling. All experiments were conducted 7−10 days after the
third immunization.
Antibody. ELISA plates were coated with 5 ng/well of BSA
conjugates or unconjugated protein control in carbonate buffer at pH
9.6 and blocked with 1% gelatin. Primary antibodies were incubated
with goat antirat IgG antibodies conjugated to horseradish peroxidase
or rabbit antimouse IgG antibodies to measure immunized rat and
mouse sera, as described previously.5d Competitive binding ELISA to
determine IC50 was performed as described previously.5d
Protein Binding of Oxycodone and Hydrocodone in Serum.
Serum protein binding of oxycodone and hydrocodone were measured
as described previously.6b Protein binding of oxycodone was measured
in five rats randomly chosen in each vaccination group receiving
oxycodone sc.
of 25 and 100 μg of the 6 immunogen did not differ in rats.6b After
completion of the hot plate behavioral end point, trunk blood and
brain were collected to measure hydrocodone concentration.
As a specificity control for the hot plate test, fentanyl
antinociception was tested using a dose of 0.3 mg/kg sc, in a separate
cohort of rats vaccinated with 6 or unconjugated KLH.
Evaluation of Vaccination on Oxycodone Antinociception
after sc Dosing in Mice. The effects of immunization with 25 μg of
either the 6, 8, 9, or KLH control on oxycodone distribution, and its
effect on the hot plate assay was measured in BALB/c mice (n = 5−8
per group) receiving 2.25 mg/kg oxycodone administered sc, the same
dose as used for rats. Antinociception tests in mice were performed as
with rats at 30 min. Hind paw lifts, hind paw licks and jumps were the
predetermined behavioral end points to calculate the % MPE. The
effect of immunization on oxycodone antinociception with the
immunogen 6 was compared to several immunogen 8 conjugates to
determine the most efficient sulfhydryl−maleimide conjugation
conditions in BALB/c mice (n = 5−7 per group). Mice received
2.25 mg/kg oxycodone sc.
ASSOCIATED CONTENT
* Supporting Information
■
S
Characterization of serum antibody affinity, serum protein
binding of oxycodone or hydrocodone in rats, evaluation of
immunogen 8 conjugate conditions. This material is available
free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Phone: 612-232 7017. Fax: 612-337-7189. E-mail: prave001@
umn.edu. Address: Minneapolis Medical Research Foundation,
701 Park Avenue, Minneapolis, MN, 55415.
■
Author Contributions
Marco Pravetoni designed and performed studies and wrote the
manuscript. Morgan Le Naour synthesized haptens and assisted
with manuscript preparation. Ashli M. Tucker assisted with
ELISA and animal studies. Theresa M. Harmon performed
analytical chemistry. Tara M. Hawley performed protein
binding assays. Philip S. Portoghese assisted with study design,
manuscript preparation, and data interpretation. Paul R. Pentel
assisted with study design, manuscript preparation, and data
interpretation.
Oxycodone and Hydrocodone Assay. Serum and brain drug
concentrations were measured by gas chromatography coupled to
mass spectrometry as previously described.6b The reported drug
concentrations represent the total drug (protein or antibody-bound as
well as free) in each sample.
Statistical Analysis. Group differences were analyzed by one-way
analysis of variance followed by Bonferroni post hoc test using Prism
5.0 (Graph Pad, La Jolla Ca).
Effects of Vaccination on Oxycodone Distribution after iv
Dosing in Rats. The effects of immunization with 100 μg of the 6, 8,
9, 10, or unconjugated KLH control on oxycodone distribution were
first measured in rats (n = 5 per group) receiving 0.1 mg/kg
oxycodone administered iv. One week after the final vaccine dose, rats
were anesthetized with ketamine/xylazine and an indwelling catheter
was placed in their right external jugular vein. Blood was withdrawn for
ELISA assays and oxycodone administered as a 10 s infusion. Rats
were decapitated 5 min later and trunk blood and brain collected. The
oxycodone dose was chosen to compare effects with previous reports
from our laboratory.5d,6b
Effect of Vaccination on Oxycodone and Hydrocodone
Distribution and Antinociception after sc Dosing in Rats. The
effects of immunization with 100 μg of the 6, 9, or KLH control on
oxycodone distribution and antinociception were measured at 30 min
in rats (n = 10 per group) receiving 2.25 mg/kg oxycodone
administered sc. This dose was previously shown to elicit a near-
maximal analgesic effect that was blocked by vaccination with 6.6b Hot
plate antinociception tests were performed at 54 °C with 60 s cutoffs
to prevent thermal damage; hind paw licks and jumps were the
predetermined behavioral end points to calculate % maximal possible
effect (MPE).6b
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by NIH NIDA grant DA026300 and
by a Minneapolis Medical Research Foundation Career
Development Award (M.P.). The authors thank Danielle
Burroughs for technical assistance. We thank Mallinckrodt
(St. Louis, MO) for the generous gift of oxycodone.
ABBREVIATIONS USED
KLH, keyhole limpet hemocyanin; BSA, bovine serum albumin
■
REFERENCES
■
(1) (a) 2008 Department of Defense Survey of Health Related Behaviors
Among Active Duty Military Personnel; U.S. Department of Defence,
2009; (b) National Survey on Drug Use and Health: National Findings;
U.S. Department of Health and Human Services Substance Abuse and
Mental Health Services Administration Office of Applied Studies,
2011; (c) Compton, W. M.; Volkow, N. D. Abuse of prescription
drugs and the risk of addiction. Drug Alcohol Depend. 2006, 83 (Suppl
1), S4−S7. (d) Compton, W. M.; Volkow, N. D. Major increases in
opioid analgesic abuse in the United States: concerns and strategies.
In rats immunized with either 25 μg of the 6, 7, or KLH control (n
= 7−12 per group), hydrocodone antinociception was assessed after
administration of 6.75 mg/kg hydrocodone sc; this dose was chosen
̈
through pilot studies in naive rats to elicit a near-maximal response. In
previous studies, serum antibody titers elicited from immunogen doses
922
dx.doi.org/10.1021/jm3013745 | J. Med. Chem. 2013, 56, 915−923

Journal of Medicinal Chemistry
Article
Drug Alcohol Depend. 2006, 81, 103−107. (e) Maxwell, J. C. The
prescription drug epidemic in the United States: a perfect storm. Drug
Alcohol Rev. 2011, 30, 264−270.
(2) (a) Oxymorphone Abuse: A Growing Threat Nationwide; EWS
Report 000011; U.S. Department of Justice, 2011; (b) Epidemic:
Responding to America’s Prescription Drug Abuse Crisis; Executive Office
of the President of the United States, 2011.
oxycodone conjugate vaccine elicits oxycodone-specific antibodies that
reduce oxycodone distribution to brain and hot-plate analgesia. J.
Pharmacol. Exp. Ther. 2012, 341, 225−232.
(7) Lewenstein, M. J., Morphine and codeinone derivatives. Patent
GB945909 19640108, 1964.
(8) Glaser, M.; Karlsen, H.; Solbakken, M.; Arukwe, J.; Brady, F.;
Luthra, S. K.; Cuthbertson, A. 18F-fluorothiols: a new approach to
label peptides chemoselectively as potential tracers for positron
emission tomography. Bioconjugate Chem. 2004, 15, 1447−1453.
(9) Sondergaard, K.; Kristensen, J. L.; Palner, M.; Gillings, N.;
Knudsen, G. M.; Roth, B. L.; Begtrup, M. Synthesis and binding
studies of 2-arylapomorphines. Org. Biomol. Chem. 2005, 3, 4077−
4081.
(10) (a) Kotick, M. P.; Leland, D. L.; Polazzi, J. O.; Schut, R. N.
Analgesic narcotic antagonists. 1. 8-beta-Alkyl-, 8-beta-acyl-, and 8-
beta-(tertiary alcohol)dihydrocodeinones and -dihydromorphinones. J.
Med. Chem. 1980, 23, 166−174. (b) Sagara, T.; Okamura, M.;
Shimohigashi, Y.; Ohno, M.; Kanematsu, K. Specificity affinity labeling
of μ opioid receptor in rat brain by S-activated sulfydryldihydromor-
phine analogs. Bioorg. Med. Chem. Lett. 1995, 5, 1609−1614. (c) Kolev,
T.; Bakalska, R.; Seidel, R. W.; Mayer-Figge, H.; Oppel, I. M.; Spiteller,
M.; Sheldrick, W. S.; Koleva, B. B. Novel codeinone derivatives via
Michael addition of barbituric acids. Tetrahedron: Asymmetry 2009, 20,
327−334.
(11) (a) Carroll, F. I.; Blough, B. E.; Pidaparthi, R. R.; Abraham, P.;
Gong, P. K.; Deng, L.; Huang, X.; Gunnell, M.; Lay, J. O., Jr.; Peterson,
E. C.; Owens, S. M. Synthesis of mercapto-(+)-methamphetamine
haptens and their use for obtaining improved epitope density on
(+)-methamphetamine conjugate vaccines. J. Med. Chem. 2011, 54,
5221−5228. (b) Pravetoni, M.; Keyler, D. E.; Pidaparthi, R. R.;
Carroll, F. I.; Runyon, S. P.; Murtaugh, M. P.; Earley, C. A.; Pentel, P.
R. Structurally distinct nicotine immunogens elicit antibodies with
non-overlapping specificities. Biochem. Pharmacol. 2012, 83, 543−550.
(12) Janda, K. D.; Benkovic, S. J.; Lerner, R. A. Catalytic antibodies
with lipase activity and R or S substrate selectivity. Science 1989, 244,
437−440.
(13) Raleigh M. D., Pravetoni M., Harris A. C., Birnbaum A. K.,
Pentel P. R., Selective effects of a morphine conjugate vaccine on
heroin and metabolite distribution and heroin-induced behaviors in
rats. J. Pharmacol. Exp. Ther. 2012, DOI: 10.1124/jpet.112.201194.
(14) Pravetoni, M.; Keyler, D. E.; Pidaparthi, R. R.; Carroll, F. I.;
Runyon, S. P.; Murtaugh, M. P.; Earley, C. A.; Pentel, P. R. Structurally
distinct nicotine immunogens elicit antibodies with non-overlapping
specificities. Biochem. Pharmacol. 2011, 83, 543−550.
(3) (a) Butler, S. F.; Black, R. A.; Cassidy, T. A.; Dailey, T. M.;
Budman, S. H. Abuse risks and routes of administration of different
prescription opioid compounds and formulations. Harm Reduction J.
2011, 8, 29. (b) Dodrill, C. L.; Helmer, D. A.; Kosten, T. R.
Prescription pain medication dependence. Am. J. Psychiatry 2011, 168,
466−471. (c) Lofwall, M. R.; Moody, D. E.; Fang, W. B.; Nuzzo, P. A.;
Walsh, S. L. Pharmacokinetics of Intranasal Crushed OxyContin and
Intravenous Oxycodone in Nondependent Prescription Opioid
Abusers. J. Clin. Pharmacol. 2011, 52, 600−606. (d) Stoops, W. W.;
Hatton, K. W.; Lofwall, M. R.; Nuzzo, P. A.; Walsh, S. L. Intravenous
oxycodone, hydrocodone, and morphine in recreational opioid users:
abuse potential and relative potencies. Psychopharmacology (Berlin)
2010, 212, 193−203. (e) Walsh, S. L.; Nuzzo, P. A.; Lofwall, M. R.;
Holtman, J. R., Jr. The relative abuse liability of oral oxycodone,
hydrocodone and hydromorphone assessed in prescription opioid
abusers. Drug Alcohol Depend. 2008, 98, 191−202. (f) Wu, L. T.;
Woody, G. E.; Yang, C.; Blazer, D. G. How do prescription opioid
users differ from users of heroin or other drugs in psychopathology:
results from the National Epidemiologic Survey on Alcohol and
Related Conditions. J. Addict. Med. 2011, 5, 28−35. (g) Wu, L. T.;
Woody, G. E.; Yang, C.; Mannelli, P.; Blazer, D. G. Differences in
onset and abuse/dependence episodes between prescription opioids
and heroin: results from the National Epidemiologic Survey on
Alcohol and Related Conditions. Subst. Abuse Rehabil. 2011, 2011,
77−88.
(4) (a) Haney, M.; Gunderson, E. W.; Jiang, H.; Collins, E. D.;
Foltin, R. W. Cocaine-specific antibodies blunt the subjective effects of
smoked cocaine in humans. Biol. Psychiatry 2010, 67, 59−65.
(b) 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. (c) Martell, B. A.; Orson, F. M.; Poling, J.; Mitchell, E.; Rossen, R.
D.; Gardner, T.; Kosten, T. R. Cocaine vaccine for the treatment of
cocaine dependence in methadone-maintained patients: a randomized,
double-blind, placebo-controlled efficacy trial. Arch. Gen. Psychiatry
2009, 66, 1116−1123.
(5) (a) Anton, B.; Leff, P. A novel bivalent morphine/heroin vaccine
that prevents relapse to heroin addiction in rodents. Vaccine 2006, 24,
3232−3240. (b) Bonese, K. F.; Wainer, B. H.; Fitch, F. W.; Rothberg,
R. M.; Schuster, C. R. Changes in heroin self-administration by a
rhesus monkey after morphine immunisation. Nature 1974, 252, 708−
710. (c) Li, Q. Q.; Luo, Y. X.; Sun, C. Y.; Xue, Y. X.; Zhu, W. L.; Shi,
H. S.; Zhai, H. F.; Shi, J.; Lu, L. A morphine/heroin vaccine with new
hapten design attenuates behavioral effects in rats. J. Neurochem. 2011,
119, 1271−1281. (d) Pravetoni, M.; Raleigh, M. D.; Le Naour, M.;
Tucker, A. M.; Harmon, T. M.; Jones, J. M.; Birnbaum, A. K.;
Portoghese, P. S.; Pentel, P. R. Co-administration of morphine and
oxycodone vaccines reduces the distribution of 6-monoacetylmorphine
and oxycodone to brain in rats. Vaccine 2012, 30, 4617−4624.
(e) Stowe, G. N.; Vendruscolo, L. F.; Edwards, S.; Schlosburg, J. E.;
Misra, K. K.; Schulteis, G.; Mayorov, A. V.; Zakhari, J. S.; Koob, G. F.;
Janda, K. D. A vaccine strategy that induces protective immunity
against heroin. J. Med. Chem. 2011, 54, 5195−5204. (f) Wainer, B. H.;
Fitch, F. W.; Rothberg, R. M.; Fried, J. Morphine-3-succinyl−bovine
serum albumin: an immunogenic hapten−protein conjugate. Science
1972, 176, 1143−1145.
(6) (a) Findlay, J. W.; Butz, R. F.; Jones, E. C. Relationships between
immunogen structure and antisera specificity in the narcotic alkaloid
series. Clin. Chem. 1981, 27, 1524−1535. (b) Pravetoni, M.; Le Naour,
M.; Harmon, T.; Tucker, A.; Portoghese, P. S.; Pentel, P. R. An
923
dx.doi.org/10.1021/jm3013745 | J. Med. Chem. 2013, 56, 915−923