A heroin/morphine vaccine: mechanism of action and extending its use to other abused opioids

University of Minnesota Ph.D. dissertation. July 2013. Major: Pharmacology. Advisor: Paul Pentel. 1 computer file (PDF); xiii, 134 pages.Heroin is more widely used than any other illicit opioid and mortality rates among heroin users are an average of 13 times higher than the general population. Intravenous heroin use is associated with crime, social disruption, and transmission of blood-born pathogens such as human immunodeficiency virus and hepatitis C. Effective pharmacotherapies are available to treat heroin abuse but have been largely unsuccessful because they require frequent dosing, have a high abuse potential, or have low compliance. Vaccines against heroin and its metabolites (e.g. morphine) are being considered as a complementary treatment for heroin abuse because they are long-acting, selective, have no abuse potential, and may benefit those unwilling to take the current pharmacotherapies. Vaccination with morphine-conjugate vaccines can elicit a strong immune response that reduces the behavioral effects of heroin in animals, presumably by morphine-specific antibodies binding opioids in blood and reducing their distribution to brain. This thesis explores the use of M-KLH, a morphine hapten conjugated to keyhole limpet hemocyanin (KLH) using a tetraglycine linker and mixed with either Freund's or alum adjuvant for increasing the immune response. Morphine vaccines present many challenges that make translation to clinical use difficult. Heroin is sequentially metabolized to its active intermediates 6-monoacetylmorphine (6-MAM), morphine, and then to morphine-6-glucuronide (in vivo and ex vivo). Heroin enters brain and is rapidly converted to 6-MAM, which is presumed to mediate most of heroin's early effects. With regard to the mechanism of action of morphine vaccines, it is unclear whether the antibodies they generate must bind heroin, its downstream metabolites, or both to prevent opioid distribution from plasma to brain and reduce heroin's behavioral effects. However, because analytical assays to measure heroin and metabolite concentrations in tissues have used a wide range of conditions and varying degrees of stability have been reported, studying the effect of vaccination on heroin distribution is not straightforward. In addition, heroin and metabolite distribution after i.v. heroin administration, the most common route of abuse by humans, has not been well characterized in non-vaccinated rodents. Finally, blockade of heroin by vaccination may not prevent the abuse of structurally distinct opioids. The overall goal of this thesis was to better understand the mechanism of action of morphine vaccines and to extend their use to other abused opioids. The specific aims were to stabilize heroin in blood and brain tissues for subsequent pharmacokinetic studies, study distribution of heroin and its metabolites in non-vaccinated and vaccinated rats, explore the effects of vaccination on heroin-induced behaviors, and determine if vaccine efficacy is retained when combined with a vaccine targeting oxycodone, another commonly abused opioid. These aims were explored using clinically relevant drug doses. Heroin and metabolite degradation was significantly reduced by 1) the addition of ice-cold sodium fluoride (a general esterase inhibitor) and formate buffer (pH 3.0) in heroin-spiked tissues, 2) rapid removal of red blood cells via centrifugation, and 3) drying opioids after extraction from tissues prior to measuring their levels. Using these conditions heroin and its metabolites were stabilized in tissues for subsequent distribution studies. In non-vaccinated rats 6-MAM was the predominant metabolite in brain as early as one minute after administration of 0.26 mg/kg i.v. heroin, which is consistent with previous studies that suggest that 6-MAM mediates heroin's early behavioral effects. Vaccination with a morphine-conjugate vaccine (M-KLH) led to a reduction of 6-MAM and morphine, but not heroin, distribution to brain after heroin administration, suggesting that morphine vaccines reduce accumulation of 6-MAM in brain. The mechanism by which this occurs is likely through antibody binding of 6-MAM in plasma to prevent its distribution to brain and is consistent with very high plasma 6-MAM concentrations in vaccinated rats after i.v. heroin or 6-MAM administration. Vaccination with M-KLH led to a reduction of heroin-induced anti-nociception and locomotor activity and remained effective for up to 16 days after repeated dosing suggesting that heroin vaccines may have long-lasting efficacy. These results are consistent with findings from the distribution studies and support the hypothesis that morphine vaccines function by retaining 6-MAM in plasma and prevent its accumulation in brain. To determine whether opioid vaccines could be combined without reducing individual vaccine efficacy and prevent heroin addicts from abusing structurally distinct opioids, rats were vaccinated with M-KLH, an oxycodone-conjugate vaccine (Oxy-KLH), or the bivalent vaccine (both M-KLH and Oxy-KLH). Total morphine- and oxycodone-specific antibody titers were significantly increased in rats that received the bivalent vaccine compared to rats that received individual vaccines. Concurrent i.v. administration of 6-MAM and oxycodone in M-KLH vaccinated rats led to increased 6-MAM retention in plasma and reduced 6-MAM distribution in brain. A similar effect on oxycodone distribution was seen in Oxy-KLH vaccinated rats. There was a trend towards greater efficacy in altering both 6-MAM and oxycodone distribution in the bivalent group compared to individual vaccine groups. These data suggest that combining opioid vaccines will retain, and possibly enhance, individual vaccine efficacy and might be a viable option to prevent addicts from abusing structurally distinct opioids. These findings contribute to the understanding of how morphine vaccines elicit their effects on heroin-induced behaviors and suggest that morphine vaccines, alone or in combination with other pharmacotherapies, may benefit those seeking treatment for heroin addiction