Elsevier

Toxicology Letters

Volume 166, Issue 1, 30 September 2006, Pages 11-18
Toxicology Letters

Toxicological analysis in rats subjected to heroin and morphine overdose

https://doi.org/10.1016/j.toxlet.2006.05.007Get rights and content

Abstract

In heroin overdose deaths the blood morphine concentration varies substantially. To explore possible pharmacokinetic explanations for variable sensitivity to opiate toxicity we studied mortality and drug concentrations in male Sprague-Dawley rats. Groups of rats were injected intravenously (i.v.) with heroin, 21.5 mg/kg, or morphine, 223 mg/kg, causing a 60–80% mortality among drug-naïve rats. Additional groups of rats were pre-treated with morphine for 14 days, with or without 1 week of subsequent abstinence. Brain, lung and blood samples were analyzed for 6-acetylmorphine, morphine, morphine-3-glucuronide and morphine-6-glucuronide. i.v. morphine administration to drug-naïve rats resulted in both rapid and delayed deaths. The brain morphine concentration conformed to an exponential elimination curve in all samples, ruling out accumulation of morphine as an explanation for delayed deaths. This study found no support for formation of toxic concentration of morphine-6-glucuronide. Spontaneous death among both heroin and morphine rats occurred at fairly uniform brain morphine concentrations. Morphine pre-treatment significantly reduced mortality upon i.v. morphine injection, but the protective effect was less evident upon i.v. heroin challenge. The morphine pre-treatment still afforded some protection after 1 week of abstinence among rats receiving i.v. morphine, whereas rats given i.v. heroin showed similar death rate as drug-naïve rats.

Introduction

About 8000 acute drug-related deaths are recorded annually in the EU (http://www.emcdda.eu.int/). An evaluation of the reported deaths during several years in 10 countries shows that approximately 80% of these deaths are related to opiate overdose. Typically, such opiate toxicity deaths strike suddenly and unexpectedly even among experienced users.

Postmortem toxicological findings in deceased heroin addicts do not support that heroin “overdose” deaths result from the intake of a large dose. In a majority of these victims the blood morphine concentration is actually found to be lower than that seen in heroin users that did not die from overdose but from other causes (Darke and Zador, 1996). Further, blood morphine concentration varies substantially, and cannot be used in isolation to diagnose heroin overdose deaths (Meissner et al., 2002). By performing segmental hair analysis, we have recently found that abstinent overdose victims presented with similar blood morphine concentration as did overdose victims with evidence of continuous opioid drug use prior to death (Druid et al., 2006).

In humans, heroin (3,6-diacetylmorphine) is rapidly degraded to the metabolites 6-acetylmorphine (6-AM) and morphine. Most of the morphine is further converted to morphine-3-glucuronide (M3G; ∼50%) and morphine-6-glucuronide (M6G; ∼10%) (Aderjan and Skopp, 1998). Heroin quickly enters the brain through the blood–brain barrier, however, due to its rapid metabolism, it is generally assumed that heroin itself has only minor pharmacological effects. Most of the pharmacological effects, including respiratory depression, are instead caused by morphine, or to some extent, by 6-AM and M6G (White and Irvine, 1999). Since the demonstration in the late 1960s that M6G possesses analgesic properties (Kamata et al., 1969) it has been proposed that the glucuronides play an important role for the toxicity of heroin and morphine. However, the main metabolite, M3G, does not seem to have any agonistic effect either in vivo or in cell cultures (Shimomura et al., 1971, Hemstapat et al., 2003). In contrast, there is some evidence that M3G may exert antagonistic effects and to some extent compete with morphine and M6G at binding sites (Gardmark et al., 1998). When M6G is directly administered to humans, some studies have shown that side effects like nausea, vomiting and respiratory depression are attenuated compared with the administration of analgesically equipotent doses of morphine, whereas others have found no such differences (Lötsch, 2005). In rats, the formation of M6G is limited (Milne et al., 1996); hence, heroin and morphine administration to rats is not expected to produce toxic concentration of this metabolite. Therefore, rat studies offer good conditions for the evaluation of the inherent toxicity of morphine and heroin.

Since blood morphine concentration in apparent heroin overdose deaths vary substantially, we decided to explore the possible correlation between the concentrations of several heroin metabolites and the outcome in an animal model, where rats were given high doses of either morphine or heroin. We hypothesized that a gradual accumulation of either morphine or M6G in the brain together with a drop in blood morphine concentration might explain low blood morphine concentration at death. Further, we also wanted to address the question of whether accumulation of opiate metabolites in the brain could account for delayed deaths. In order to assess the impact of tolerance, we included groups of rats that were pre-treated with intraperitoneal (i.p.) injections of morphine, and then challenged them with an acute intravenous (i.v.) injection of heroin or morphine, with or without a preceding period of abstinence.

Section snippets

Animals

Male Sprague-Dawley rats (Scanbur BK AB, Sollentuna, Sweden) initially weighing 216–376 g were used. Rats were allowed to acclimatize for 1 week prior to experiment. Rats were allocated in groups of four in each cage and allowed fresh water and standard rat chow ad libitum throughout the whole experimental period. Rats were kept in a 12:12 h light:dark cycle synchronous with daylight (lights on at 7.00 a.m.). All experiments were performed in strict accordance with the guidelines and the consent

Results

i.v. doses causing approximately 70% mortality among drug-naïve rats was established prior to this study (21.5 mg/kg for heroin and 223 mg/kg for morphine). In the present study these doses produced 67% mortality among drug-naïve rats given i.v. heroin and 79% mortality among drug-naïve rats given i.v. morphine (Table 1). In comparison, animals receiving 2 weeks of pre-treatment with i.p. morphine showed a lower mortality after i.v. heroin as well as after i.v. morphine injection. Even after 1

Discussion

We developed a rat model to mimic morphine and heroin overdose death in humans. Our major findings were: (1) death occurred at different times after i.v. injection, but there was no evidence of accumulation of morphine or other opiate compounds in the brain to explain for delayed deaths; (2) pre-treatment with morphine significantly reduced mortality after i.v. morphine injection, and some protective effect was still observed after 1 week of abstinence; however, pre-treatment with morphine did

Acknowledgements

We thank Wenche Rødseth Brede, Department of Clinical Pharmacology, St. Olav University Hospital, Trondheim, Norway, for technical assistance with the drug analysis. This study was funded by grants from the Swedish National Drug Policy Coordinator, the Swedish National Board of Forensic Medicine, the Swedish Medical Society, Lennanders Foundation and the Magn. Bergvall Foundation.

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