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of individual drugs to cause harmful increases in core temperature. Keywords Cocaine . Methamphetamine . MDMA . Para-methoxyamphetamine . Ecstasy .
Psychopharmacology (2007) 194:41–52 DOI 10.1007/s00213-007-0825-9

ORIGINAL INVESTIGATION

Pharmacological and behavioral determinants of cocaine, methamphetamine, 3,4-methylenedioxymethamphetamine, and para-methoxyamphetamine-induced hyperthermia Emily Joy Jaehne & Abdallah Salem & Rodney James Irvine

Received: 5 March 2007 / Accepted: 6 May 2007 / Published online: 27 May 2007 # Springer-Verlag 2007

Abstract Rationale Cocaine, methamphetamine, 3,4-methylenedioxymethamphetamine (MDMA, ecstasy), and para-methoxyamphetamine (PMA) disrupt normal thermoregulation in humans, with PMA being associated with more severe cases of hyperthermia. Harm minimization advice on how to prevent overheating depends on appropriate thermoregulatory behavior by drug users. Objectives The purpose of the current study was to establish dose–response relationships for the effects of a number of commonly used illicit stimulants and investigate the behavioral response to increased core temperature. Materials and methods Sprague–Dawley rats with telemetry implants were administered either saline or 4, 12, 26, 40 or 80 μmol/kg of cocaine, methamphetamine, MDMA, or PMA and confined to an ambient temperature of 30°C for 30 min, before being able to choose their preferred temperature on a thermally graded runway (11–41°C). Results The increased core temperature caused by administration of cocaine, methamphetamine, and MDMA treatment led to the animals seeking the cool end of the runway to correct their core temperature, although this did not occur in PMA-treated rats. The order of potency for increasing core temperature was methamphetamine >PMA = MDMA > cocaine. This differed to the slopes of the dose–response curves where MDMA and PMA showed the steepest slope for the doses used followed by methamphetamine then cocaine. E. J. Jaehne (*) : A. Salem : R. J. Irvine Discipline of Pharmacology, School of Medical Sciences, University of Adelaide, Level 5 Medical School North, Adelaide, South Australia 5005, Australia e-mail: [email protected]

Conclusions These results suggest that behavioral aspects of thermoregulation are important in assessing the potential of individual drugs to cause harmful increases in core temperature. Keywords Cocaine . Methamphetamine . MDMA . Para-methoxyamphetamine . Ecstasy . Thermoregulation, Behavior

Introduction A common feature of the most popular illicit stimulant drugs is their ability to induce hyperthermia in human users (Kalant 2001). Case reports appear to indicate that the incidence and severity of these events vary between drugs, but experimental evidence to support this is sparse. The drugs most often implicated are cocaine, methamphetamine, 3,4-methylenedioxymethamphetamine (MDMA), and para-methoxyamphetamine (PMA). Cocaine is one of the most commonly used illicit stimulants in the USA (United Nations 2006), and case studies show that it can lead to fatalities involving hyperthermia (Marzuk et al. 1998). Methamphetamine use is rising in many countries and has also been linked to cases of fatal hyperthermia (Kojima et al. 1984). However, most of the recent focus on hyperthermia induced by illicit stimulants has been on MDMA (Gowing et al. 2002; Green et al. 2004; Williamson et al. 1997) and PMA, the latter of which appears to be the most toxic of these substances and has had serious consequences for people thinking they are taking ‘ecstasy’ (MDMA) (Ling et al. 2001). Although the incidences of acute extreme hyperthermia are low in human users, the events are unpredictable, and the impact on the individual is severe (Gowing et al. 2002; Williamson et al. 1997).

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The increasing use of these drugs is also of concern when one considers the probable role of hyperthermia in the development of stimulant-induced neurotoxicity (Clemens et al. 2004; Malberg and Seiden 1998). Animal studies have shown that hyperthermic doses of methamphetamine can lead to decreased brain concentrations of both serotonin (5-hydroxytryptamine [5-HT]) and dopamine (DA) (Bowyer et al. 1992, 1994). Animal studies have additionally shown a link between MDMA and PMA administration leading to acute hyperthermia and long-term changes in brain concentration of 5-HT or related structures in both animals (Callaghan et al. 2006, 2007; Malberg and Seiden 1998; Wang et al. 2004) and humans (McCann et al. 1998; Volkow et al. 2001). There is also evidence that ‘ecstasy’ use may be associated with long-term psychobiological problems in human users (Parrott 2002), suggesting that this may be particularly harmful with increased ambient and perceived body temperature (Parrott et al. 2006). In view of the fact hyperthermia may be critical in the health of stimulant users, there has been difficulty in predicting when these adverse effects will occur. Wide ranges of plasma concentrations have been seen in fatal cases (Caldicott et al. 2003; Gowing et al. 2002), and these concentrations often overlap with those associated with a minor change in core temperature and other physiological effects (Irvine et al. 2006). There are also many contributing factors to hyperthermia including environment, poly drug use, and behavior, and the full extent of the combined role each factor plays has not yet been fully elucidated. Although extrapolating results obtained from animal studies must be made with caution (Easton and Marsden 2006), it can also be a useful tool in establishing possible problems associated with particular drugs (de la Torre and Farre 2004; Easton and Marsden 2006). Examples of this are animal studies that have shown that changes in body temperature induced by stimulant drugs are dependent on ambient temperature (Jaehne et al. 2005; Lomax and Daniel 1990; Stanley et al. 2007; Xie et al. 2000), which is important for human users taking these drugs in warm clubs, although additional factors may also be important. Although behavior has been recognized as important in the overall control of core body temperature (Attia 1984; Sessler 1997), its role in stimulant-induced hyperthermia has only recently been studied (Jaehne et al. 2005). That study placed rats in a thermal gradient after their body temperature had been increased or decreased by treatment with MDMA (10 mg/kg) and showed that rats chose appropriate ambient temperatures to bring their core temperature back to normal (Jaehne et al. 2005). However, rats treated with PMA (10 mg/kg) chose much warmer areas on the runway despite being hyperthermic. We hypothesized that this may partially explain the much greater incidence of fatal hyperthermia for PMA compared to other more

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popular stimulant drugs. Thus, the ability to behaviorally thermoregulate may play a major role in reducing harms associated with stimulant-induced hyperthermia. These drugs are often used in hot ambient temperature as experienced in nightclubs (Parrott et al. 2006), and current harm minimization advice is to move to cooler “chill out” areas to prevent the onset of severe hyperthermia. This strategy assumes that the users perceive that they are warm and respond appropriately. Furthermore, the majority of previous studies have used a limited number of doses, which has not given adequate information on the pharmacology of these compounds. This is important as case studies have so far failed to reveal a clear dose–response relationship. The current study, therefore, aims to address these problems by extending our model to define the relative potencies of the most popular illicit drugs associated with hyperthermia in humans and to assess both the physiological and thermoregulatory behavior after challenge with a range of popular illicit stimulants.

Materials and methods Animals Thirty-two male Sprague–Dawley rats, weighing 310±15 g at the start of experiments, were used for all testing. The rats were housed in groups of two or three during the experimental period, with food and water available ad libitum. Ambient temperature of the laboratory was in the range 20–23°C. All behavioral testing was conducted between 1000 and 1500 hours, when the core body temperature of rats varies little under normal conditions (Gordon 1990). All experimentation was approved by the University of Adelaide Animal Ethics Committee and followed the Australian code of practice for the care and use of animals for scientific purposes. Equipment The apparatus used was the same as we have used previously (Jaehne et al. 2005) and based on previous studies (Florez-Duquet et al. 2001; Gordon 1987). It consists of an insulated aluminum floor (120 cm) with an actual runway length of 72 cm divided into five zones with dimensions 14.5 by 30 cm. The runway is split into two 15 cm wide sides so that two rats can be observed simultaneously. The ends and center divide are aluminum (28 cm high), and there are transparent plexiglass sidewalls for observations. The confinement areas have dimensions 14.5 by 15 cm, with a lid 14 cm above the floor. At one end of the floor during experiments is a metal container filled with ice, and at the other end underneath the floor is a heat box set at 62°C. Thermocouple wires are attached between the under side of the floor and a layer of Styrofoam insulation, at the center of each zone. The equipment was allowed to equilibrate to the required floor temperatures for at least 1 h before

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each experiment. The floor temperature for the five zones were 11±1, 17.5±1, 22.5±1, 29±1.5, and 39.5±1.5°C and were measured continuously throughout each experiment. Preparation and administration of drugs All drugs were given as the hydrochloride salt and were dissolved in 0.9% saline to give concentrations of 4, 12, 26, or 40 mM cocaine, methamphetamine, MDMA, and PMA. Cocaine was also made up to a concentration of 80 mM. Doses of each drug were administered at 1 ml/kg via intraperitoneal injection at doses of 4, 12, 26, 40, or 80 μmol/kg. These doses equate to 1.36, 4.08, 8.83, 13.59, and 27.18 mg/kg for cocaine, 0.74, 2.23, 4.84, and 7.44 mg/kg for methamphetamine, 0.92, 2.76, 5.97, and 9.19 mg/kg for MDMA, and 0.81, 2.24, 5.25, and 8.08 mg/kg for PMA. These doses were chosen based on our previous work and to determine dose–response relationships. Control treatment consisted of rats receiving the same dose volume of saline only. Each group of rats was administered only one drug at each dose with 1 week between administration of each dose to allow sufficient time for each drug to be cleared from their system (Law and Moody 1994). The lower doses were given first in each case, as these are doses that have been shown to not be neurotoxic in rats (Cappon et al. 1998; Green et al. 2003; Kita et al. 2003). All drugs were obtained from The Australian Government Analytical Laboratories (Sydney, Australia). Data acquisition Rats were surgically implanted with telemetry devices (TA11CTA-F40, Data Sciences International), which measure core body temperature, activity, and electrocardiogram, as reported previously (Bexis et al. 2004). The implants were placed into the rats’ abdominal cavity under anesthesia (sodium pentobarbital, 60 mg/kg). Two weeks recovery from surgery was allowed before rats underwent any injection treatments. Radio receivers, placed to the side of the runway, received information from the implants and transferred it to a computer which recorded the data using Dataquest LabPro software (Data Sciences International). Data were recorded every 2 min over the experimental period. Experimental protocol During the week before testing began, rats were habituated to the apparatus by being placed in the warm area with an ambient temperature of 30±1°C for 30 min on two separate occasions. They were also placed in the thermal gradient on two separate occasions, once directly from the home cage and once after being in the warm ambient temperature for 30 min. On experimental days, rats were taken from their home cage, administered either saline or drug, and placed in to the warm ambient temperature for 30 min. Our previous study showed that 30 min was an appropriate length of time to elicit a

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significant but not dangerous change in body temperature after 10 mg/kg MDMA or PMA at this high ambient temperature. Eight rats were used for each drug tested. At the end of the 30 min (time [t]=0), rats were allowed access to the thermal gradient for 4 h to choose their preferred floor temperature. Temperature preference (T P ) was recorded as the zone each rat was in at the end of every 2-min period. Core body temperature (TC), locomotor activity, and heart rate (HR) were recorded via telemetry every 2 min. Grooming behavior was rated for 30 s as an all or nothing response every 5 min. Neurotransmitter analysis To confirm the doses of stimulants used were not neurotoxic, concentrations of 5-HT, 5-hydroxyindole acetic acid (5-HIAA), DA, and dihydroxyphenyl acetic acid (DOPAC) in the cortex were measured. One week after the final dose of drug, rats were anesthetized (sodium pentobarbital, 60 mg/kg), decapitated, and their brain removed and frozen. The cerebral cortex was dissected from the brain, and tissue samples were prepared for highperformance liquid chromatography (HPLC) as described by Callaghan et al. (2006). The HPLC-electrochemical detector system consisted of a BAS LC-4B fitted with a working electrode potential set at 0.7 V with a range of 1 nA. The mobile phase composed of: NaH2PO4 14.20 g/l, octanesulphonic acid 108.2 mg/l, ethylenediamine tetraacetic acid 37.2 mg/l, and 12.5% methanol and was adjusted to pH 3.8 with phosphoric acid. The mobile phase was delivered at a flow rate of 1 ml/min. Compounds of interest were separated using a 250×4.6 mm 5 μ C18 column (Allinta), and sampling was recorded using an ICI DP800 Chromatograph Data Station (Version 2.5). Statistical analysis All calculations and analysis were done using Graph Pad Prism software. Area under the curve values for TC and TP over time were calculated for the time periods: 0–30, 30–60, 60–120, 120–180, and 180–240 min and analyzed between treatments for each drug group. Dose–response curves were constructed for the maximum increase in temperature elicited by each drug in the warm environment and effective dose, 50% (ED50) values compared between drugs. Percentage increase was calculated by setting a 4°C increase as a 100% response, and the mean increase in saline-treated animals considered a 0% response. A 4°C increase in TC was considered the maximum response possible as evidenced by the fact two of the rats treated with 40 μmol/kg PMA died after their TC exceeded this change and could not be used for any analysis of effects on rats during their time in the thermal gradient. Linear regression calculations were performed to determine the slopes of the dose–response curves and the rate of decrease in core temperature of rats after they were let out of the heat. The mean percentage of rats grooming at

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Fig. 1 Core body temperature after administration of cocaine at 4, 12, 26, 40, and 80 μmol/kg while confined to a warm environment (30±1°C) followed by 4 h in a thermal gradient. Core temperature was measured every 2 min but time points for only every 6 min are shown for clarity. Significant differences between doses are also not shown for clarity, these are summarized in the text. The lower panel shows the mean preferred temperature on the thermal gradient of the rats over time periods: 0–30, 30–60, 60–120, 120–180, and 180– 240 min. Each time period has been analyzed separately using a one-way ANOVA with Tukey’s post hoc test between doses. *, #, ^, $, + Significant difference compared with saline, 4, 12, 26, and 40 μmol/kg, respectively. *P