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Prophylactic vaccination protects against the development of oxycodone addiction

27 Oct 2017-bioRxiv (Cold Spring Harbor Laboratory)-pp 210369

AbstractAbuse 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 IVSA 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 antinociceptive and reinforcing effects of oxycodone. Anti-drug 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 increase the difficulty of drug use or incentivize other behaviors.

Topics: Oxycodone (59%)

Summary (2 min read)

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.
  • 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.
  • 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.

2. Materials and Methods

  • Adult male Wistar (N=48; Charles River, New York) rats were housed in humidity and temperature-controlled vivaria (23+1oC) on 12:12 hour light:dark cycles.
  • The solvents from the organic layer were dried with sodium sulfate (Na2SO4), filtered and evaporated.
  • After completion of the reaction, NaBH(OAc)3 was quenched with water and the reaction was washed with saturated sodium bicarbonate (2 x 10 mL).

2.2.3 Synthesis of activated oxycodone hapten 7 and conjugation to carrier protein tetanus toxoid (TT) or

  • The deprotection was allowed to proceed for several hours and was monitored by TLC (9:1 CH2Cl2:MeOH) and LCMS.
  • To begin a session, the catheter fittings on the animals’ backs were connected to polyethylene tubing contained inside a protective spring suspended into the operant chamber from a liquid swivel attached to a balance arm.
  • The eluted sample was evaporated using GENEVAC and taken up in MeOH for LCMS analysis.

3.1 Conjugation of Oxy-TT Vaccine

  • The oxycodone hapten (Oxy) and Oxy-TT immunoconjugate were prepared as described previously (Kimishima et al, 2016) with minor modification detailed in the Experimental Section .
  • MALDI-ToF analysis of Oxy-BSA confirmed successful conjugation with approximately 16 moles of Oxy hapten per mole of carrier protein.
  • Following the vaccination regimen, rats in the Oxy-TT groups established plasma antibody titer .

3.2 Vaccination Protects Against Self-Administration of Oxycodone

  • Using a threshold per infusion dose of oxycodone (0.06 mg/kg/inf; Cohort 1) under fixed-ratio (FR) schedule of reinforcement, rats steadily increased intake across the first 18 sessions of training .
  • Only 58% of the Oxy-TT group met the acquisition criterion (average of 7 infusions across two sessions), whereas all of the TT animals met criterion .
  • Under a higher per infusion dose (0.15 mg/kg/inf; Cohort 2), rats increased oxycodone intake over the first 18 sessions of acquisition, with the Oxy-TT group eventually obtaining more infusions .

3.3 Oxy-TT Vaccine Decreases Motivation for Oxycodone Self-Administration

  • By increasing the workload required for reinforcement, the first PR probe (See details of PR schedule and j values in Materials and Methods) in Cohort 1 produced a significant reduction in the number of infusions earned for the Oxy-TT rats only ; there was a significant effect of schedule condition [F(1,21)=9.35; p=0.006] but not of vaccine Group.
  • Oxy-TT rats trained under a higher per infusion dose (Cohort 2) also self-administered less oxycodone under the higher workload conditions of a PR schedule .
  • In the subsequent FR, PRJ2, PRJ3, FR transitions fewer infusions were obtained in both PRJ2 and PRJ3 relative to each of the FR conditions within the Oxy-TT group .

3.4 Vaccine Attenuates Antinociceptive Effects of Oxycodone

  • The effects of oxycodone on nociception were first determined prior to self-administration training in Cohort 1 (N=11-12) and Cohort 2 (N=12) rats.
  • The dose-dependent attenuation of the antinociceptive effects of oxycodone in Oxy-TT rats persisted throughout the study .
  • Post hoc analysis confirmed increased tail withdrawal latency in TT rats compared to Oxy-TT during the 30 and 120 min time points.
  • Finally, Oxy-TT vaccination did not protect against heroin (1.0 mg/kg, s.c.)-induced antinociception which was assessed in Cohort 2 .

3.5 Vaccine Decreases Brain Penetration of Oxycodone

  • Plasma oxycodone (ng/mL) was significantly higher in the Cohort 1 Oxy-TT rats compared to the corresponding TT group following injection of oxycodone , which was confirmed by an unpaired t-test [t(20)=7.708, p<0.0001].
  • The Tukey post hoc test confirmed that Oxy-TT and TT oxycodone concentration levels found in plasma under all dose and time post-injection conditions were significantly different.

4. Discussion

  • This study provides evidence that active vaccination with Oxy-TT provides functional protection against the reinforcing effects of oxycodone.
  • The tail-withdrawal test demonstrated that the Oxy-TT groups were less sensitive to the antinociceptive effect of injected oxycodone in a dose-dependent manner.
  • This was observed in Cohort 2 despite the fact that more infusions were obtained by Oxy-TT rats when only one response was required.
  • This ratio is cut in half for seniors endorsing past month use, suggesting a change in relative risk from prescription to illicit opioids in the more-frequently using population which would potentially pivot health care priorities from single drug prophylaxis to multi-drug prevention/therapy depending on exposure history.
  • The increased intake of oxycodone in the Upper halves of each of the Oxy-TT vaccinated groups might be misinterpreted in a human clinical context as a significant failure, since drug-taking is usually the main outcome measure of clinical trials (Hoogsteder et al, 2014; Martell et al, 2009).

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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

Figures (5)
Citations
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TL;DR: This novel synthetic route to (-)-oxycodone, a semisynthetic opioid analgesic, features a palladium-catalyzed direct intramolecular arylation of an aryl bromide, oxidative dearomatization of a dihydrophenanthrenol, formation of a benzylic quaternary carbon by an intramolescular Michael addition of a malonate moiety.
Abstract: Our novel synthetic route to (-)-oxycodone, a semisynthetic opioid analgesic, features a palladium-catalyzed direct intramolecular arylation of an aryl bromide, oxidative dearomatization of a dihydrophenanthrenol, formation of a benzylic quaternary carbon by an intramolecular Michael addition of a malonate moiety, and construction of the morphinan skeleton via a Hofmann rearrangement/lactamization cascade.

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Patent
16 May 2019
Abstract: The invention provides haptens and methods of preparation thereof, for producing immunoconjugates of oxycodone and hydrocodone suitable for raising antibodies in animals specific for the opioid drugs. Adminislration of the opioid-speeific antibodies to humans having the opioid drugs in a body fluid such as blood serum, binds the opioid drugs to the antibody, making the opioids unavailable for producing a drug effect. Accordingly the antibodies can be used to reverse the effects of a drug overdose of oxycodone and hydrocodone, or to reverse a perceived mental effect of the. drugs, such as euphoria.

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Frequently Asked Questions (2)
Q1. What contributions have the authors mentioned in the paper "Prophylactic vaccination protects against the development of oxycodone self-administration" ?

Janda et al. this paper used a vaccine incorporating an oxycodone-hapten conjugated to fixmetetanus toxoid ( Oxy-TT ) to reduce voluntary drug-seeking in a rat model. 

Such consideration might include the likelihood of the individual requiring opioid medications in the near future and the availability of alternatives, as well as the relative risks of potentially using a higher dose of, e. g., oxycodone, in a very short course of pain management. This study was funded by grants from the USPHS R01 DA035281 ( M. A. T. ), R01 DA024705 ( M. A. T. ), UH3 DA041146 ( K. D. J. ) and F32 AI126628 ( C. S. H. ), which had no further input on the conduct of the work or the decision to publish results. Conversely it also underlines the potential need for multiple vaccines to address differing risk profiles with respect to abuse of multiple opioids, e. g. as modelled by Hwang and colleagues ( Hwang et al, 2018b ). This ratio is cut in half for seniors endorsing past month use, suggesting a change in relative risk from prescription to illicit opioids in the more-frequently using population which would potentially pivot health care priorities from single drug prophylaxis to multi-drug prevention/therapy depending on exposure history.