Originally developed as an anti-depressant and later available as a ‘party-pill’ in New Zealand, methylone is currently classed as an illegal drug. This is due to findings of its similarity in chemical structure to that of Ecstasy (MDMA). Methylone is a relatively new drug into which little research has been conducted. Consequently, no known study has investigated the long-term effects on behavioural development arising from exposure during adolescence. The present thesis therefore aimed to identify long-term effects of chronic adolescent exposure to methylone on adult anxiety-like behaviours. This was achieved by the use of 80 rats (40 males: 40 females) and exposing them to either a methylone or saline treatment for ten consecutive days. Two different treatment age groups (early versus late adolescence) were examined and to ensure adequate comparisons could be made, two control groups were utilised. All rats were tested during adulthood in four specifically selected anxiety-measure tests; the open-field, preference for the light side of a light-dark box, acoustic startle and responsiveness to the novel arm of a Y-maze. The results suggested methylone-exposed rats displayed more anxiolytic behaviours than saline-treated rats. In the open field methylone exposed rats exhibited less ambulation than controls and those treated in early adolescence defecated more while rats treated in late adolescence occupied the corners of the apparatus more exhibiting higher anxiety-like behaviours. Exploratory behaviours in the Y-maze were decreased in methylone-treated rats, and those exposed in early adolescence entered the novel arm less often. However, acoustic startle results suggested methylone-exposed rats were less anxious as evidenced by a lower startle amplitude than controls. Overall, the results suggested differences in anxiety-like behaviours between methylone-exposed rats and controls. It did not appear that being exposed to methylone in early adolescence resulted in vast differences in anxiety-like behaviours than if exposure began in late adolescence.

abstract: Globally, addiction to stimulants such as methamphetamine (METH) remains a significant public health problem. Despite decades of research, no approved anti-relapse medications for METH or any illicit stimulant exist, and current treatment approaches suffer from high relapse rates. Recently, synthetic cathinones have also emerged as popular abused stimulants, leading to numerous incidences of toxicity and death. However, contrary to traditional illicit stimulants, very little is known about their addiction potential. Given the high relapse rates and lack of approved medications for METH addiction, chapters 2 and 3 of this dissertation assessed three different glutamate receptor ligands as potential anti-relapse medications following METH intravenous self-administration (IVSA) in rats. In chapters 4 through 7, using both IVSA and intracranial self-stimulation (ICSS) procedures, experiments assessed abuse liability of the popular synthetic cathinones 3,4-Methylenedioxypyrovalerone (MDPV) , methylone, α-pyrrolidinovalerophenone (α-PVP) and 4-methylethylcathinone (4-MEC). Results from these seminal studies suggest that these drugs possess similar abuse potential to traditional illicit stimulants such as METH, cocaine, and 3,4-methylenedioxymethamphetamine (MDMA). Finally, studies outlined in chapter 8 assessed the potential neurotoxic or adverse cognitive effects of METH and MDPV following IVSA procedures for the purpose of identifying potential novel pharmacotherapeutic targets. However, results of these final studies did not reveal neurotoxic or adverse cognitive effects when using similar IVSA procedural parameters that were sufficient for establishing addiction potential, suggesting that these parameters do not allow for sufficient drug intake to produce similar neurotoxicity or cognitive deficits reported in humans. Thus, these models may be inadequate for fully modeling the adverse neural and psychological consequences of stimulant addiction. Together, these studies support the notion for continued research into the abuse liability and toxicity of METH and synthetic cathinones and suggest that refinements to traditional IVSA models are needed for both more effective assessment of potential cognitive and neural deficits induced by these drugs and screening of potentially clinically efficacious pharmacotherapeutics. / Dissertation/Thesis / Doctoral Dissertation Psychology 2014

“Bath salts” and “plant food”, which were legally marketed synthetic cathinones, have a high potential for abuse. Several recent studies indicate that 3,4-methylenedioxypyrovalerone (MDPV) and 3,4-methylenedioxymethcathinone (methylone), two common drugs of this type, have similar pharmacology to controlled psychostimulants such as cocaine, methamphetamine, and methylphenidate. MDPV acts as a norepinephrine (NE) and dopamine (DA) reuptake inhibitor via blockade of their transporters (DAT and NET), whereas methylone is a substrate for the NE, DA, and serotonin (5-HT) transporters, increasing the non-vesicular release of these monoamines. Both drugs cause significant increases in the levels of these neurotransmitters in the cleft. Increases in DA are associated with euphoric effects and thus promote drug abuse and addiction, hence the high addiction potential of MDPV and methylone. Indeed, MDPV is 50 times more potent at the DAT and 10 times more potent at the NET than cocaine. Here, we examined the pharmacokinetics of MDPV and methylone in the brain and plasma, following intraperitoneal injection in mice. These types of injections have similar pharmacokinetics to insufflation (snorting), which is the manner in which MDPV and methylone are commonly abused. Briefly, adolescent male Swiss-Webster mice were injected intraperitoneally with either 10 mg/kg MDPV or 10 mg/kg methylone, and brains and plasma were collected at the following time points: 1, 10, 15, 30, 60, and 120 minutes. Samples were then flash-frozen and stored at -70°C until analysis. Samples were spiked with deuterium-labeled MDPV or methylone (internal standards), and the drugs were extracted from tissue using a previously published solid phase extraction method. Chromatographic separation of the compounds was achieved using a HILIC column with a gradient elution of acetonitrile and 5 mM ammonium formate at a flow rate of 0.2 mL/min. Mass spectrometric detection utilized a Shimadzu IT-TOF system with the electrospray source running in positive mode. Data acquisition utilized a direct MS-MS method using a precursor ion of 276.3 m/z for MDPV and methylone. The calibration curve ranged from 100 ng/ml to 0.1 ng/ml. These conditions allowed for a lower limit of detection (LLOD) of less than or equal to 1 ng/mL and a lower limit of quantification (LLOQ) of less than or equal to 5 ng/mL for MDPV and methylone. MDPV and methylone peak concentrations in plasma were seen immediately at 1 min, while brain concentrations peaked at 15 min; however, MDPV reached higher concentrations in the brain the methylone. This is consistent with MDPV’s higher lipophilicity (logP value). In conclusion, the pharmacokinetic profile of these drugs reflects a quick uptake and distribution of the drugs to the brain, followed by the quick distribution out of the brain, which likely contributes to the binge use of these drugs.

Earlier this decade, “bath salts” were popularized as legal alternatives to the pyschostimulants cocaine and the amphetamines. These products contained synthetic cathinones including 3,4-methylenedioxypyrovalerone (MDPV), 4-methylmethcathinone (mephedrone), and 3,4-methylenedioxymethcathinone (methylone). Studies indicate that the cathinones have similar pharmacology to controlled psychostimulants, increasing levels of dopamine (DA) in the synaptic cleft. Most preclinical investigations have only assessed the effect of these synthetic cathinones independently; however, case reports and DEA studies indicate that “bath salts” often contain mixtures of these substances. Therefore, in a recent study by our laboratory, we examined effects of individual versus combined exposure to MDPV, mephedrone, and methylone. Interestingly, an enhanced effect on the levels of DA was observed, as well as significant alterations in locomotor activity following co-exposure to the cathinones. In this study, we examine whether the enhanced effects of the drug combination were due to pharmacokinetic (PK) interactions. It is known that many of the same cytochrome P450 (CYP) isoenzymes metabolize each of these three drugs. Therefore, it is probable that the drugs’ PK would differ when administered individually as compared to in combination. We hypothesize that combined exposure to MDPV, mephedrone, and methylone will result in increased drug concentrations and enhanced total drug concentrations when compared to individual administration. The pharmacokinetics of MDPV, mephedrone, and methylone in the brain and plasma were examined following intraperitoneal injection in mice. Briefly, adolescent male Swiss-Webster mice were injected intraperitoneally with either 10 mg/kg MDPV, 10 mg/kg mephedrone, 10 mg/kg methylone, or 10 mg/kg combined MDPV, mephedrone, and methylone. Following injection, brains and plasma were collected at the following time points: 1, 10, 15, 30, 60, and 120 minutes. Samples were then flash-frozen and stored at -70°C until analysis. Drugs were extracted via solid-phase extraction and concentrations were determined using a previously validated and published high pressure-liquid chromatography tandem mass spectrometry (LC-MS/MS) method. Following intraperitoneal administration, all drugs quickly crossed the blood-brain barrier and entered the brain. Peak drug concentrations, time to peak concentration, drug half-lives, and total drug exposure (as measured by area under the curve) are compared when drugs were given individually versus in combination. These data provide insight into the consequences of co-exposure to popular “bath salt” products.

An unprecedented rise in the availability of new synthetic drugs of abuse has been observed in the recent years. One of the most noted cases is that of a popularized designer drug mixture known as ‘bath salts’. Commonly obtained from various shops and on the internet, “bath salts” often contain the synthetic cathinones 3,4 methylenedioxypyrovalerone (MDPV), mephedrone, and methylone in diverse combinations. Individually, the synthetic cathinones are known to have similar pharmacology to controlled psychostimulants such as cocaine and the amphetamines, increasing the levels of dopamine (DA) in the synaptic cleft. DA is an important neurotransmitter that regulates a variety of behaviors and functions; neurons within the mesolimbic DA pathway (ventral tegmental area to nucleus accumbens) are involved in reward and motivation and are activated by these drugs of abuse. Additionally, psychostimulant-induced increases in DA in the nigrostriatal pathway (substantia nigra to corpus striatum) lead to increases in locomotor behavior. However, the majority of preclinical investigations have only assessed the effects of individual bath salt constituents and have provided little information regarding the possibility of significant drug interactions with the co-exposure of MDPV, mephedrone, and methylone. This study sought to evaluate and compare the effects of individual versus combined MDPV, mephedrone, and methylone on dopamine (DA) levels in discrete brain regions as well as motor stimulant responses in mice. Male adolescent Swiss-Webster mice received intraperitoneal injections of saline, MDPV, mephedrone, methylone (1.0 or 10.0 mg/kg), or the cathinone cocktail (MDPV + mephedrone + methylone at 1.0, 3.3, or 10 mg/kg). The effect of each treatment on DA and DA metabolite levels in mesolimbic and nigrostriatal brain tissue was quantified 15 min after a single exposure utilizing high pressure liquid chromatography with electrochemical detection (HPLC-ECD). Additionally, locomotor activity was recorded in mice after acute (day 1) and chronic intermittent (day 7) dosing. The results demonstrate that MDPV, mephedrone, and methylone produce dose-related increases in the mesolimbic and nigrostriatal DA levels that are significantly enhanced following their co-administration. Additionally, a decrease in locomotor activity on day 1 that was exacerbated by day 7 was noted in mice treated with the cathinone cocktail and was not observed with any of the single agents. The decrease in locomotor activity was accompanied by an increase in stereotypic-like behavior including excessive grooming and even self-mutilation. Our findings demonstrate a significantly enhanced effect of MDPV, mephedrone, and methylone on both DA and its metabolites resulting in significant alterations in locomotor activity. This work provides insight into the potential enhanced risk of the use of these combination synthetic cathinone products.

Designer drug mixtures popularized as “bath salts” often contain the synthetic cathinones 3,4 methylenedioxypyrovalerone (MDPV), mephedrone, and methylone in various combinations. However, most preclinical investigations have only assessed the effects of individual bath salt constituents, and little is known about whether co-exposure to MDPV, mephedrone, and methylone produces significant neuropharmacological interactions. This study evaluated and compared how MDPV, mephedrone, and methylone influence discrete brain tissue dopamine (DA) levels and motor stimulant responses in mice when administered alone and as a ternary mixture. Male adolescent Swiss-Webster mice received intraperitoneal injections of saline or 1 or 10 mg/kg doses of MDPV, mephedrone, or methylone, or a cocktail of all three cathinones at doses of 1, 3.3, or 10 mg/kg each. The effect of each treatment on DA and DA metabolite levels in mesolimbic and nigrostriatal brain tissue was quantified 15 min after a single exposure using HPLC-ECD. Additionally, locomotor activity was recorded in mice after acute (day 1) and chronic intermittent (day 7) dosing. MDPV, mephedrone, and methylone produced dose-related increases in mesolimbic and nigrostriatal DA levels that were significantly enhanced following their co-administration. In addition, mice treated with the cathinone cocktail displayed decreased locomotor activity on day 1 that was exacerbated by day 7 and not observed with any of the drugs alone. Our findings demonstrate a significant enhanced effect of MDPV, mephedrone, and methylone on both DA, and these effects on DA result in significant alterations in locomotor activity.

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