We investigated the relative importance of heroin and its metabolites in eliciting a behavioral response in mice by studying the relationship between concentrations of heroin, 6-monoacetylmorphine (6MAM), and morphine in brain tissue and the effects on locomotor activity. Low doses (subcutaneous) of heroin (Յ5 mol/kg) or 6MAM (Յ15 mol/kg) made the mice run significantly more than mice given equimolar doses of morphine. There were no differences in the response between heroin and 6MAM, although we observed a shift to the left of the dose-response curve for the maximal response of heroin. The behavioral responses were abolished by pretreatment with 1 mg/kg naltrexone. Heroin was detected in brain tissue after injection, but the levels were low and its presence too shortlived to be responsible for the behavioral response observed.The concentration of 6MAM in brain tissue increased shortly after administration of both heroin and 6MAM and the concentration changes during the first hour roughly reflected the changes in locomotor activity. Both the maximal and the total concentration of 6MAM were higher after administration of heroin than after administration of 6MAM itself. The morphine concentration increased slowly after injection and could not explain the immediate behavioral response. In summary, the locomotor activity response after injection of heroin was mediated by 6MAM, which increased shortly after administration. Heroin acted as an effective prodrug. The concentration of morphine was too low to stimulate the immediate response observed but might have an effect on the later part of the heroin-induced behavioral response curve.

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BACKGROUND AND PURPOSEHeroin, with low affinity for μ-opioid receptors, has been considered to act as a prodrug. In order to study the pharmacokinetics of heroin and its active metabolites after i.v. administration, we gave a bolus injection of heroin to rats and measured the concentration of heroin and its metabolites in blood and brain extracellular fluid (ECF). EXPERIMENTAL APPROACHAfter an i.v. bolus injection of heroin to freely moving Sprague-Dawley rats, the concentrations of heroin and metabolites in blood samples from the vena jugularis and in microdialysis samples from striatal brain ECF were measured by ultraperformance LC-MS/MS. KEY RESULTSHeroin levels decreased very fast, both in blood and brain ECF, and could not be detected after 18 and 10 min respectively. 6-Monoacetylmorphine (6-MAM) increased very rapidly, reaching its maximal concentrations after 2.0 and 4.3 min, respectively, and falling thereafter. Morphine increased very slowly, reaching its maximal levels, which were six times lower than the highest 6-MAM concentrations, after 12.6 and 21.3 min, with a very slow decline during the rest of the experiment and only surpassing 6-MAM levels at least 30 min after injection. CONCLUSIONS AND IMPLICATIONSAfter an i.v. heroin injection, 6-MAM was the predominant opioid present shortly after injection and during the first 30 min, not only in the blood but also in rat brain ECF. 6-MAM might therefore mediate most of the effects observed shortly after heroin intake, and this finding questions the general assumption that morphine is the main and most important metabolite of heroin. Abbreviations6-MAM, 6-monoacetylmorphine; AUCLast, area under the concentration-time curve from time zero to last sample time; Cmax, maximum concentration; BBB, blood-brain barrier; Cl, clearance; Cu, concentration of unbound analyte in brain

Immunotherapy against drugs of abuse is being studied as an alternative treatment option in addiction medicine and is based on antibodies sequestering the drug in the bloodstream and blocking its entry into the brain. Producing an efficient vaccine against heroin has been considered particularly challenging because of the rapid metabolism of heroin to multiple psychoactive molecules. We have previously reported that heroin's first metabolite, 6-monoacetylmorphine (6-MAM), is the predominant mediator for heroin's acute behavioral effects and that heroin is metabolized to 6-MAM primarily prior to brain entry. On this basis, we hypothesized that antibody sequestration of 6-MAM is sufficient to impair heroin-induced effects and therefore examined the effects of a monoclonal antibody (mAb) specific for 6-MAM. In vitro experiments in human and rat blood revealed that the antibody was able to bind 6-MAM and block the metabolism to morphine almost completely, whereas the conversion of heroin to 6-MAM remained unaffected. Mice pretreated with the mAb toward 6-MAM displayed a reduction in heroin-induced locomotor activity that corresponded closely to the reduction in brain 6-MAM levels. Intraperitoneal and intravenous administration of the anti-6-MAM mAb gave equivalent protection against heroin effects, and the mAb was estimated to have a functional half-life of 8 to 9 days in mice. Our study implies that an antibody against 6-MAM is effective in counteracting heroin effects.

After injection, heroin is rapidly metabolized to 6-monoacetylmorphine (6-MAM) and further to morphine. As morphine has been shown to increase striatal dopamine, whereas 6-MAM has not been studied in this respect, we gave i.v. injections of 3 μmol 6-MAM, morphine or heroin to rats. Opioids were measured in blood, and dopamine and opioids in microdialysate from brain striatal extracellular fluid (ECF), by UPLC-MS/MS. After 6-MAM injection, 6-MAM ECF concentrations increased rapidly, and reached Cmax of 4.4 μM after 8 min. After heroin injection, 6-MAM increased rapidly in blood and reached Cmax of 6.4 μM in ECF after 8 min, while ECF Cmax for heroin was 1.2 μM after 2 min. T max for morphine in ECF was 29 and 24 min following 6-MAM and heroin administration, respectively, with corresponding Cmax levels of 1 and 2 μM. Dopamine levels peaked after 8 and 14 min following 6-MAM and heroin administration, respectively. The dopamine responses were equal, indicating no dopamine release by heroin per se. Furthermore, 6-MAM, and not morphine, appeared to mediate the early dopamine response, whereas morphine administration, giving rise to morphine ECF concentrations similar to those observed shortly after 6-MAM injection, did not increase ECF dopamine. 6-MAM appeared accordingly to be the substance responsible for the early increase in dopamine observed after heroin injection. As 6-MAM was formed rapidly from heroin in blood, and was the major substance reaching the brain after heroin administration, this also indicates that factors influencing blood 6-MAM concentrations might change the behavioural effects of heroin.

High blood-brain permeability and effective delivery of morphine to the brain have been considered as explanations for the high potency of heroin. Results from Andersen et al. indicate that 6-monoacetylmorphine (6-MAM), and not morphine, is the active metabolite responsible for the acute effects observed for heroin. Here, we use pharmacokinetic modeling on data from the aforementioned study to calculate parameters of the distribution of heroin, 6-MAM and morphine in blood and brain tissue after subcutaneous heroin administration in mice. The estimated pharmacokinetic parameters imply that the very low heroin and the high 6-MAM levels observed both in blood and brain in the original experiment are likely to be caused by a very high metabolic rate of heroin in blood. The estimated metabolic rate of heroin in brain was much lower and cannot account for the low heroin and high 6-MAM levels in the brain, which would primarily reflect the concentrations of these compounds in blood. The very different metabolic rates for heroin in blood and brain calculated by the model were confirmed by in vitro experiments. These results show that heroin's fast metabolism in blood renders high concentrations of 6-MAM which, due to its relatively good blood-brain permeability, results in high levels of this metabolite in the brain. Thus, it is the high blood metabolism rate of heroin and the blood-brain permeability to 6-MAM, and not to heroin, which could account for the highly efficient delivery of active metabolites to the brain after heroin administration.

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