Michael D. Raleigh

Co-administration of morphine and oxycodone vaccines reduces the distribution of 6-monoacetylmorphine and oxycodone to brain in rats

Opioid conjugate vaccines have shown promise in animal models as a potential treatment for opioid addiction. Individual vaccines are quite specific and each targets only a limited number of structurally similar opioids. Since opioid users can switch or transition between opioids, we studied a bivalent immunization strategy of combining 2 vaccines that could target several of the most commonly abused opioids; heroin, oxycodone and their active metabolites. Morphine (M) and oxycodone (OXY) haptens were conjugated to keyhole limpet hemocyanin (KLH) through tetraglycine (Gly)(4) linkers at the C6 position. Immunization of rats with M-KLH alone produced high titers of antibodies directed against heroin, 6-monoacetylmorphine (6-MAM) and morphine. Immunization with OXY-KLH produced high titers of antibodies against oxycodone and oxymorphone. Immunization with the bivalent vaccine produced consistently high antibody titers against both immunogens. Bivalent vaccine antibody titers against the individual immunogens were higher than with the monovalent vaccines alone owing, at least in part, to cross-reactivity of the antibodies. Administration of a single concurrent intravenous dose of 6-MAM and oxycodone to rats immunized with the bivalent vaccine increased 6-MAM, morphine and oxycodone retention in serum and reduced the distribution of 6-MAM and oxycodone to brain. Vaccine efficacy correlated with serum antibody titers for both monovalent vaccines, alone or in combination. Efficacy of the individual vaccines was not compromised by their combined use. Consistent with the enhanced titers in the bivalent group, a trend toward enhanced pharmacokinetic efficacy with the bivalent vaccine was observed. These data support the possibility of co-administering two or more opioid vaccines concurrently to target multiple abusable opioids without compromising the immunogenicity or efficacy of the individual components.

Selective Effects of a Morphine Conjugate Vaccine on Heroin and Metabolite Distribution and Heroin-Induced Behaviors in Rats

Morphine conjugate vaccines have effectively reduced behavioral effects of heroin in rodents and primates. To better understand how these effects are mediated, heroin and metabolite distribution studies were performed in rats in the presence and absence of vaccination. In non-vaccinated rats 6-monoacetylmorphine (6-MAM) was the predominant opioid in plasma and brain as early as 1 minute after i.v. administration of heroin and for up to 14 minutes. Vaccination with morphine conjugated to keyhole limpet hemocyanin (M-KLH) elicited high titers and concentrations of antibodies with high affinity for heroin, 6-MAM, and morphine. Four minutes after heroin administration vaccinated rats showed substantial retention of all three opioids in plasma compared to controls and reduced 6-MAM and morphine, but not heroin, distribution to brain. Administration of 6-MAM rather than heroin in M-KLH vaccinated rats showed a similar drug distribution pattern. Vaccination reduced heroin-induced analgesia and blocked heroin-induced locomotor activity throughout 2 weeks of repeated testing. Higher serum opioid-specific antibody concentrations were associated with higher plasma opioid concentrations, lower brain 6-MAM and morphine concentrations, and lower heroin-induced locomotor activity. Serum antibody concentrations over 0.2 mg/ml were associated with substantial effects on these measures. These data support a critical role for 6-MAM in mediating the early effects of i.v. heroin and suggest that reducing 6-MAM concentration in brain is essential to the efficacy of morphine conjugate vaccines.

Vaccination against nicotine alters the distribution of nicotine delivered via cigarette smoke inhalation to rats.

Preclinical models of nicotine vaccine pharmacology have relied on i.v. or s.c. administration of nicotine. Models using cigarette smoke inhalation might more accurately simulate nicotine exposure in smokers. Nicotine vaccine effects were examined in rats using two cigarette smoke exposure models: a 10 min nose-only exposure (NSE) producing serum nicotine levels equivalent to the nicotine boost from 1 cigarette in a smoker, and a 2h whole-body exposure (WBE) producing serum nicotine levels similar to those associated with regular mid-day smoking. Vaccination prior to 10min smoke NSE reduced nicotine distribution to brain by 90%, comparable to its effect on nicotine administered i.v. Vaccination prior to 2 h smoke WBE reduced nicotine distribution to brain by 35%. The nicotine concentration in broncheoalveolar lavage (BAL) fluid obtained after 2 h WBE was increased by 230% in vaccinated rats but was also increased in rats passively immunized with a nicotine-specific monoclonal antibody, and so was likely due to transfer of antibody from serum rather than local production at the pulmonary mucosa. Nicotine-specific IgA was not detectable in BAL fluid, but titers in serum were appreciable at 21-25% of the IgG titer and could contribute to vaccine efficacy. Both vaccination and passive immunization are effective in reducing nicotine distribution to brain in rats when nicotine is delivered via inhaled cigarette smoke. These data validate results previously obtained in rodents for nicotine vaccines using i.v. or s.c. nicotine dosing and provide a quantitative method for studying aspects of nicotine exposure which are unique to cigarette smoke inhalation.

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.

Engineering of a hybrid nanoparticle-based nicotine nanovaccine as a next-generation immunotherapeutic strategy against nicotine addiction: A focus on hapten density

Although vaccination is a promising way to combat nicotine addiction, most traditional hapten-protein conjugate nicotine vaccines only show limited efficacy due to their poor recognition and uptake by immune cells. This study aimed to develop a hybrid nanoparticle-based nicotine vaccine with improved efficacy. The focus was to study the impact of hapten density on the immunological efficacy of the proposed hybrid nanovaccine. It was shown that the nanovaccine nanoparticles were taken up by the dendritic cells more efficiently than the conjugate vaccine, regardless of the hapten density on the nanoparticles. At a similar hapten density, the nanovaccine induced a significantly stronger immune response against nicotine than the conjugate vaccine in mice. Moreover, the high- and medium-density nanovaccines resulted in significantly higher anti-nicotine antibody titers than their low-density counterpart. Specifically, the high-density nanovaccine exhibited better immunogenic efficacy, resulting in higher anti-nicotine antibody titers and lower anti-carrier protein antibody titers than the medium- and low-density versions. The high-density nanovaccine also had the best ability to retain nicotine in serum and to block nicotine from entering the brain. These results suggest that the hybrid nanoparticle-based nicotine vaccine can elicit strong immunogenicity by modulating the hapten density, thereby providing a promising next-generation immunotherapeutic strategy against nicotine addiction.

Pharmacokinetic Correlates of the Effects of a Heroin Vaccine on Heroin Self-Administration in Rats

The purpose of this study was to evaluate the effects of a morphine-conjugate vaccine (M-KLH) on the acquisition, maintenance, and reinstatement of heroin self-administration (HSA) in rats, and on heroin and metabolite distribution during heroin administration that approximated the self-administered dosing rate. Vaccination with M-KLH blocked heroin-primed reinstatement of heroin responding. Vaccination also decreased HSA at low heroin unit doses but produced a compensatory increase in heroin self-administration at high unit doses. Vaccination shifted the heroin dose-response curve to the right, indicating reduced heroin potency, and behavioral economic demand curve analysis further confirmed this effect. In a separate experiment heroin was administered at rates simulating heroin exposure during HSA. Heroin and its active metabolites, 6-acetylmorphine (6-AM) and morphine, were retained in plasma and metabolite concentrations were reduced in brain in vaccinated rats compared to controls. Reductions in 6-AM concentrations in brain after vaccination were consistent with the changes in HSA rates accompanying vaccination. These data provide evidence that 6-AM is the principal mediator of heroin reinforcement, and the principal target of the M-KLH vaccine, in this model. While heroin vaccines may have potential as therapies for heroin addiction, high antibody to drug ratios appear to be important for obtaining maximal efficacy.

A nanoparticle-based nicotine vaccine and the influence of particle size on its immunogenicity and efficacy

Traditional hapten-protein conjugate nicotine vaccines have shown less than desired immunological efficacy due to their poor recognition and internalization by immune cells. We developed a novel lipid-polymeric hybrid nanoparticle-based nicotine vaccine to enhance the immunogenicity of the conjugate vaccine, and studied the influence of particle size on its immunogenicity and pharmacokinetic efficacy. The results demonstrated that the nanovaccines, regardless of size, could induce a significantly stronger immune response against nicotine compared to the conjugate vaccine. Particularly, a significantly higher anti-nicotine antibody titer was achieved by the 100 compared to the 500nm nanovaccine. In addition, both the 100 and 500nm nanovaccines reduced the distribution of nicotine into the brain significantly. The 100nm nanovaccine exhibited better pharmacokinetic efficacy than the 500nm nanovaccine in the presence of alum adjuvant. These results suggest that a lipid-polymeric nanoparticle-based nicotine vaccine is a promising candidate to treat nicotine dependence.

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.

Vaccines for Opioid Addiction

Agonist and antagonist medications are available for the treatment of opioid addiction, but interest in their use is limited by their real as well as their perceived limitations. Opioid vaccines represent an attractive alternative or additional treatment option because their distinct mechanism of action circumvents many of these limitations. Opioid addiction is a particularly challenging target for vaccines because a variety of both illegal and prescription opioids can be abused, most have one or more active metabolites, and opioid abusers commonly switch among opioids. In addition, the single and daily doses of abused opioids are generally high compared to the drug-binding capacity of antibodies that can be generated by vaccination. Nevertheless, several types of heroin vaccines have shown considerable efficacy in animals for binding heroin or its active metabolites in serum, reducing or slowing their distribution to the brain, and attenuating addiction-relevant behaviors including heroin self-administration and reinstatement. The antibodies elicited by vaccines directed at heroin and its metabolites do not appreciably cross-react with methadone, buprenorphine, or naltrexone and so have the potential to be used in combination with these to enhance treatment efficacy. Heroin vaccines do not appreciably bind commonly abused prescription opioids such as oxycodone and hydrocodone, but vaccines directed specifically at these opioids are also highly effective in animals. It is possible to coadminister heroin and oxycodone vaccines to provide activity against a wide range of abusable opioids. Clinical trials of these vaccines, particularly for relapse prevention, are warranted by the strength and consistency of these preclinical data.

Prescreening of Nicotine Hapten Linkers in Vitro To Select Hapten-Conjugate Vaccine Candidates for Pharmacokinetic Evaluation in Vivo

Since the demonstration of nicotine vaccines as a possible therapeutic intervention for the effects of tobacco smoke, extensive effort has been made to enhance nicotine specific immunity. Linker modifications of nicotine haptens have been a focal point for improving the immunogenicity of nicotine, in which the evaluation of these modifications usually relies on in vivo animal models, such as mice, rats or nonhuman primates. Here, we present two in vitro screening strategies to estimate and predict the immunogenic potential of our newly designed nicotine haptens. One utilizes a competition enzyme-linked immunoabsorbent assay (ELISA) to profile the interactions of nicotine haptens or hapten-protein conjugates with nicotine specific antibodies, both polyclonal and monoclonal. Another relies on computational modeling of the interactions between haptens and amino acid residues near the conjugation site of the carrier protein to infer linker-carrier protein conjugation effect on antinicotine antibody response. Using these two in vitro methods, we ranked the haptens with different linkers for their potential as viable vaccine candidates. The ELISA-based hapten ranking was in an agreement with the results obtained by in vivo nicotine pharmacokinetic analysis. A correlation was found between the average binding affinity (IC50) of the haptens to an anti-Nic monoclonal antibody and the average brain nicotine concentration in the immunized mice. The computational modeling of hapten and carrier protein interactions helps exclude conjugates with strong linker-carrier conjugation effects and low in vivo efficacy. The simplicity of these in vitro screening strategies should facilitate the selection and development of more effective nicotine conjugate vaccines. In addition, these data highlight a previously under-appreciated contribution of linkers and hapten-protein conjugations to conjugate vaccine immunogenicity by virtue of their inclusion in the epitope that binds and activates B cells.