1
Prophylactic vaccination protects against the development of oxycodone
self-administration
Jacques D. Nguyen
1*
, Candy S. Hwang
2*
, Yanabel Grant
1
, Kim D. Janda
2
, Michael A. Taffe
1
1
Department of Neuroscience;
2
Departments of Chemistry and Immunology, The Skaggs Institute for
Chemical Biology, Worm Institute for Research and Medicine (WIRM); The Scripps Research Institute;
La Jolla, CA, USA
* these authors contributed equally
Address Correspondence to: Dr. Michael A. Taffe, Department of Neuroscience, SP30-2400; 10550 North Torrey
Pines Road; The Scripps Research Institute, La Jolla, CA 92037; USA; Phone: +1.858.784.7228; Fax:
+1.858.784.7405; Email: mtaffe@scripps.edu
certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was notthis version posted May 22, 2018. ; https://doi.org/10.1101/210369doi: bioRxiv preprint
2
Abstract
Abuse of prescription opioids is a growing public health crisis in the United States, with drug overdose
deaths increasing dramatically over the past 15 years. Few preclinical studies exist on the reinforcing
effects of oxycodone or on the development of therapies for oxycodone abuse. This study was
conducted to determine if immunopharmacotherapy directed against oxycodone would be capable of
altering oxycodone-induced antinociception and intravenous self-administration. Male Wistar rats were
administered a small-molecule immunoconjugate vaccine (Oxy-TT) or the control carrier protein, tetanus
toxoid (TT), and trained to intravenously self-administer oxycodone (0.06 or 0.15 mg/kg/infusion). Brain
oxycodone concentrations were 50% lower in Oxy-TT rats compared to TT rats 30 minutes after injection
(1 mg/kg, s.c.) whereas plasma oxycodone was 15-fold higher from drug sequestration by circulating
antibodies. Oxy-TT rats were also less sensitive to 1-2 mg/kg, s.c. oxycodone on a hot water nociception
assay. Half of the Oxy-TT rats failed to acquire intravenous self-administration under the 0.06
mg/kg/infusion training dose. Oxycodone self-administration of Oxy-TT rats trained on 0.15
mg/kg/infusion was higher than controls; however under progressive ratio (PR) conditions the Oxy-TT
rats decreased their oxycodone intake, unlike TT controls. These data demonstrate that active
vaccination provides protection against the reinforcing effects of oxycodone. Anti-oxycodone vaccines
may entirely prevent repeated use in some individuals who otherwise would become addicted.
Vaccination may also reduce dependence in those who become addicted and therefore facilitate the
effects of other therapeutic interventions which either increase the difficulty of drug use or incentivize
other behaviors.
Keywords: Oxycodone; vaccine; self-administration
certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was notthis version posted May 22, 2018. ; https://doi.org/10.1101/210369doi: bioRxiv preprint
3
1. Introduction
Drug overdose deaths in the US have increased dramatically over the past 15 years with most of
these attributable to opioids, including prescription drugs. Despite an increase in nonmedical prescription
opioid abuse, with the use of heroin and other illicit drugs most often presaged by prescription opioids in
new users (Dertadian and Maher, 2014; Mars et al, 2014), preclinical research into the addictive
properties of oxycodone has been infrequent in comparison with studies on heroin or morphine. Limited
studies in mice (Enga et al, 2016; Mayer-Blackwell et al, 2014; Zhang et al, 2015; Zhang et al, 2009) and
rats (Leri and Burns, 2005; Mavrikaki et al, 2017; Secci et al, 2016; Wade et al, 2015) affirm that
oxycodone will reinforce operant responding in intravenous self-administration (IVSA) models, thus,
traditional pre-clinical measures of opioid addiction are useful to study the effects of potential
therapeutics.
Anti-drug vaccination alters the effects of many drugs of abuse (Bremer and Janda, 2017;
Lockner and Janda, 2013; Ohia-Nwoko et al, 2016); vaccines against cocaine (Haney et al, 2010; Kosten
et al, 2002; Martell et al, 2009) and nicotine (Cornuz et al, 2008; Hatsukami et al, 2011) have advanced
to clinical trials. Initial studies by Pravetoni and colleagues showed that an oxycodone-specific vaccine
could induce antibodies capable of sequestering oxycodone in the blood and lowering brain penetration
of oxycodone in rats (Pravetoni et al, 2012; Pravetoni et al, 2014). Vaccination decreased the
antinociceptive effects of a single dose of oxycodone and reduced oxycodone intake during the
acquisition of IVSA. Vaccination has also been shown to prevent oxycodone-induced respiratory
depression and cardiovascular effects in rats (Raleigh et al, 2018; Raleigh et al, 2017). This success
encourages more comprehensive research on anti-oxycodone vaccines, to further general understanding
of how anti-drug vaccinations function in addition to supporting an eventual clinical vaccine for
oxycodone abuse.
Janda and colleagues created a vaccine incorporating an oxycodone-hapten conjugated to
tetanus toxoid (Oxy-TT) that has shown efficacy in attenuating antinociceptive effects and oxycodone
overdose in mice (Kimishima et al, 2017). Therefore, it is of interest to determine if the Oxy-TT can
reduce voluntary drug-seeking in a rat model. Prior evaluation of anti-drug vaccination efficacy in drug
certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was notthis version posted May 22, 2018. ; https://doi.org/10.1101/210369doi: bioRxiv preprint
4
self-administration has typically focused on the effects of vaccination after the acquisition of drug taking
behavior, e.g., for vaccines directed against cocaine (Carrera et al, 2000; Evans et al, 2016; Kantak et al,
2000), nicotine (Lindblom et al, 2002; Moreno et al, 2010) and morphine or heroin (Anton and Leff, 2006;
Hwang et al, 2018a; Hwang et al, 2018b; Schlosburg et al, 2013). Although there have been a few
studies of the effects of prophylactic vaccination prior to self-administration of cocaine (Wee et al, 2012),
nicotine (LeSage et al, 2006), oxycodone (Pravetoni et al, 2014), heroin (Stowe et al, 2011) and
methamphetamine (Duryee et al, 2009; Miller et al, 2015), the effects of vaccination in the early stages of
addiction are not well understood. Prior studies of IVSA of oxycodone (Pravetoni et al, 2014) and
methamphetamine (Duryee et al, 2009) suggest that increasing the workload (i.e., the number of lever
responses required for each infusion) may decrease intake in the vaccinated groups relative to controls.
Another finding with an anti-methamphetamine vaccine suggests, however, that initial higher drug intake
may be gradually extinguished over the first several IVSA sessions with no change in workload (Miller et
al, 2015). Cocaine IVSA is lower in vaccinated animals than in controls under a Progressive Ratio (PR)
schedule of reinforcement and increased workload (Wee et al, 2012), whereas nicotine IVSA increases
in the vaccinated rats (LeSage et al, 2006). The present study was conducted to determine if the novel
Oxy-TT vaccine directed against oxycodone would be capable of altering the IVSA of oxycodone.
2. Materials and Methods
2.1 Animals. Adult male Wistar (N=48; Charles River, New York) rats were housed in humidity and
temperature-controlled vivaria (23+1
o
C) on 12:12 hour light:dark cycles. Cohort 1 (N=24; lower training
dose) animals were 10 weeks old (mean weight 288.3 g; SD 12.8) at the start of the study. Cohort 2
(N=24; higher training dose) animals were 13 weeks old (mean weight 408.9 g; SD 30.6) at the start of
the study. Animals had ad libitum access to food and water in their home cages. All procedures were
conducted under protocols approved by the Institutional Animal Care and Use Committees of The
Scripps Research Institute and in a manner consistent with the National Institutes of Health Guide for the
Care and Use of Laboratory Animals.
certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was notthis version posted May 22, 2018. ; https://doi.org/10.1101/210369doi: bioRxiv preprint
5
2.2 Hapten Synthesis. The oxycodone hapten (Oxy) was designed with an activated linker extending
from the bridgehead nitrogen to directly react with the surface lysines of carrier protein tetanus toxoid
(TT, Figure 1A) or BSA. Compound 7 was synthesized according to previously published methods in our
laboratory with slight modification in the reductive amination and amide bond formation steps (Kimishima
et al, 2016). Although decreasing the equivalents of NaBH(OAc)
3
to 1.1 equivalents avoids formation of
the undesired alcohol, the reaction results in an incomplete mixture of 2 and 4 after several hours.
Instead, 3 equivalents of NaBH(OAc)
3
rapidly led to the conversion of the reductive amination and
reduced C-6 ketone product. The secondary C-6 alcohol can be simply and mildly oxidized to the desired
ketone (4) using DMP.(Kimishima et al, 2014) After preparation and confirmation of the product (4) by
1
H
and
13
C NMR, 4 was deprotected and condensed with 5 using HATU as a catalyst, resulting in a 61%
yield of purified 6. Additional details can be found in the Supplementary Information.
2.2.1 Synthesis of tert-butyl(4-((4R,4aS,7aR,12bS)-4a-hydroxy-9-methoxy-7-oxo-1,2,4,4a,5,6,7,7a-
octahydro-3H-4,12-methanobenzofuro[3,2-e]isoquinolin-3-yl)butyl)carbamate (4)
Oxycodone hydrochloride (100 mg, 0.28 mmol) was dissolved in dichloromethane (CH
2
Cl
2
) and
washed with saturated sodium bicarbonate (2 x 10 mL). The solvents from the organic layer were dried
with sodium sulfate (Na
2
SO
4
), filtered and evaporated. The white powder was dissolved in 4 mL of dry,
1,2-dichloroethane. Sodium bicarbonate (235 mg, 2.8 mmol, 10 equiv) and ACE-Cl (245 μL, 2.3 mmol, 8
equiv) were added at room temperature (rt). The mixture was then heated to reflux overnight under argon
and checked by TLC (5:1 CH
2
Cl
2
:methanol). The reaction was then cooled and solvent was removed
under reduced pressure. The residue was then dissolved in 10 mL CH
2
Cl
2
and washed with saturated
bicarbonate (2 x 10 mL). The aqueous layers were combined and washed with ethyl acetate (EtOAc, 1 x
10 mL). The organic layers were combined and dried with Na
2
SO
4
. The solution was filtered and solvents
were evaporated. The oil was then dissolved in a portion of methanol (MeOH), stirred at rt for several
hours and monitored by TLC (5:1 CH
2
Cl
2
:MeOH). The solvents were evaporated and the residue was
purified by flash chromatography using 10% MeOH in CH
2
Cl
2
to give 36 mg of 2 (43% crude yield) as a
colorless oil. ESI-MS: MS (m/z): calcd for C
17
H
20
NO
4
+
: 302.1, found: 302.2 [M + H]
+
.
certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was notthis version posted May 22, 2018. ; https://doi.org/10.1101/210369doi: bioRxiv preprint