Jannike M. Andersen

Increased Locomotor Activity Induced by Heroin in Mice: Pharmacokinetic Demonstration of Heroin Acting as a Prodrug for the Mediator 6-Monoacetylmorphine in Vivo

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 (≤5 μmol/kg) or 6MAM (≤15 μmol/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 short-lived 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.

Effects of scopolamine and d-cycloserine on non-spatial reference memory in rats

The aim of the present work was to use a three-choice simultaneous brightness discrimination test to examine the retention of non-spatial reference memory in rats treated with scopolamine (0.5 mg/kg) alone and in combination with various concentrations of D-cycloserine (DCS) (5, 15 and 50 mg/kg). Scopolamine given 1 h before testing for retention was found to increase both the number of errors and the number of trials necessary to reach criterion. The three doses of DCS reduced this increase significantly and resulted in a U-shaped dose-response curve, where 15 mg/kg had a stronger compensatory effect than both 5 and 50 mg/kg. Control experiments were performed to confirm that the observed response was not due to non-mnemonic factors. Our results indicate that scopolamine and DCS affect non-spatial reference memory in a similar manner as shown for spatial reference memory.

Discussion of the role of the extracellular signal-regulated kinase-phospholipase A2 pathway in production of reactive oxygen species in Alzheimer's disease

In this paper we show that exposure of a rat brain synaptosome fraction to the amyloid beta peptide fragment βA(25-35), but not the inverted peptide βA(35-25), stimulated production of reactive oxygen species (ROS) in a concentration- and time-dependent manner. The ROS formation was attenuated by the tyrosine kinase inhibitor genistein, the mitogen-activated protein kinase inhibitor U0126, and the phospholipase A2 (PLA2) inhibitor 7,7-dimethyl-(5Z,8Z)-eicosadienoic acid. This strongly suggests that βA(25-35) stimulated ROS production through an extracellular signal-regulated kinase-PLA2-dependent pathway. The interaction between these enzymes and their possible involvement in free radical formation in Alzheimers disease are discussed.

Pharmacokinetic modeling of subcutaneous heroin and its metabolites in blood and brain of mice

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 heroins 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.

Methadone does not alter key parameters of adult hippocampal neurogenesis in the heroin-naïve rat

Methadone is a synthetic opiate that is useful in a variety of clinical settings, including in maintenance therapy of heroin dependence and as an analgesic. However, methadone can have negative effects on cognition in humans and in rodents. The mechanisms underlying methadone-induced disruption in cognition are unknown. One possibility is that methadone disrupts adult hippocampal neurogenesis, a form of hippocampal plasticity involved in cognition that is disrupted by other opiates, like morphine. The goal of this study was to determine if methadone alters key parameters of hippocampal neurogenesis in the adult rat. Four groups of male rats were injected with saline (Saline, n=11) or methadone (Escalating, Short Term, Acute, n=10-11/group) over the course of three weeks. Weight gain, locomotor activity, and neurogenesis data were collected. Consistent with prior results, Escalating rats had slower weight gain (-4% vs. Saline). Also consistent with prior results, methadone did not alter locomotor activity over the course of a 90 min test. However, closer analysis revealed that methadone - irrespective of the dose or duration - led to a decrease in locomotor activity (-11 to -20% vs. saline) when examined during the first 5 min of the locomotor test. Surprisingly, methadone did not alter any of three quantified parameters relevant to adult hippocampal neurogenesis (number of Ki67-, doublecortin-, or BrdU-immunoreactive cells [BrdU given prior to saline/methadone exposure]). These results suggest that - unlike other opiates such as morphine - experimenter-delivered methadone does not alter hippocampal plasticity by decreasing the number of adult-generated neurons.

Interference with visual memory in rats following infusion of the functional NMDA receptor antagonist, HA-966, into temporal regions

Results from lesion studies show that selective damage to the temporal cortex or lateral entorhinal cortex impairs visual memory, whereas damage to the hippocampal region does not affect retention of a visual discrimination task. Major input pathways of the above structures use glutamate as neurotransmitter. The glutamate NMDA receptor appears to play an important role for cognitive functions. The objective of the present study was to examine whether microinjections of the functional NMDA receptor antagonist, 3-amino-1-hydroxy-2-pyrrolidinone ((+)-HA-966), might result in effects mimicking those seen in lesion studies. The results show that infusion of HA-966 into the temporal cortex or lateral entorhinal cortex 1.5-3 h after the learning criterion had been obtained led to an impeded visual memory when tested 13 days later, whereas HA-966 infused into the hippocampal region did not affect memory. A similar retention deficit with HA-966 infusions in the temporal cortex or lateral entorhinal cortex was seen when testing took place 23 days later, whereas a markedly weaker effect was observed when the retention period was reduced to 3 days. It is suggested that the hippocampal region is a temporary storing site for nonspatial memory engrams, and later posttraining memory consolidation involves the temporal and lateral entorhinal cortices. Furthermore, the degree of the effect of HA-966 is related to the length of the retention period.

A Monoclonal Antibody Specific for 6-Monoacetylmorphine Reduces Acute Heroin Effects in Mice

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.

Long‐term methadone treatment reduces phosphorylation of CaMKII in rat brain

Objectives  To reveal a possible relationship between a previously reported impairment of novelty seeking in rats exposed to methadone and changes in intracellular molecules related to learning and memory.

D-Serine alleviates retrograde amnesia of a visual discrimination task in rats with a lesion of the perirhinal cortex.

D-Serine has been suggested to be a potent endogenous glycine-site agonist on the N-methyl-D-aspartate receptor, thereby having a potential role in the process of learning and memory. In rats, perirhinal cortex (PC) constitutes a particularly important structure for mnemonic processing, and damage to this area induces both anterograde and retrograde amnesia. In the present work, we show that intraperitoneal administration of 1000 mg/kg D-serine immediately after bilateral lesion of PC produced complete restoration of retrograde memory in rats, measured by a visual brightness discrimination task, while a higher dose (3000 mg/kg) did not show any reliable effect. Uptake of the drug into the brain was confirmed using high performance liquid chromatography (HPLC).

Pharmacokinetic interactions between ethanol and heroin: A study on post-mortem cases

INTRODUCTION Ethanol and heroin are both depressant drugs on the central nervous system, and combined use is known to be dangerous due to pharmacodynamic interactions, leading to an even higher risk of respiratory depression and death. In addition, previous studies have suggested a pharmacokinetic interaction between ethanol and the metabolism of heroin. The aim of the present study was to investigate if there was a pharmacokinetic interaction between heroin and ethanol, by comparing concentrations of heroin metabolites in cases with and without ethanol, as detected in blood samples collected from a large material of forensic autopsy cases. METHODS The material consisted of 1583 forensic autopsy cases, all containing 6-monoacetylmorphine (6-MAM), as evidence of heroin intake, in either blood or urine samples, from the time period between the 1st of January 2000 and the 31st of December 2012. Due to the high risk of post-mortem ethanol formation in cases revealing blood ethanol concentrations between 0.1 and 0.3‰, these cases were excluded from the study, along with cases where the analysis for ethanol was missing. After this exclusion of cases, the material (n=1474) was divided into two groups; one group where ethanol was not detected in blood (n=1160), and another group where ethanol was detected in blood at or above the concentration of 0.4‰ (n=314). Furthermore, the material was also divided into two other subgroups; one group where 6-MAM was detected in blood samples, indicating a very recent intake of heroin, and another group where 6-MAM was detected in the urine, but not in blood, indicating a less recent heroin intake. RESULTS The concentration ratios of morphine/6-MAM, morphine-3-glucuronide (M3G)/morphine, and morphine-6-glucuronide (M6G)/morphine in blood samples, were all significantly lower in the ethanol positive cases compared with that of the ethanol negative cases. For the subgroup of cases revealing a very recent intake of heroin (n=645), only the morphine/6-MAM ratio was significantly lower in the ethanol positive cases than in the ethanol negative cases. For the subgroup of cases with a less recent heroin intake (n=817), lower M3G/morphine and M6G/morphine ratios were found among the ethanol positive cases. CONCLUSIONS The results indicate that ethanol inhibits two steps in the heroin metabolism; the hydrolysis of 6-MAM to morphine, and the glucuronidation of morphine to M3G and M6G. This pharmacokinetic interaction could further complicate the outcome after combined use of heroin and ethanol, in addition to the already well-known pharmacodynamic interactions.