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.

Effect of Adjuvant Release Rate on the Immunogenicity of Nanoparticle-Based Vaccines: A Case Study with a Nanoparticle-Based Nicotine Vaccine.

Adjuvants are a critical component for vaccines, especially for a poorly immunogenic antigen, such as nicotine. However, the impact of adjuvant release rate from a vaccine formulation on its immunogenicity has not been well illustrated. In this study, we fabricated a series of hybrid-nanoparticle-based nicotine vaccines to study the impact of adjuvant release rate on their immunological efficacy. It was found that the nanovaccine with a medium or slow adjuvant release rate induced a significantly higher anti-nicotine antibody titer than that with a fast release rate. Furthermore, the medium and slow adjuvant release rates resulted in a significantly lower brain nicotine concentration than the fast release rate after nicotine challenge. All findings suggest that adjuvant release rate affects the immunological efficacy of nanoparticle-based nicotine vaccines, providing a potential strategy to rationally designing vaccine formulations against psychoactive drugs or even other antigens. The hybrid-nanoparticle-based nicotine vaccine with an optimized adjuvant release rate can be a promising next-generation immunotherapeutic candidate against nicotine.

Alum as an adjuvant for nanoparticle based vaccines: A case study with a hybrid nanoparticle-based nicotine vaccine.

The treatment efficacy of a nicotine vaccine largely relies on its ability to induce high titers of nicotine-specific antibodies. Due to its strong immune-potentiating effects, aluminum salt (Alum) has been commonly used as an adjuvant in various nicotine vaccine formulations. In this study, we attempted to improve the immunological performance of a hybrid nanoparticle-based nicotine vaccine (NanoNicVac) by co-administering it with Alum. It was found that Alum severely restricted the release of NanoNicVac at the site of injection. Moreover, Alum damaged the hybrid structure of the vaccine. In the animal trial, mice immunized with NanoNicVac alone achieved an anti-nicotine IgG titer of 3.5 ±â€¯0.2 × 104 after three injections. Unexpectedly, Alum with quantities of 125, 250, 500, and 1000 µg did not enhance the immunogenicity of NanoNicVac. In addition, Alum did not improve the ability of the vaccine to reduce the entry of nicotine into the brain.

Hybrid nanoparticle-based nicotine nanovaccines: Boosting the immunological efficacy by conjugation of potent carrier proteins.

A series of hybrid nanoparticle-based nicotine nanovaccines (NanoNicVac) were engineered in this work by conjugating potent carrier protein candidates (Keyhole limpet hemocyanin (KLH) multimer, KLH subunit, cross-reactive material 197 (CRM197), or tetanus toxoid (TT)) for enhanced immunological efficacy. NanoNicVac with CRM197 or TT were processed by dendritic cells more efficiently than that with KLH multimer or subunit. NanoNicVac carrying CRM197 or TT exhibited a significantly higher immunogenicity against nicotine and a considerably lower immunogenicity against carrier proteins than NanoNicVac carrying KLH multimer or subunit in mice. The in vivo results revealed that NanoNicVac with CRM197 or TT resulted in lower levels of nicotine in the brain of mice after nicotine challenge. All findings suggest that an enhanced immunological efficacy of NanoNicVac can be achieved by using CRM197 or TT instead of KLH or KLH subunit as carrier proteins, making NanoNicVac a promising next-generation immunotherapeutic candidate against nicotine addiction.

Paradox of PEGylation in fabricating hybrid nanoparticle-based nicotine vaccines.

Polyethylene glycol (PEG) has long been used in nanoparticle-based drug or vaccine delivery platforms. In this study, nano-nicotine vaccines (NanoNicVac) were PEGylated to different degrees to investigate the impact of PEG on the immunological efficacy of the vaccine. Hybrid nanoparticles with various degrees of PEGylation (2.5%-30%) were assembled. It was found that 30% PEGylation resulted in a hybrid nanoparticle of a compromised core-shell structure. A higher concentration of PEG also led to a slower cellular uptake of hybrid nanoparticles by dendritic cells. However, increasing the quantity of the PEG could effectively reduce nanoparticle aggregation during storage and improve the stability of the hybrid nanoparticles. Subsequently, nicotine vaccines were synthesized by conjugating nicotine haptens to the differently PEGylated hybrid nanoparticles. In both in vitro and in vivo studies, it was found that a nicotine vaccine with 20% PEGylation (NanoNicVac 20.0) was significantly more stable than the vaccines with lower PEGylation. In addition, NanoNicVac 20.0 induced a significantly higher anti-nicotine antibody titer of 3.7 ±â€¯0.6 × 104 in mice than the other NanoNicVacs with lower concentrations of PEG. In a subsequent pharmacokinetic study, the lowest brain nicotine concentration of 34 ±â€¯11 ng/g was detected in mice that were immunized with NanoNicVac 20.0. In addition, no apparent adverse events were observed in mice immunized with NanoNicVac. In summary, 20% PEGylation confers NanoNicVac with desirable safety, the highest stability, and the best immunological efficacy in mice.

Rational incorporation of molecular adjuvants into a hybrid nanoparticle-based nicotine vaccine for immunotherapy against nicotine addiction.

Current clinically-tested nicotine vaccines have yet shown enhanced smoking cessation efficacy due to their low immunogenicity. Achieving a sufficiently high immunogenicity is a necessity for establishing a clinically-viable nicotine vaccine. This study aims to facilitate the immunogenicity of a hybrid nanoparticle-based nicotine vaccine by rationally incorporating toll-like receptor (TLR)-based adjuvants, including monophosphoryl lipid A (MPLA), Resiquimod (R848), CpG oligodeoxynucleotide 1826 (CpG ODN 1826), and their combinations. The nanoparticle-delivered model adjuvant was found to be taken up more efficiently by dendritic cells than the free counterpart. Nanovaccine particles were transported to endosomal compartments upon cellular internalization. The incorporation of single or dual TLR adjuvants not only considerably increased total anti-nicotine IgG titers but also significantly affected IgG subtype distribution in mice. Particularly, the nanovaccines carrying MPLA+R848 or MPLA+ODN 1826 generated a much higher anti-nicotine antibody titer than those carrying none or one adjuvant. Meanwhile, the anti-nicotine antibody elicited by the nanovaccine adjuvanted with MPLA+R848 had a significantly higher affinity than that elicited by the nanovaccine carrying MPLA+ODN 1826. Moreover, the incorporation of all the selected TLR adjuvants (except MPLA) reduced the brain nicotine levels in mice after nicotine challenge. Particularly, the nanovaccine with MPLA+R848 exhibited the best ability to reduce the level of nicotine entering the brain. Collectively, rational incorporation of TLR adjuvants could enhance the immunological efficacy of the hybrid nanoparticle-based nicotine vaccine, making it a promising next-generation immunotherapeutic candidate for treating nicotine addiction.

Rationalization of a nanoparticle-based nicotine nanovaccine as an effective next-generation nicotine vaccine: A focus on hapten localization.

A lipid-polymeric hybrid nanoparticle-based next-generation nicotine nanovaccine was rationalized in this study to combat nicotine addiction. A series of nanovaccines, which had nicotine-haptens localized on carrier protein (LPKN), nanoparticle surface (LPNK), or both (LPNKN), were designed to study the impact of hapten localization on their immunological efficacy. All three nanovaccines were efficiently taken up and processed by dendritic cells. LPNKN induced a significantly higher immunogenicity against nicotine and a significantly lower anti-carrier protein antibody level compared to LPKN and LPNK. Meanwhile, it was found that the anti-nicotine antibodies elicited by LPKN and LPNKN bind nicotine stronger than those elicited by LPKN, and LPNK and LPNKN resulted in a more balanced Th1-Th2 immunity than LPKN. Moreover, LPNKN exhibited the best ability to block nicotine from entering the brain of mice. Collectively, the results demonstrated that the immunological efficacy of the hybrid nanoparticle-based nicotine vaccine could be enhanced by modulating hapten localization, providing a promising strategy to combatting nicotine addiction.

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

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

Stability of heroin, 6-monoacetylmorphine, and morphine in biological samples and validation of an LC-MS assay for delayed analyses of pharmacokinetic samples in rats.

Degradation of heroin to 6-monoacetylmorphine (6-MAM) and then morphine happens rapidly in vivo and in vitro. The rates of heroin and 6-MAM degradation depend on the type of biological samples, and the duration and conditions of storage. In order to optimize conditions for measuring heroin and its metabolites in samples collected for pharmacokinetic studies in rats, we investigated the time course of degradation of heroin, 6-MAM, and morphine in four biological matrices: rat blood, rat brain homogenate, bovine serum, and human plasma under various conditions. Analyte concentrations were measured by LC-MS. The goal was to identify conditions that allow maximum flexibility in scheduling sample collection and analysis, as well as gain more information on the stability of heroin in blood and tissue samples. A solid-phase extraction method with ice-cold solvents, sodium fluoride (NaF) and a low pH (3.0) maintained sample stability. Quality controls were within 94.0-105% of the target value. Variability was 4.0-8.9% for all analytes within the range of 5-200 ng/mL for heroin, 5-1000 ng/mL for 6-MAM, and 10-200 ng/mL for morphine. Heroin degradation to 6-MAM was faster in rat whole blood than in plasma, and faster in rat plasma than in rat brain homogenate. Maintaining NaF at 4 mg/mL throughout processing enhanced stability; higher NaF concentrations added to whole blood caused hemolysis. Samples processed through solid phase extraction and stored as dried pellets at 80°C constituted the most stable environment for heroin, and was superior to the storing of samples in solution prior to or after extraction. Nevertheless, post-extraction heroin and 6-MAM levels declined by 6.7-8.3% over one week in rat plasma under these conditions, and by <1-4.7% in bovine serum or human plasma.