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. Author manuscript; available in PMC: 2022 Sep 1.
Published in final edited form as: Neuropharmacology. 2021 Jun 11;195:108653. doi: 10.1016/j.neuropharm.2021.108653

Pharmacological mechanisms underlying the efficacy of antibodies generated by a vaccine to treat oxycodone use disorder

MD Raleigh 1,2, SJ King 2, F Baruffaldi 2, A Saykao 2, FA Hamid 1, S Winston 3, MG LeSage 2,4, PR Pentel 2,4, M Pravetoni 1,2,4,5
PMCID: PMC8410661  NIHMSID: NIHMS1714837  PMID: 34126123

Abstract

Therapeutic vaccines offer a viable strategy to treat opioid use disorders (OUD) complementary to current pharmacotherapies. The candidate Oxy(Gly)4-sKLH vaccine targeting oxycodone displayed pre-clinical proof of efficacy, selectivity and safety, and it is now undergoing clinical evaluation. To further support its implementation in the clinic, this study tested critical in vivo neuropsychopharmacological properties of the Oxy(Gly)4-sKLH vaccine in rats. While repeated immunizations with Oxy(Gly)4-sKLH were necessary to maintain the antibody response overtime, exposure to free oxycodone did not boost oxycodone-specific antibody levels in vaccinated rats, limiting concerns of immune-related side effects. Immunization with Oxy(Gly)4-sKLH achieved sustained antibody titers over a period of five months following initial vaccination, supporting its potential for providing long-lasting protection. In vivo studies of selectivity showed that vaccination prevented oxycodone-induced but not methadone-induced antinociception, while still preserving the opioid antagonist naloxone’s pharmacological effects. Vaccination did not interfere with fentanyl-induced antinociception or fentanyl distribution to the brain. These in vivo data confirm the previously reported in vitro selectivity profile of Oxy(Gly)4-sKLH. Vaccination extended oxycodone’s half-life up to 25 hr compared to control. While vaccination reduced the reinforcing efficacy of oxycodone in an intravenous self-administration model, signs of toxicity were not observed. These rodent studies confirm that active immunization with Oxy(Gly)4-sKLH induces highly specific and long-lasting antibodies which are effective in decreasing the reinforcing effects of oxycodone while preserving the efficacy of medications used to treat OUD and overdose.

1.0. Introduction

Opioid use disorders (OUD) and opioid-related fatal overdoses are a public health concern worldwide (CDC, 2020; UNODC, 2020), which have been exacerbated during the novel coronavirus SARS-CoV-2 pandemic (CDC, 2020; Singh et al., 2019). Vaccines targeting heroin, oxycodone, fentanyl, and its analogs are being developed to augment existing medications to treat OUD and prevent overdose (Anton and Leff, 2006; Bonese et al., 1974; Bremer et al., 2016; Hwang et al., 2018; Kimishima et al., 2017; Kosten et al., 2013; Li et al., 2014; Nguyen et al., 2018; Raleigh et al., 2019; Raleigh et al., 2013; Stowe et al., 2011; Torten et al., 1975). Pre-clinical studies identified a candidate vaccine targeting oxycodone (Baruffaldi et al., 2019), which is currently being evaluated in Phase I clinical trials (NCT04458545). This vaccine formulation consists of an oxycodone-based hapten containing a tetraglycine linker [Oxy(Gly)4] conjugated to the subunit keyhole limpet hemocyanin (sKLH) carrier protein (Oxy(Gly)4-sKLH), and adsorbed to aluminum adjuvant. Pre-clinical studies showed that immunization with Oxy(Gly)4-sKLH triggers CD4+ T cell-dependent activation of B cells to generate high serum concentrations of oxycodone-specific polyclonal IgG antibodies that bind oxycodone in blood, reduce its distribution to the brain, and reduce oxycodone-induced locomotor activity, respiratory depression, bradycardia, antinociception, and acquisition of oxycodone intravenous self-administration (Laudenbach et al., 2015; Pravetoni et al., 2012a; Pravetoni et al., 2013; Pravetoni et al., 2014; Pravetoni et al., 2012b; Raleigh et al., 2018; Raleigh et al., 2017). To support regulatory approval and clinical implementation of Oxy(Gly)4-sKLH, this study sought to identify key in vivo characteristics of vaccine-generated oxycodone-specific polyclonal antibodies, and their efficacy against pharmacokinetic and pharmacodynamic effects of oxycodone in rats.

While small molecules such as oxycodone are not expected to elicit adaptive immune responses unless conjugated to larger immunogenic carriers, the effect of the free drug on drug-specific antibody secreting B cells in immunized individuals has not been fully explored. Clinical studies of vaccines targeting nicotine and cocaine have not characterized nor reported boosts in drug-specific antibody levels following exposure to the target drug in immunized (Hatsukami et al., 2011; Hatsukami et al., 2005; Kosten et al., 2002). In the context of clinical use of Oxy(Gly)4-sKLH, there is a remote possibility that oxycodone could act as an immunogen itself in immunized individuals boosting B cells into extending the antibody response beyond the intended vaccine dosing schedule and extend compliance long after ending treatment. In support of clinical implementation, it will also be important to determine the persistence of antibodies at completion of the immunization regimen to optimize the vaccination schedule and maximize vaccine efficacy and duration of protection.

In the clinic, other opioids may be needed for pain management or as part of medication-assisted treatment (MAT) for OUD. While in vitro analyses may provide a first screening for antibody binding to target and off target compounds, demonstrating selectivity and retention of in vivo efficacy of key opioids is critical. As an example, vaccine-induced heroin-specific antibodies displayed low in vitro cross-reactivity towards methadone but vaccination blocked methadone’s in vivo antinociceptive effects (Raleigh et al., 2013). A first-generation oxycodone vaccine containing the native KLH did not interfere with fentanyl-induced antinociception (Pravetoni et al., 2013) and naloxone (Laudenbach et al., 2018; Pravetoni et al., 2012a; Raleigh et al., 2017). Antibodies generated by Oxy(Gly)4-sKLH are selective towards oxycodone, but not to methadone or fentanyl in vitro (Raleigh et al., 2017), so in vivo cross-reactivity is not expected. However, it is important to establish whether the current GMP-grade Oxy(Gly)4-sKLH formulation preserves the effects of methadone, fentanyl, and naloxone in vivo.

Antibodies elicited by Oxy(Gly)4-sKLH have been shown to retain oxycodone in serum, decrease the concentration of unbound (free) oxycodone, and reduce its distribution to brain at specific time points after single or multiple acute dose challenges (Baruffaldi et al., 2018; Pravetoni et al., 2013; Pravetoni et al., 2012b; Raleigh et al., 2018). However, only limited data are available regarding vaccine effects on oxycodone’s pharmacokinetic parameters (Kimishima et al., 2017; Pravetoni et al., 2013). A better understanding of the pharmacokinetic effects of Oxy(Gly)4-sKLH could help interpret results from clinical studies in individuals receiving oxycodone.

Self-administration remains the gold standard model for measuring vaccine effects on a drug’s reinforcing efficacy. Reduction of acquisition and maintenance of oxycodone self-administration (OSA) has been previously demonstrated in rats prophylactically vaccinated against oxycodone (Nguyen et al., 2018; Pravetoni et al., 2014). Although this finding indicates a reduction in oxycodone’s reinforcing effects that engender its abuse, patients in clinical studies would either currently be using oxycodone or attempting to quit during vaccination. It is therefore critical to understand whether vaccination can alter the maintenance of OSA in rats that have a history of self-administration prior to being vaccinated, and whether vaccination during ongoing drug use may trigger any observable toxicity.

The current study demonstrated that vaccine-induced oxycodone-specific antibodies generated by this oxycodone vaccine persist in rats for at least 2-3 months following immunization, antibodies did not interfere with the effects of methadone, fentanyl, or naloxone, and oxycodone exposure in rats immunized after vaccination with Oxy(Gly)4-sKLH did not boost antibody production. Vaccination markedly altered the pharmacokinetics of oxycodone and decreased its reinforcing effects in OSA, but did not elicit signs of toxicity. These data provide advanced proof of efficacy, selectivity and safety for anti-opioid vaccines, and can inform the design and expectations of clinical studies.

2.0. Materials and Methods

2.1. Bioethics.

Studies were performed in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. Animal protocols were approved by the Hennepin Healthcare Research Institute and University of Minnesota Institutional Animal Care and Use Committees. Surgery was performed under i.m. ketamine (75–90 mg/kg) and dexmedetomidine (0.25 mg/kg) anesthesia, animals were euthanized by CO2 inhalation using AAALAC approved chambers, and all efforts were made to minimize suffering.

2.2. Animals.

Studies used male Sprague Dawley (SD) rats (Envigo, Madison, WI) weighing between 225-250g at arrival. In Experiments 1, 2, 3, and 4 rats were double housed under a 12/12-hr standard light/dark cycle, free-fed, and testing occurred during the light phase. In Experiment 5, SD rats were individually housed under a 12/12-hr light/dark cycle, food was restricted to 18-20 g/day, and self-administration sessions occurred during the dark phase of the light cycle. Independent cohorts of SD rats were used to conduct each experiment, and experiment-specific details are provided below.

2.3. Drugs.

Oxycodone, methadone, fentanyl, and naloxone were obtained through the National Institute on Drug Abuse Drug Supply Program (Bethesda, MD) or Sigma-Aldrich (St. Louis, MO). Drug doses and concentrations are expressed as the weight of the base.

2.4. Vaccine.

This study tested the efficacy of a GMP-grade vaccine formulation consisting of oxycodone-based hapten (Cambrex, Duram, NC) conjugated via a tetraglycine linker (Oxy(Gly)4) to subunit keyhole limpet hemocyanin monomer (sKLH, Biosyn, Carlsbad, CA) via carbodiimide coupling chemistry, and adsorbed on aluminum hydroxide (Alhydrogel, Brenntag Biosector, Denmark) at Goodwin Biotechnology (Plantation, FL) as described (Baruffaldi et al., 2018; Pravetoni et al., 2012a; Pravetoni et al., 2012b; Raleigh et al., 2013). Animals were vaccinated i.m. once every three weeks with either 60 μg Oxy(Gly)4-sKLH or sKLH adsorbed to 90 μg aluminum for a total of 4 vaccinations on days 0, 21, 42, and 63. In self-administration studies, rats were vaccinated once every 2 weeks with either 60 μg Oxy(Gly)4-sKLH or 90 μg aluminum alone for a total of 6 vaccine doses to maintain antibody levels during this protocol.

2.5. Analysis of drug concentration in tissue.

Briefly, trunk blood was centrifuged at 3100 x g for 3 min at 4°C and serum collected. Brain was rinsed with distilled H2O and patted dry. Serum and brain were stored at −20°C until analysis. Oxycodone and fentanyl concentrations were measured by gas chromatography/mass spectrometry as previously described (Pravetoni et al., 2013; Raleigh et al., 2019).

2.6. Drug protein binding.

The percentage of oxycodone bound to protein in serum was measured by ultrafiltration as previously described (Raleigh et al., 2018). In this assay, sera were centrifuged at 10,000 x g for 1 hr in 10 kDa molecular weight cutoff ultrafiltration tubes (Nanosep 10K Omega, PALL Laboratory, Port Washington, NY). The total oxycodone concentration was measured from an aliquot of serum before ultrafiltration, and the unbound concentration was measured in the ultrafiltrate. Bound oxycodone was determined as the total minus unbound concentrations.

2.7. Antibody characterization: titers and affinity.

Oxycodone-specific serum IgG antibody titers were measured by ELISA as previously described (Pravetoni et al., 2012b; Raleigh et al., 2017; Raleigh et al., 2013). Competitive binding ELISA was used to measure the relative affinity (cross-reactivity) of oxycodone-specific serum IgG antibodies for a panel of target and off target drugs as previously described (Raleigh et al., 2017). Relative affinity (%) was reported as the ratio of the IC50 value (μM) for oxycodone to the competitor multiplied by 100.

2.8. Experimental design

2.8.1. Experiment 1: Effect of oxycodone administration on antibody levels following vaccination.

Rats were vaccinated with either Oxy(Gly)4-sKLH (n=8/group) or sKLH (n=4/group). Two months after completion of the immunization regimen, antibody levels were measured and a day later rats were treated s.c. with either 2.25 mg/kg oxycodone (or saline) was administered s.c. Antibody titers were measured one week later to determine whether oxycodone administration boosted antibody titers. Rats were then challenged on two separate days with three daily doses of 1 mg/kg oxycodone s.c. (3 mg/kg/day) and antibody titers measured the following week. Rats were then boosted i.m. with either Oxy(Gly)4-sKLH or sKLH, and antibody titers were measured one week later.

2.8.2. Experiment 2: Decay of oxycodone-specific serum IgG titers following vaccination.

Rats (n=10) were vaccinated with OXY(Gly)4-sKLH. Blood was collected via the tail vein on Days 70, 97, 129, 159, 190, and 220 after the first immunization to measure antibody titers.

2.8.3. Experiment 3: Effect of vaccination on altering antinociceptive or respiratory depressive effects of oxycodone, methadone, and fentanyl, and preserving naloxone’s efficacy in vivo.

Rats (n=10/group) were vaccinated with either Oxy(Gly)4-sKLH or sKLH. One week after the final vaccination, half of the rats from each group were challenged with increasing doses of either oxycodone or methadone (up to a total dose of 9 mg/kg) every 17 min. Fifteen min after drug administration, rats were placed on the hotplate to test for drug-induced antinociception and then placed in a chamber with a collar placed around the rats neck to measure oxygen saturation (%SaO2). For antinociceptive testing, rats were habituated for 1 hr to the testing environment and nociception assessed on a hot plate (Columbus Instruments, Columbus, OH) set to 54°C ± 0.2°C (Raleigh et al., 2013). A nociceptive response was measured as the latency to perform a hindpaw lick or jumping. A maximum cutoff of 60 sec was used to avoid injury. Immediately following the final test, rats received a 0.1 mg/kg dose of naloxone s.c. and were tested 15 min later on the hotplate and oximeter assays. One week later, rats that received oxycodone on the previous week were given escalating doses of methadone (and vice versa) and the experiment was repeated. One week later, rats were tested on the hotplate at baseline and at 30 min following s.c administration of 0.05 mg/kg fentanyl and brain tissue collected for measurement of fentanyl.

2.8.4. Experiment 4: Effect of vaccination on half-life of oxycodone in serum.

Male (n=5/group) and female (n=5/group) SD rats age-matched to 8 weeks on arrival were vaccinated with either Oxy(Gly)4-sKLH (n=10) or sKLH (n=10). One week following the final immunization, rats were anesthetized i.m. with 75 mg/kg ketamine and 0.25 mg/kg dexmedetomidine and an indwelling catheter was placed in the right external jugular vein. Two days after recovery, awake rats were challenged with 5 mg/kg oxycodone administered over a 10-minute i.v. infusion, followed by 1 mL of saline to flush the catheter. Blood was drawn via indwelling catheter at 0.17, 0.5, 1, 2, 3, and 5 hr in sKLH vaccinated rats and at 0.17, 2, 5, 8, 12, and 20 hr in Oxy(Gly)4-sKLH vaccinated rats. Blood taken at 5 hr in both groups was assessed for protein binding as described above. One female rat vaccinated with Oxy(Gly)4-sKLH died following surgery, and two male rats vaccinated with Oxy(Gly)4-sKLH required tail vein sampling in lieu of catheter sampling) at t=5 hr and t=8 hr, with no sampling at t=12 hr due to suboptimal patency. One male rat vaccinated with Oxy(Gly)4-sKLH was removed from calculating the 2-compartment model curve because its data did not fit a 2-compartment model. A non-compartmental analysis was performed on this rat instead to produce its pharmacokinetic parameters.

2.8.5. Experiment 5: Effect of vaccination on maintenance of oxycodone self-administration (OSA) and potential signs of toxicity.

One week after arrival, rats were implanted with a chronic indwelling catheter according to our standard procedures (LeSage et al., 2003). Briefly, rats were anesthetized with i.m. ketamine (75–90 mg/kg) and dexmedetomidine (0.25 mg/kg), and a catheter was inserted into the right jugular vein. The opposite end of the catheter exited the body between the scapulae and attached to a vascular-access harness (see Supplementary Materials). Rat recovered from surgery for at least three days, during which catheters were flushed daily with a heparinized (30 units/ml) saline/glycerol (25%) solution and ceftriaxone (5.25 mg), and a s.c. injection of buprenorphine (0.05 mg/kg; first two days only) was given for analgesia. Over the course of the experiment, daily infusions of heparinized saline/glycerol continued and infusions of methohexital (0.1 ml of 10 mg/ml, i.v.) were given on Fridays after the session to determine catheter patency (indicated by anesthesia within 5 sec). If a catheter lost patency or was otherwise compromised (e.g. the rat pulled out its catheter), data from that week were excluded from analysis and another catheter was implanted in the femoral vein. The majority of these reimplants occurred during training before vaccination began. Two rats lost patency in week three of vaccination (resulting in missing data for that week). These rats were reimplanted with new catheters, and performance was reacquired in week four, allowing them to remain in the study. One rat lost patency and another pulled out its catheter in week seven. Performance was reacquired in the former rat after being reimplanted (resulting in missing data only for week seven), but the latter rat could not be reimplanted because it had been reimplanted previously. This rat was removed from the rest of the study, but its data through week six were used for analysis.

Rats were trained to self-administer oxycodone at a dose of 0.06 mg/kg/kg/infusion under a fixed-ratio (FR) 3 schedule during 2 hr sessions in standard operant conditioning chambers (see Supplemental Materials and Figure S3). Sessions were conducted five days per week. Once OSA was stable, rats were vaccinated i.m. with either Oxy(Gly)4-sKLH (n=8 males, 4 females) or aluminum (n=8 males, 5 females). The majority of rats were vaccinated every 2 weeks on Fridays for a total of 6 vaccine doses to maintain high antibody titers during this protocol. To obtain oxycodone dose-response curves, rats started an oxycodone dose reduction schedule at week 8 (after the 5th vaccine dose) where the oxycodone unit dose was progressively reduced at weekly intervals (i.e., the oxycodone dose was reduced every Monday of every week) using the following unit doses: 0.015, 0.0075, 0.0038, 0.0019, and 0.0 mg/kg/infusion. The final (6th) vaccine dose was administered after completing the last session at the 0.0075 mg/kg unit dose to maintain antibody levels over the remainder of the dose-reduction schedule.

Seven rats received an additional vaccination during assessment of the 0.0019 mg/kg unit dose because the Thanksgiving holiday allowed only three sessions that week (i.e. a seventh vaccine dose was needed to conduct a total of five sessions at this dose). One rat received eight vaccinations because it pulled out its catheter during the dose-reduction phase (between the fifth and sixth vaccination), which required implanting a new catheter and recovering baseline self-administration at the 0.06 mg/kg unit dose before redoing the dose-reduction schedule. Blood samples were collected via the tail vein one week after the fourth vaccine injection to measure antibody titers.

At completion of each self-administration session during the maintenance and dose-reduction protocols, rats were observed in an isolated self-administration chamber for 3 minutes for signs of oxycodone toxicity to address the potential concern that vaccination might elicit a compensatory increase in OSA that may result in oxycodone toxicity. A description of signs of toxicity is provided in Table 1. Trained observers scored these signs from zero to two (0 = none, 1 = intermittent, 2 = continuous). Male and female rat data were combined and averaged. Experimenters were blinded to treatment group.

Table 1. Signs of opioid toxicity during oxycodone self-administration during Experiment 5.

Toxicity was scored as 0 = none, 1 = intermittent, 2 = continuous.

Sign of toxicity Description of sign
Respiration Slow, rapid, irregular or gasping respiration
Hyperactivity Increased movement or exploration
Hypoactivity Decrease movement or exploration, or decreased muscle tone
Gait and posture Unsteady gait, swaying of head, abnormal posture
Drooling
Pica Chewing or eating bedding, other objects
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After completion of the dose-reduction phase, rats were returned to the 0.06 mg/kg oxycodone unit dose and allowed to reacquire OSA (mean 29±1.9 sessions). Rats were then euthanized 24 hr after their last self-administration session and their brains collected to explore long-term effects of vaccination on oxycodone-induced changes in brain mRNA. Brains were sliced along the coronal plane into 5 mm sections using a 1.0mm Alto brain matrix (CellPoint Scientific, MD), and placed in RNAlater (Thermo Scientific, MA) stabilization solution and refrigerated until processed. Sections (“punches”) of brain slice 4, which included the ventral tegmental area and nucleus accumbens, were collected and RNA was extracted using RNAqueous phenol-free total RNA isolation kit (Thermo Scientific, MA). Nanodrop (2000c, Thermo Scientific, MA) confirmed RNA concentrations between 200 – 470 ng/mL. Stranded mRNA was measured using an Illumina Platform PE150 poly-A RNA sequencing with Nova6000. 2 x 150bp FastQ paired end reads (n=21.8 Million average per sample) were trimmed using Trimmomatic (v 0.33) enabled with the optional “-q” option; 3bp sliding-window trimming from 3’ end requiring minimum Q30. Quality control of raw sequence data for each sample was performed with FastQC. Read mapping was performed via Hisat2 (v2.1.0) using the rat genome (Rnor_6.0) as a reference. Gene quantification was done via Feature Counts for raw read counts. Gene expression was normalized to counts per million by multiplying (number of reads mapped to a selected gene plus 0.1) times (one divided by total number of mapped reads in a sample times 106). The addition of 0.1 to the number of reads mapped to a selected gene was introduced to avoid issues with zero read counts. Differentially expressed genes (DEG) were identified using the edgeR (negative binomial) feature in CLCGWB (Qiagen, Redwood City, CA) using raw read counts. The generated list was filtered based on a minimum 2x Absolute Fold Change and FDR corrected p < 0.05.

2.9. Statistical analysis.

Geometric mean antibody titers, opioid concentrations, and individual pharmacokinetic parameters were compared using unpaired t tests. When variances were significantly different, Welsh’s correction was applied. To examine differences in oxycodone self-administration, the mean number infusions per session were normalized as % baseline (see Supplementary Material for analysis of absolute values) and compared between groups via a mixed-effects model using the maximum likelihood method and Geisser-Greenhouse correction, with group as a non-repeated factor and session or unit dose as a repeated factor, followed by Sidak’s or Dunnett’s multiple comparison tests. The percentage of rats with infusion rates below baseline range (pre-vaccination) at each unit dose was compared using Fisher’s exact test with Bonferroni’s correction for multiple comparisons (i.e., a p value of 0.0125 was used for significance for each of four tests). To determine whether vaccination reduced the reinforcing efficacy of oxycodone per se (i.e., independent of reducing oxycodone potency), exponential demand curve analysis was performed on oxycodone intake during the unit dose reduction phase as a percentage of baseline as previously described (Raleigh et al., 2014). The primary parameter of interest, α, is estimated from the best-fit function and refers to the rate of reduction in consumption with increases in unit price, which is an index of elasticity of demand or reinforcing efficacy (Hursh and Silberberg, 2008). Secondary parameters of interest included Q0 (maximal consumption at zero price (i.e., demand intensity)), Pmax (the price at which consumption becomes relatively elastic) and Omax (maximum level of responding). Curves were fit to individual subject data and mean parameter values were compared between groups using unpaired t-tests with Welch’s correction. Trends (i.e. slopes) in OSA during the first four vaccinations were compared between groups using linear regression. Mean SaO2 levels, heart rate, and latency to respond on the hotplate were compared using two-way ANOVA (using the Geisser-Greenhouse correction) and Sidak’s multiple comparison test. The effect of naloxone on latency to respond, heart rate, or SaO2 within a single group used a two sided paired t test with values before and after naloxone, while unpaired t tests with Welch’s correction were used to compare sKLH versus Oxy(Gly)4-sKLH groups. Analyses were performed using Prism v9.0 (GraphPad, La Jolla, CA).

3.0. Results

3.1. Relative affinity of vaccine-induced antibodies towards target and off target compounds.

The relative affinities of oxycodone-specific serum IgG antibodies for a panel of target and off target drugs from rats vaccinated with Oxy(Gly)4-sKLH is shown in Table S1. Antibodies showed high relative affinity towards Oxy(Gly)4 hapten (0.074 μM, 127%), oxycodone (0.094 μM, 100%), and oxymorphone (0.12 μM, 77%). Oxycodone-specific serum IgG titers had very low relative affinity (less than 1% cross-reactivity) towards off-target competitors including endogenous opioids, naloxone, naltrexone, nalmefene, morphine, fentanyl, buprenorphine, methadone, nicotine, cocaine, opioids, and other drugs used in treatment of human immunodeficiency virus (HIV) and hepatitis B virus infections (Table S1).

3.2. Experiment 1: Effect of oxycodone administration on eliciting antibody production following vaccination.

Compared to saline treatment as control, no main effect of oxycodone treatment on oxycodone-specific serum IgG titers was detected in the Oxy(Gly)4-sKLH group (F(1,14)=0.3, p=0.59) (Figure 1A). As expected, boosting with Oxy(Gly)4-sKLH significantly increased oxycodone-specific serum IgG titers (t(7)=4.85, p=0.0019), while oxycodone-specific serum IgG titers continued to decrease in rats boosted with sKLH alone (t(7)=4.29, p=0.0036) (Figure 1B).

Figure 1. Oxycodone does not boost oxycodone-specific antibody titers and antibody titer decay after vaccination.

Figure 1.

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Rats were vaccinated with 60 μg OXY(Gly)4-sKLH (n=8/group) or sKLH (n=4/group) and 90 μg aluminum i.m. once every three weeks for a total of 4 vaccinations. (A) Two months later antibody levels were measured and 2.25 mg/kg oxycodone (or saline) was administered s.c (designated as O1). A week later, antibody titers were measured. Rats were then challenged on two separate days with 3X daily doses of 1 mg/kg oxycodone s.c. (designated as O2) and antibody titers measured the following week. (B) To demonstrate that antibody could be elicited, rats that had been vaccinated with Oxy(Gly)4-sKLH were boosted i.m. with either OXY(Gly)4-sKLH or sKLH and antibody titers were measured one week later. (C) Oxycodone-specific antibody titers after completion of vaccination. Rats (n=10) were vaccinated with 60 μg OXY(Gly)4-sKLH adsorbed to 90 μg aluminum i.m. as above. Blood was taken on Days 70, 97, 129, 159, 190, and 220 post-1st vaccination to measure antibody levels. Antibody titers expressed as geometric mean ± SD. ** p<0.01 compared to pre-boost titers and *** p<0.001 compared to Day 70 titers.

3.3. Experiment 2: Decay of oxycodone-specific serum IgG titers following vaccination.

Seven days following the 4th vaccination of Oxy(Gly)4-sKLH, oxycodone-specific serum IgG titers were 156 ± 21 x 103 (mean ± SD, Figure 1C). There was a significant effect of time on oxycodone-specific serum IgG titers [F(1.073, 9.660)=15.20, p=0.0028]. Oxycodone-specific serum IgG titers were not significantly lower on Day 66, but were lower from Day 96 and on (p<0.001 at each time point).

3.4. Experiment 3: Effect of vaccination on oxycodone, methadone, fentanyl, and naloxone in vivo.

Oxycodone-specific serum IgG titers in Oxy(Gly)4-sKLH vaccinated rats that received methadone or oxycodone on week 1 were 198 ± 24 x 103 and 188 ± 37 x 103, respectively (mean ± SD), which did not differ (t(6.9)=0.51, p=0.62). Vaccination with Oxy(Gly)4-sKLH significantly reduced oxycodone-induced antinociception at the 1.125 mg/kg oxycodone dose (p<0.05, Figure 2A). There was no effect of vaccination (F(1, 18)=0.33, p=0.57) on oxycodone-induced respiratory depression although there was a significant interaction (dose x vaccination group) (F(4, 72)=5.48, p<0.001, Figure 2B). Similarly for oxycodone-induced bradycardia, there was no effect of vaccination (F(1, 18)=1.14, p=0.30), but there was a significant interaction (F(4, 72)=3.16, p<0.05, Figure 2C). Vaccination did not affect naloxone’s ability to reverse oxycodone-induced antinociception, respiratory depression, or bradycardia and there were no differences between sKLH and Oxy(Gly)4-sKLH vaccinated rats following naloxone administration (Figures 2A–2C). Vaccination with Oxy(Gly)4-sKLH had no effect on methadone-induced antinociception, respiratory depression, or bradycardia (Figure 2D–2F). Vaccination also did not affect naloxone’s ability to reverse methadone-induced antinociception, respiratory depression, or bradycardia (Figure 2D–2F). However, there was a significant difference between sKLH and Oxy(Gly)4-sKLH vaccinated rats following naloxone administration during antinociception and respiratory depression (t(10.4)=3.12, p<0.05 and t(11.3)=2.69, p<0.05, respectively) (Figure 2D and 2E). Vaccination with Oxy(Gly)4-sKLH had no effect on fentanyl-induced antinociception or brain fentanyl levels compared to control (Figure 3A and 3B).

Figure 2. Oxy(Gly)4-sKLH does not interfere with methadone.

Figure 2.

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Rats (n=10/group) were immunized 4 times every 3 weeks with Oxy(Gly)4-sKLH or sKLH. One week after the final vaccination, rats were challenged with increasing doses of either oxycodone or methadone (up to a total dose of 9 mg/kg) every 17 min. Fifteen min after drug administration, rats were placed on the hotplate to test for antinociception and then placed in a chamber with a collar placed around the rats neck to measure oxygen saturation (%SaO2). Immediately following the final test, rats received a 0.1 mg/kg dose of naloxone s.c. and were tested 15 min later on the hotplate and oximeter. Latency to respond to noxious stimuli (A, C), oxygen saturation (B, E), and heart rate (C, F) are shown following oxycodone or methadone dosing, respectively. Measurements were expressed as mean ± SD. * p<0.05 compared to sKLH. ## p<0.01 and ### p<0.001 for the difference (within a single group) between values measured after the highest opioid dose and after naloxone.

Figure 3. Effect of vaccination with Oxy(Gly)4-sKLH on fentanyl-induced antinociception and fentanyl distribution in tissues.

Figure 3.

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Rats (n=10) were vaccinated with 60 μg OXY(Gly)4-sKLH adsorbed to 90 μg aluminum i.m. once every three weeks for a total of 4 vaccinations. Rats received 0.05 mg/kg fentanyl s.c. 30 min later rats were tested on the hotplate and brain collected immediately after testing. No differences were detected between groups. Data are expressed as mean ± SD.

3.5. Experiment 4: Effect of vaccination on half-life of oxycodone in serum.

Oxycodone-specific IgG antibody titers in sKLH and Oxy(Gly)4-sKLH vaccinated rats were 0.4 ± 0.5 x 103 and 167 ± 61 x 103 (mean ± SD), respectively. In the Oxy(Gly)4-sKLH group, male and female rats displayed oxycodone-specific antibody titers of 121 ± 37 x 103 and 212 ± 42 x 103 (mean ± SD), respectively, which were significantly different (t(8)=3.618, p<0.01). The percent of bound oxycodone at 5 hr post-dosing was 77% in the sKLH control group compared to 99% in Oxy(Gly)4-sKLH rats.

The elimination phase half-life of oxycodone following a 5 mg/kg i.v. infusion over 10 min in rats vaccinated with sKLH and Oxy(Gly)4-sKLH was 0.65 ± 0.14 hr and 25.4 ± 30.0 hr (mean ± SD) (Figure 4A), respectively, which was significantly different (t(8)=2.476, p<0.05). Male and female rats vaccinated with sKLH had elimination phase half-lives of oxycodone of 0.61 ± 0.04 hr and 0.68 ± 0.19 hr (mean ± SD), respectively, which did not differ (Figure 4B). Male and female rats vaccinated with Oxy(Gly)4-sKLH displayed elimination phase half-lives of oxycodone of 15.0 ± 13.2 hr and 38.5 ± 42.0 hr (mean ± SD), respectively, which also did not differ. Other estimates for pharmacokinetic parameters in combined or separated male and female rats are shown in Table 2.

Figure 4. Effect of vaccination with Oxy(Gly)4-sKLH on oxycodone half-life in serum.

Figure 4.

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Rats (n=10/group, 5 male and 5 female per group) were vaccinated i.m. with either 60 ug sKLH or Oxy(Gly)4-sKLH in 90 ug aluminum on days 0, 21, and 42, and then challenged with an intravenous bolus dose of 5 mg/kg oxycodone administered over a 10-minute infusion. After the oxycodone infusion, serum oxycodone was monitored for up to 24 hr by multiple blood collection via jugular indwelling catheters. Male and female rat data was shown (A) combined and (B) separated. Serum oxycodone was expressed as mean ± SD.

Table 2.

Pharmacokinetic parameters of oxycodone in sKLH and Oxy(Gly)4-sKLH vaccinated rats following a 10 minute infusion of 5 mg/kg oxycodone.

sKLH Oxy(Gly)4-sKLH
Parameter Male Female Combined Male Female Combined
T1/2α (h)
 Mean ± SD 0.73±0.32 0.57±0.16   0.65±0.25
 Range  0.5-1.2 0.5-0.8   0.5-1.2
T 1/2 terminal (h)
 Mean ± SD 0.61±0.04 0.68±0.19 0.65±0.14* 15.0±13.2 38.5±42.0   25.4±30.0
 Range 0.57-0.67 0.52-1.01 0.52-1.01 4.4-30.2 5.0-96.9   4.4-96.9
AUC0-inf. (h * ug/mL)
 Mean ± SD 2.1±0.4 2.8±1.5* 2.5±1.1* 17.9±8.0 42.6±33.9   28.9±25.1
 Range 1.5-2.4 1.8-5.5 1.5-5.5 9.3-26.1 15.5-91.4   9.3-91.4
C0 (ng/mL)
 Mean ± SD 2.3±0.4* 3.2±2.3 2.8±1.6** 8.4±6.2 11.1±3.5   9.6±5.1
 Range 1.7-2.8 1.9-7.3 1.7-7.3 3.7-18.8 8.0-16.0   3.7-18.8
MRT0-inf (h)
 Mean ± SD 0.9±0.1 1.0±0.3 0.9±0.2 13.7±13.0 47.7±57.3   28.8±40.5
 Range 0.8-1.0 0.7-1.5 0.7-1.5 3.4-33.8 4.0-128.2   3.4-128.2
CI total (ml/min * kg)
 Mean ± SD 41.6±8.8* 34.4±12.6*** 38.0±10.9*** 11.0±6.5 9.1±1.9   10.1±4.8
 Range 34.9-55.7 15.3-47.1 15.0-55.7 3.2-18.6 7.2-11.4   3.2-18.6
Vss (L/kg)
 Mean ± SD 2.2±0.4 2.0±0.8 2.1±0.6 4.5±4.1 4.1±2.9   4.3±3.4
 Range 1.8-2.9 0.7-2.6 0.7-2.9 0.8-10.4 1.3-7.0   0.8-10.4
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*,

p<0.05;

**,

p<0.01;

***,

p<0.001 compared to Oxy(Gly)4-sKLH vaccinated rats.

Data are expressed as mean ± SD.

3.6. Experiment 5: Effect of vaccination on maintenance of oxycodone self-administration (OSA).

Oxycodone-specific IgG antibody titers in aluminum and Oxy(Gly)4-sKLH vaccinated rats were 3.6 ± 4.2 x 103 and 581 ± 260 x 103 (mean ± SD), respectively. In the Oxy(Gly)4-sKLH group, male and female rats had oxycodone-specific antibody titers of 591 ± 284 x 103 and 553 ± 233 x 103 (mean ± SD), respectively, which were not significantly different (t(4.5)=0.229, p=0.83).

Figure 5A shows the mean number of oxycodone infusions during the last 3 days of each week during the initial 4 vaccinations, expressed as a percentage of baseline. The baseline number of infusions was 36.9 ± 3.5 and 38.7 ± 4.1 (mean ± SEM) for rats given Oxy(Gly)4-sKLH or aluminum, respectively (see also Figure S1 for baseline active and inactive responding). During this phase, there was a main effect of vaccination (F=4.56, p<0.05) and time (F=5.70, p<0.001), while the vaccination x time interaction approached significance (F=1.70, p=0.1). The Oxy(Gly)4-sKLH group, but not the aluminum group, exhibited a significant compensatory increase in mean infusions over this initial course of vaccination (slope = 5.41, F=13.05, p<0.001). Figure 5B shows the mean number of infusions at each unit dose for rats that completed the dose-reduction phase (n=10 and 12 for Oxy(Gly)4-sKLH and aluminum, respectively). The baseline for this figure (100%) represents the self-administration rate for each group on the final week at the 0.06 mg/kg oxycodone unit dose. There was a main effect of vaccination (F=4.84, p<0.05) and dose (F=16.68, p<0.0001), and a significant vaccination x dose interaction (F=2.52, p<0.05). Overall, OSA was significantly lower in the Oxy(Gly)4-sKLH group compared to aluminum. Only the 0.06 mg/kg training dose maintained OSA infusion rates above saline in the Oxy(Gly)4-sKLH group, whereas all unit doses except 0.0019 mg/kg/infusion maintained infusion rates above saline in the aluminum group. A significantly higher percentage of rats exhibited infusion rats below baseline (data not shown) in the Oxy(Gly)4-sKLH group compared to aluminum at the 0.0075 and 0.0038 mg/kg unit doses (70% vs 0%, p=0.0007; and 90% vs 33%, p=0.0115; respectively). Finally, the α parameter from exponential demand curves (Figure 5C and Table 3) was significantly higher (i.e. consumption was more elastic and sensitive to unit price) in the Oxy(Gly)4-sKLH group (α=10.48e-006) compared to aluminum (α=6.09e-006, t=2.45, p<0.05). The Q0 parameter was significantly higher in the Oxy(Gly)4-sKLH group compared to aluminum control (t=2.44, p<0.05). Pmax and Omax, were significantly lower in the Oxy(Gly)4-sKLH compared to aluminum (t=4.28 and 2.6, p<0.001 and p<0.05, respectively). See Figure S4 in Supplementary Materials for absolute values of the data in Figure 5.

Figure 5. Effect of Oxy(Gly)4-sKLH vaccination on maintenance of oxycodone self-administration.

Figure 5.

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Oxycodone mean (± SEM) infusions expressed as a percentage of baseline (B) during the initial vaccination phase (panel A) and during the unit dose reduction phase (panel B). Exponential demand curve fits to log mean (±SEM) oxycodone consumption expressed as a percentage of baseline during the dose-reduction phase (panel C). Data at the 0.06 mg/kg dose in Panel B is the baseline prior to dose reduction. Cumulative mean (±SEM) scores of somatic signs throughout entire oxycodone self-administration protocol (panel D). Arrows indicate when vaccinations occurred. Panel A: Different from baseline, *p < 0.05, ^p < 0.06. Panel B: Different from saline, Oxy(Gly)4-sKLH ++p < 0.01, aluminum **p < 0.01, ***p < 0.001. Panel C: Different from aluminum p < 0.05.

Table 3.

Exponential demand curve parameters (95% CI) from curves fits in Figure 5C. Range constant k = 2.643.

Parameter Aluminum (control) Oxy(Gly)4-sKLH
α 6.5e-006 (± 0.69e-006) 11.8e-006 (± 2.0e-006)*
Q0 130.6 (± 6.9) 221.1 (± 36.4)*
Pmax 274.5 (± 25.6) 117.8 (± 23.5)***
Omax 11,747 (± 1,292) 7,237 (± 1,149)
R2 0.96 (± 0.01) 0.92 (± 0.02)
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Different from aluminum

**

p < 0.01

***

p < 0.001.

During the OSA study rats were observed for gross signs of oxycodone toxicity (Table 1). Individual signs overall were only minimally elevated, the greatest increase being in “Pica” but there were no differences between vaccine and control groups in any cumulative or individual score (Figure 5D and S3, respectively).

No differences in gene expression were detected between mu, delta, or kappa opioid receptors in the self-administration study between aluminum and Oxy(Gly)4-sKLH vaccinated rats (Figure S4). There was a significant increase (p<0.05) in glycoprotein hormone alpha chain, thyrotropin subunit beta, and NADH dehydrogenase, and a significant decrease (p<0.05) in RNA-binding protein 1 in the Oxy(Gly)4-sKLH group compared to controls. Non-vaccinated compared to vaccinated male rats showed significant differences in 11 genes, while non-vaccinated compared to vaccinated female rats showed significant differences in 120 genes.

4.0. Discussion

The main findings from this study were: 1) oxycodone exposure does not boost antibody levels in vaccinated rats; 2) antibodies persist for 2-3 months following vaccination; 3) vaccination does not alter the effects of methadone, fentanyl, or naloxone; 4) vaccination alters the pharmacokinetic parameters of oxycodone; 5) vaccination reduced the reinforcing efficacy of oxycodone in a self-administration model; and 6) vaccination does not elicit signs of toxicity. These data highlight the in vivo characteristics of Oxy(Gly)4-sKLH and support its use in clinical studies.

Exposure to oxycodone did not boost oxycodone-specific antibody production in vaccinated rats, confirming that unconjugated (free) oxycodone itself does not act as an antigen able to stimulate adaptive immunity. Although opioids, in the absence of vaccination, may elicit low levels of IgM and IgE anti-opioid antibodies (Biagini et al., 1990; Gamaleya et al., 1993), small molecules are not effective in triggering adaptive immune responses (Dintzis et al., 1976). Furthermore, pre-clinical studies have demonstrated that vaccination against drugs of abuse requires use of immunogenic carriers to trigger CD4+ T cell-dependent B cell activation and that intact T cell receptor signaling is required for production of antibodies as shown by either depletion of either CD4+ T cells or use of TCR-deficient mice and by use of oxycodone-based haptens conjugated to T cell-independent carriers such as dextran or ficoll (Baruffaldi et al., 2018; Laudenbach et al., 2015). Other studies have shown that antibody levels in vaccinated rats and non-human primates increased following vaccine boosts, but not during opioid self-administration (Nguyen et al., 2018; Tenney et al., 2019). Secondarily, antibodies persisted for 2-3 months following the final vaccination, which is consistent with findings using other conjugate vaccines in rodents and humans (Anton and Leff, 2006; Hatsukami et al., 2005; Maurer, 2005). Finally, peak oxycodone-specific antibody titers were consistent across experiments, except following OSA (Experiment 5), and equivalent or slightly higher to titers in previous studies using similar vaccine formulations (Baruffaldi et al., 2019; Raleigh et al., 2018; Raleigh et al., 2017). Titers in Experiment 5 were much higher than in the other experiments. However, this was likely due to antibody titers from Experiment 5 being measured at another site (University of Minnesota) which utilized different lots of materials. Taken together, these data confirm that immunization with Oxy(Gly)4-sKLH is required for eliciting antibody titers, whereas exposure to the targeted opioids is not immunogenic per se, and that antibody titers will decay slowly after vaccination is ceased.

Vaccination with Oxy(Gly)4-sKLH selectively reduced oxycodone, but not methadone, effects in vivo, confirming both current and previous in vitro analysis of oxycodone-specific antibodies (Raleigh et al., 2017). Importantly, vaccination with Oxy(Gly)4-sKLH did not hinder naloxone’s ability to reverse methadone-induced respiratory depression, demonstrating that the life-saving effects of naloxone remain intact. However, while naloxone was able to completely reverse oxycodone-induced antinociception, naloxone could not completely reverse methadone-induced antinociception. One possible explanation for this finding is that methadone has 7.5 times higher affinity towards opioid receptors than oxycodone (Volpe et al., 2011), which suggests that higher naloxone doses may be required to fully reverse the antinociceptive effects of methadone compared to oxycodone.

Fentanyl-induced antinociception was also preserved in Oxy(Gly)4-sKLH vaccinated rats in the current study. Cross-reactivity was not expected due to the different structures of oxycodone (a phenanthrene) and fentanyl (a phenylpiperidine), as well as in vitro observations from another oxycodone vaccine (Kimishima et al., 2017). Since fentanyl could be used for emergency pain management (Farahmand et al., 2014; Vahedi et al., 2019), it is advantageous to preserve its antinociceptive effects in vaccinated individuals.

Vaccination with Oxy(Gly)4-sKLH greatly altered the pharmacokinetics of oxycodone. The increase in half-life suggests that oxycodone is held tightly in the serum compartment of immunized animals. In another oxycodone vaccine study, oxycodone’s half-life was extended from 0.2 hr in controls to 12 hr in vaccinated mice (Kimishima et al., 2017). One potential concern that will need to be monitored in any clinical trials of this vaccine is that, if the half-life of oxycodone is extended too long, antibody saturation could occur should an individual consume more drug before previous doses have been cleared, reducing vaccine efficacy during that time. Another concern would be that oxycodone:antibody complexes may trigger rare immune-related side effects related to activation of antibody-mediated effector functions involving complement, or phagocytic cells via Fc gamma receptors. However, no evidence of side effects was observed in the OSA study, which involved between 110 – 138 days of oxycodone exposure in vaccinated rats, nor in a recently completed GLP toxicology study of Oxy(Gly)4-sKLH in rats concurrently exposed to oxycodone (data not shown, personal communication).

Immunization also markedly increased the area under the curve (AUC) of oxycodone compared to controls. Although increases in AUC suggest greater drug exposure, most of the oxycodone in this study was bound by antibody. A similar finding regarding AUC was seen with another oxycodone vaccine (Kimishima et al., 2017). Finally, clearance was greatly reduced in vaccinated rats, which is likely what contributed to the change in oxycodone’s half-life the most. Taken together, these data suggest that vaccination with Oxy(Gly)4-sKLH drastically altered the pharmacokinetics of oxycodone.

In a self-administration model, oxycodone intake increased at the 0.06 mg/kg maintenance dose relative to pre-vaccination intake but decreased to a greater extent at lower unit doses in Oxy(Gly)4-sKLH rats compared to control rats. This rightward shift in the unit dose response curve indicates that Oxy(Gly)4-sKLH reduced the potency of oxycodone (i.e. effectively lowering the oxycodone dose). The higher baseline intensity of demand (Q0) in Oxy(Gly)4-sKLH rats is also consistent with this interpretation. A similar shift in potency and increase in Q0 was also previously reported for an analogous heroin vaccine in a heroin self-administration model (Raleigh et al., 2014). Because the 0.06 mg/kg oxycodone dose lies on the descending limb of the oxycodone self-administration (OSA) dose-response curve, an increase in OSA at this unit dose indicates a reduction in the reinforcing effects of oxycodone similar to what would occur if the unit dose was decreased. In addition, the increase in elasticity of demand and sensitivity to unit price (α and Pmax) and decrease in maximum effort (Omax) suggests that vaccination decreased the reinforcing efficacy or motivational strength of oxycodone, in addition to decreasing its potency. This finding differs from those of the heroin vaccine mentioned above, which decreased the potency (increased Q0) but not reinforcing efficacy (α) of heroin. Even though α is thought to provide a measure of reinforcing efficacy that is independent of drug potency (Hursh and Roma, 2013), some studies have shown this isn’t necessarily the case (Kearns and Silberberg, 2016). Further studies are needed using different reinforcement schedules, training doses, and means of manipulating unit price to better assess whether vaccination with Oxy(Gly)4-sKLH decreases oxycodone’s reinforcing efficacy per se. Regardless, the present findings suggest that Oxy(Gly)4-sKLH may be effective at facilitating abstinence from oxycodone in humans by attenuating its reinforcing effects.

Previous studies of similar oxycodone vaccine formulations showed that prophylactic vaccination of rats prior to exposure to oxycodone reduced the acquisition of (Pravetoni et al., 2014) and maintenance (Nguyen et al., 2018) of OSA in rats. The present study demonstrates that therapeutic vaccination can reduce the reinforcing effects of oxycodone when vaccination occurs in rats that have already acquired stable drug intake. This is a notable extension of previous studies because it models more closely how vaccination would occur in clinical trials, where patients will already be well-established oxycodone users who continue using the drug while they are vaccinated prior to a target quit date. However, the present finding of increased oxycodone intake at the training dose during vaccination suggests that a compensatory increase in oxycodone use could occur in humans during the vaccination period prior to quitting. However, there was no evidence of toxicity in the vaccinated group, despite the compensatory increase in oxycodone intake.

No changes in opioid receptor gene expression were noted in the self-administration study, similar to previous findings in a self-administration study using a similar Oxy(Gly)4-sKLH formulation (Pravetoni et al., 2014). However, glycoprotein hormones are often grouped with opioid receptors (Abbadie and Pasternak, 2001; Jiang et al., 2014) and lower levels of gene expression were seen in controls. Further study is needed to clarify this finding. Lower NADH dehydrogenase levels in control rats could be a sign of impaired glucose metabolism and withdrawal (Jiang et al., 2007). The difference in the number of genes altered in females (120) compared to males (11) suggests a sex effect, but it is not clear whether this effect is due to vaccination or due to opioids. While these results are quite exploratory, they provide some insight into how vaccination may protect against changes in gene expression in the brain.

While this study tested the efficacy of an oxycodone vaccine in male and female rats, one limitation is that sample sizes were not powered to detect sex differences. Indeed, only Experiment 4 (oxycodone pharmacokinetics) and Experiment 5 (OSA) used both sexes, as these experiments were the most critical to demonstrate evidence of differences between sexes. While sex differences were noted with antibody titers in Experiment 4, there were no differences detected in Experiment 5 despite equal number of vaccinations, suggesting that differences are unlikely to be meaningful clinically. Sex differences in vaccines against substance use disorders have not been widely studied to date, although some literature reports have shown comparisons of male and female mice immunized with a heroin vaccine (Hwang et al., 2019), fentanyl vaccine (Crouse et al., 2020), and a cocaine vaccine (Kosten et al., 2014).

Overall, these data contribute to the understanding of the in vivo characteristics of vaccination with Oxy(Gly)4-sKLH in rats. The combined data support previous preclinical data that demonstrate that Oxy(Gly)4-sKLH elicits long lasting protection against oxycodone and reduces the reinforcing efficacy of oxycodone. Future studies of the antibody response elicited by Oxy(Gly)4-sKLH will focus on clinical studies to assess how well these findings translate to humans.

Supplementary Material

Supplementary Material

Highlights.

  • Opioid use disorder is a worldwide problem and better treatments are needed

  • This study characterizes an oxycodone vaccine currently undergoing clinical testing

  • Exposure to oxycodone does not elicit antibody production in vaccinated animals

  • Vaccine blunts oxycodone, but not methadone, fentanyl, or naloxone effects

  • Vaccine alters oxycodone self-administration; no opioid-induced toxicity observed

6.0. Acknowledgements

The authors thank Juan Abrahante Lloréns for assistance with analyzing and interpreting gene expression data and Jennifer Vigliaturo and April Huseby for technical support.

5.0. Funding and Disclosure

This work was supported by the National Institutes of Health National Institute on Drug Abuse [Grants U01DA038876 (M.P. and P.R.P and UG3DA047711 (M.P.)]. S.W. received compensation as an independent consultant on this study and is the sole proprietor of Winston Biopharmaceutical Consulting. The authors declare no conflicts of interest.

Footnotes

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