Therapeutic and Prophylactic Vaccines to Counteract Fentanyl Use Disorders and Toxicity.

The incidence of fatal overdoses has increased worldwide due to the widespread access to illicit fentanyl and its potent analogues. Vaccines offer a promising strategy to reduce the prevalence of opioid use disorders (OUDs) and to prevent toxicity from accidental and deliberate exposure to fentanyl and its derivatives. This study describes the development and characterization of vaccine formulations consisting of novel fentanyl-based haptens conjugated to carrier proteins. Vaccine efficacy was tested against opioid-induced behavior and toxicity in mice and rats challenged with fentanyl and its analogues. Prophylactic vaccination reduced fentanyl- and sufentanil-induced antinociception, respiratory depression, and bradycardia in mice and rats. Therapeutic vaccination also reduced fentanyl intravenous self-administration in rats. Because of their selectivity, vaccines did not interfere with the pharmacological effects of commonly used anesthetics nor with methadone, naloxone, oxycodone, or heroin. These preclinical data support the translation of vaccines as a viable strategy to counteract fentanyl use disorders and toxicity.

Polymer-mediated delivery of vaccines to treat opioid use disorders and to reduce opioid-induced toxicity.

Vaccines offer a potential strategy to treat opioid use disorders (OUD) and to reduce the incidence of opioid-related overdoses. Vaccines induce opioid-specific polyclonal antibodies that selectively and effectively bind the target opioid and prevent its distribution across the blood-brain barrier. Because antibody-mediated reduction of drug distribution to the brain reduces drug-induced behavior and toxicity, vaccine efficacy depends on the quantity and quality of the antibody response. This study tested whether polymer-mediated delivery could improve vaccine efficacy against opioids as well as eliminate the need for booster injections normally required for a successful immunization. A series of novel biodegradable biocompatible thermogelling pentablock co-polymers were used to formulate a candidate vaccine against oxycodone in mice and rats. Polymer-based delivery of the anti-oxycodone vaccine was equally or more effective than administration in aluminum adjuvant in generating oxycodone-specific antibodies and in reducing oxycodone-induced effects and oxycodone distribution to the brain in mice and rats. The composition and release kinetics of the polymer formulations determined vaccine efficacy. Specifically, a formulation consisting of three simultaneous injections of the anti-oxycodone vaccine formulated in three different polymers with slow, intermediate, and fast release kinetics was more effective than an immunization regimen consisting of three sequential injections with the vaccine adsorbed on aluminum. The novel three-phased polymer vaccine formulation was effective in blocking oxycodone-induced antinociception, respiratory depression and bradycardia in rats.

Interruption of continuous opioid exposure exacerbates drug-evoked adaptations in the mesolimbic dopamine system.

Drug-evoked adaptations in the mesolimbic dopamine system are postulated to drive opioid abuse and addiction. These adaptations vary in magnitude and direction following different patterns of opioid exposure, but few studies have systematically manipulated the pattern of opioid administration while measuring neurobiological and behavioral impact. We exposed male and female mice to morphine for one week, with administration patterns that were either intermittent (daily injections) or continuous (osmotic minipump infusion). We then interrupted continuous morphine exposure with either naloxone-precipitated or spontaneous withdrawal. Continuous morphine exposure caused tolerance to the psychomotor-activating effects of morphine, whereas both intermittent and interrupted morphine exposure caused long-lasting psychomotor sensitization. Given links between locomotor sensitization and mesolimbic dopamine signaling, we used fiber photometry and a genetically encoded dopamine sensor to conduct longitudinal measurements of dopamine dynamics in the nucleus accumbens. Locomotor sensitization caused by interrupted morphine exposure was accompanied by enhanced dopamine signaling in the nucleus accumbens. To further assess downstream consequences on striatal gene expression, we used next-generation RNA sequencing to perform genome-wide transcriptional profiling in the nucleus accumbens and dorsal striatum. The interruption of continuous morphine exposure exacerbated drug-evoked transcriptional changes in both nucleus accumbens and dorsal striatum, dramatically increasing differential gene expression and engaging unique signaling pathways. Our study indicates that opioid-evoked adaptations in brain function and behavior are critically dependent on the pattern of drug administration, and exacerbated by interruption of continuous exposure. Maintaining continuity of chronic opioid administration may, therefore, represent a strategy to minimize iatrogenic effects on brain reward circuits.

Sex differences in oral oxycodone self-administration and stress-primed reinstatement in rats.

The opioid epidemic has become a severe public health problem, with approximately 130 opioid-induced deaths occurring each day in the United States. Prescription opioids are responsible for approximately 40% of these deaths. Oxycodone is one of the most commonly abused prescription opioids, but despite its prevalent misuse, the number of preclinical studies investigating oxycodone-seeking behaviors is relatively limited. Furthermore, preclinical oxycodone studies that include female subjects are even more scarce, and it is critical that future work includes both sexes. Additionally, the oral route of administration is one of the most common routes for recreational users, especially in the early stages of drug experimentation. However, currently, only two studies have been published investigating operant oral oxycodone self-administration in rodents. Therefore, the primary goal of the present study was to establish an oral oxycodone operant self-administration model in adult male and female rats, as well as to examine a potential mechanism of stress-primed reinstatement. We found that females consumed significantly more oral oxycodone than males in operant self-administration sessions. We also found that active oxycodone self-administration was reduced by mu opioid receptor antagonism and by substitution of water for oxycodone solution. Lastly, we induced stress-primed reinstatement and found that this behavior was significantly attenuated by antagonism of the neurokinin-1 receptor, consistent with our prior work examining stress-induced reinstatement of alcohol- and cocaine-seeking.

A Fentanyl Vaccine Alters Fentanyl Distribution and Protects against Fentanyl-Induced Effects in Mice and Rats.

Fentanyl is an extremely potent synthetic opioid that has been increasingly used to adulterate heroin, cocaine, and counterfeit prescription pills, leading to an increase in opioid-induced fatal overdoses in the United States, Canada, and Europe. A vaccine targeting fentanyl could offer protection against the toxic effects of fentanyl in both recreational drug users and others in professions at risk of accidental exposure. This study focuses on the development of a vaccine consisting of a fentanyl-based hapten (F) conjugated to keyhole limpet hemocyanin (KLH) carrier protein or to GMP-grade subunit KLH (sKLH). Immunization with F-KLH in mice and rats reduced fentanyl-induced hotplate antinociception, and in rats reduced fentanyl distribution to the brain compared with controls. F-KLH did not reduce the antinociceptive effects of equianalgesic doses of heroin or oxycodone in rats. To assess the vaccine effect on fentanyl toxicity, rats immunized with F-sKLH or unconjugated sKLH were exposed to increasing subcutaneous doses of fentanyl. Vaccination with F-sKLH shifted the dose-response curves to the right for both fentanyl-induced antinociception and respiratory depression. Naloxone reversed fentanyl effects in both groups, showing that its ability to reverse respiratory depression was preserved. These data demonstrate preclinical selectivity and efficacy of a fentanyl vaccine and suggest that vaccines may offer a therapeutic option in reducing fentanyl-induced side effects.

Preclinical Efficacy and Characterization of Candidate Vaccines for Treatment of Opioid Use Disorders Using Clinically Viable Carrier Proteins.

Vaccines may offer a new treatment strategy for opioid use disorders and opioid-related overdoses. To speed translation, this study evaluates opioid conjugate vaccines containing components suitable for pharmaceutical manufacturing and compares analytical assays for conjugate characterization. Three oxycodone-based haptens (OXY) containing either PEGylated or tetraglycine [(Gly)4] linkers were conjugated to a keyhole limpet hemocyanin (KLH) carrier protein via carbodiimide (EDAC) or maleimide chemistry. The EDAC-conjugated OXY(Gly)4-KLH was most effective in reducing oxycodone distribution to the brain in mice. Vaccine efficacy was T cell-dependent. The lead OXY hapten was conjugated to the KLH, tetanus toxoid, diphtheria cross-reactive material (CRM), as well as the E. coli-expressed CRM (EcoCRM) and nontoxic tetanus toxin heavy chain fragment C (rTTHc) carrier proteins. All vaccines induced early hapten-specific B cell expansion and showed equivalent efficacy against oxycodone in mice. However, some hapten-protein conjugates were easier to characterize for molecular weight and size. Finally, heroin vaccines formulated with either EcoCRM or KLH were equally effective in reducing heroin-induced antinociception and distribution to the brain of heroin and its metabolites in mice. This study identifies vaccine candidates and vaccine components for further development.

Blocking interleukin-4 enhances efficacy of vaccines for treatment of opioid abuse and prevention of opioid overdose.

Vaccines offer an option to treat heroin and prescription opioid abuse and prevent fatal overdoses. Opioid vaccines elicit antibodies that block opioid distribution to the brain and reduce opioid-induced behavioral effects and toxicity. The major limitation to the translation of addiction vaccines is that efficacy is observed only in subjects achieving optimal drug-specific serum antibody levels. This study tested whether efficacy of a vaccine against oxycodone is increased by immunomodulators targeting key cytokine signaling pathways involved in B and T cell lymphocyte activation. Blockage of IL-4 signaling increased vaccine efficacy in blocking oxycodone distribution to the brain and protection against opioid-induced behavior and toxicity in mice. This strategy generalized to a peptide-protein conjugate immunogen, and a tetanus-diphtheria-pertussis vaccine. These data demonstrate that cytokine-based immunomodulators increase efficacy of vaccines against small molecules, peptides and proteins, and identify IL-4 as a pharmacological target for improving efficacy of next-generation vaccines.

Opioid Dose- and Route-Dependent Efficacy of Oxycodone and Heroin Vaccines in Rats.

Heroin and oxycodone abuse occurs over a wide range of drug doses and by various routes of administration characterized by differing rates of drug absorption. The current study addressed the efficacy of a heroin vaccine [morphine hapten conjugated to keyhole limpet hemocyanin (M-KLH)] or oxycodone vaccine [oxycodone hapten conjugated to keyhole limpet hemocyanin (OXY-KLH)] for reducing drug distribution to brain after intravenous heroin or oxycodone, or subcutaneous oxycodone. Rats immunized with M-KLH or keyhole limpet hemocyanin (KLH) control received an intravenous bolus dose of 0.26 or 2.6 mg/kg heroin. Vaccination with M-KLH increased retention of heroin and its active metabolites 6-acetylmorphine (6-AM) and morphine in plasma compared with KLH controls, and reduced total opioid (heroin + 6-AM + morphine) distribution to brain but only at the lower heroin dose. Immunization also protected against respiratory depression at the lower heroin dose. Rats immunized with OXY-KLH or KLH control received 0.22 or 2.2 mg/kg oxycodone intravenously, the molar equivalent of the heroin doses. Immunization with OXY-KLH significantly reduced oxycodone distribution to brain after either oxycodone dose, although the magnitude of effect of immunization at the higher oxycodone dose was small (12%). By contrast, vaccination with OXY-KLH was more effective when oxycodone was administered subcutaneously rather than intravenously, reducing oxycodone distribution to brain by 44% after an oxycodone dose of 2.3 mg/kg. Vaccination also reduced oxycodone-induced antinociception. These data suggest that the efficacy of OXY-KLH and M-KLH opioid vaccines is highly dependent upon opioid dose and route of administration.

The frequency of naive and early-activated hapten-specific B cell subsets dictates the efficacy of a therapeutic vaccine against prescription opioid abuse.

Translation of therapeutic vaccines for addiction, cancer, or other chronic noncommunicable diseases has been slow because only a small subset of immunized subjects achieved effective Ab levels. We hypothesize that individual variability in the number of naive and early-activated hapten-specific B cells determines postvaccination serum Ab levels and vaccine efficacy. Using a model vaccine against the highly abused prescription opioid oxycodone, the polyclonal B cell population specific for an oxycodone-based hapten (6OXY) was analyzed by flow cytometry paired with Ag-based magnetic enrichment. A higher frequency of 6OXY-specific B cells in either spleen biopsies or blood, before and after immunization, correlated to subsequent greater oxycodone-specific serum Ab titers and their efficacy in blocking oxycodone distribution to the brain and oxycodone-induced behavior in mice. The magnitude of 6OXY-specific B cell activation and vaccine efficacy was tightly correlated to the size of the CD4(+) T cell population. The frequency of enriched 6OXY-specific B cells was consistent across various mouse tissues. These data provide novel evidence that variations in the frequency of naive or early-activated vaccine-specific B and T cells can account for individual responses to vaccines and may predict the clinical efficacy of a therapeutic vaccine.