Class of 2012 Abstract / Specific Aims: The purpose of this study is to identify the incidence of symptoms associated after exposure to “bath salts,” a term for synthetic cathinones in Arizona. Methods: This is a retrospective chart review of reported exposures to synthetic cathinones to the Arizona Poison and Drug Information Center and the Banner Good Samaritan Poison and Drug Information Center. Main Results: There were 306 cases of synthetic cathinone exposures reviewed and 76.5% were males (n=234) and 23.5% were females (n=72). They were ingested, inhaled, snorted, or injected. The mean age of exposure to synthetic cathinones was 29 years old. The most common symptoms included agitation (48.7%), hallucinations (27.1%), confusion (17.6%), hypertension (21.9%), tachycardia (50.6%), CK elevation (17.3%) and chest pain (9.5%). Less frequent symptoms exhibited in synthetic cathinone abuse included other CNS effects, gastrointestinal symptoms, muscular dysfunction, visual disturbances, and respiratory issues. Conclusions: The symptoms exhibited after exposure to synthetic cathinones were mainly neurologic and cardiovascular. In most cases, symptoms were effectively resolved within 24 to 48 hours after treatment with intravenous fluids and benzodiazepines. In some reports, patients were also given oxygen, anti-emetics, sedatives and anti-psychotic medications. Medical outcomes included major (1.6%), moderate (42.2%) and minor effects (26.1%) while 92 patients were lost to follow-up.

The abuse of synthetic cathinones, formerly marketed as “bath salts”, has emerged over the last decade. Three common drugs in this class include 3,4-methylenedioxypyrovalerone (MDPV), 4-methylmethcathinone (mephedrone), and 3,4-methylenedioxymethcathinone (methylone). An LC–MS/MS method has been developed and validated for the simultaneous quantification of MDPV, mephedrone, and methylone in brain tissue. Briefly, MDPV, mephedrone, methylone, and their deuterium-labeled analogs were subjected to solid phase extraction (SPE) and separated using an HILIC Silica Column. The HPLC was coupled to a Shimadzu IT-TOF (ion trap-time of flight) system with the electrospray source running in positive mode (+ESI). The method was validated for precision, accuracy, and extraction efficiency. All inter-day and intra-day % RSD (percent relative standard deviation) and % error values were less than 15% and extraction efficiency exceeded 80%. These conditions allowed for limits of detection of 1ng/mL for MDPV, and 5 ng/mL for both mephedrone and methylone. The limits of quantification were determined to be 5ng/mL for MDPV and 10 ng/mL for mephedrone and methylone. The method was utilized to evaluate the pharmacokinetics of these drugs in adult male rats following administration of a drug cocktail including MDPV, mephedrone, and methylone. All three compounds reached peak concentrations in the brain within 15 min. Although methylone and mephedrone were administered at the same dose, the peak concentration (Cmax) of mephedrone in the brain was significantly higher than that for methylone, as was the area under the curve (AUC). In summary, this quick and sensitive method for measuring synthetic cathinones may be used for future pharmacokinetic investigations of these drugs in target tissue.

Synthetic cathinones, commonly referred to as “bath salts”, are a subgroup of novel psychoactive substances that have seen a dramatic rise in abuse worldwide over the past decade. These compounds are synthesized by clandestine drug manufacturers using basic medicinal chemistry techniques, and marketed as “legal high” alternatives to illicit psychostimulants (ie. cocaine and MDMA). Two of the most common synthetic cathinones since the emergence of this class of drugs are 4-methylmethcathinone (mephedrone, MEPH) and 3,4-methylenedioxypyrovalerone (MDPV). The novelty of these compounds in the illicit drug marketplace has limited the current understanding of synthetic cathinone neuropharmacology. Our studies, as outlined in this dissertation, aimed to further characterize the neuropharmacology of MEPH and MDPV, specifically evaluating the contributions of stereospecific mechanisms in the monoaminergic systems, as well as the role of the glutamatergic system in mitigating reward, reinforcement, and relapse to drug seeking. We first evaluated MEPH’s ability to produce behavioral sensitization (detailed in Chapter 2), a hallmark behavior of psychostimulants involving repeated, intermittent drug administration, followed by a period of drug abstinence, and a subsequent drug challenge. This evaluation of MEPH’s ability to produce behavioral sensitization was conducted across multiple treatment and dosing paradigms, withdrawal time point intervals, and drug administration contexts. A 7-day, variable-dose administration paradigm (Days 1+7= 15 mg/kg, Days 2-6= 30 mg/kg) and a 5-day, constant-dose administration paradigm (15 mg/kg) both induced enhancement of repetitive movements (i.e. stereotypy), but not ambulatory activity, during a challenge dose following 10 days of drug abstinence. Additionally, with the 7-day variable-dose design, sensitization of repetitive movements was observed following a shorter (2-day) abstinence interval, and before the initiation of MEPH abstinence on Day 7 of MEPH treatment. This sensitization was observed in both context-independent and context-dependent dosing schedules. A lower dose of MEPH (5 mg/kg) in the 5-day constant dose paradigm produced no sensitization of repetitive movements following 10 days of abstinence. Lastly, in all sensitization paradigms employed, no sensitization of ambulatory activity was observed. These data indicate that MEPH produces preferential sensitization of repetitive movements across multiple treatment paradigms, preferentially over ambulatory activity. Our findings suggest that MEPH is a unique stimulant displaying weak sensitizing properties with both amphetamine-like properties, as well as distinctive properties relative to both amphetamine and cocaine. Abusers of synthetic cathinones are often polydrug abusers who seek out compounds like MEPH as a replacement for other psychostimulants that are commonly detected on drug screenings. We investigated interactions of MEPH with cocaine (COC) and methamphetamine (METH), specifically testing the hypothesis that prior MEPH exposure enhances the locomotor-stimulant effects of COC and METH, and vice versa (detailed in Chapter 3). For cocaine experiments, rats were conditioned with saline, cocaine (15 mg/kg), or MEPH (15 mg/kg) for 5 days were given a cocaine challenge (15 mg/kg) after 10 days of drug abstinence. For METH experiments, rats conditioned with saline, METH (2 mg/kg), or MEPH (15 mg/kg) were given a METH challenge (2.0 mg/kg) after 10 days of drug absence. Cocaine challenge produced greater locomotor activity in rats conditioned with cocaine or MEPH than those conditioned with saline. METH challenge produced greater locomotor activity in METH-conditioned rats than saline-conditioned rats; however, locomotor activity in rats conditioned with MEPH or saline and then challenged with METH (0.5 or 2.0 mg/kg) was not significantly different. The locomotor response to MEPH (15 mg/kg) was not significantly affected by conditioning with cocaine (15 mg/kg) or METH (0.5, 2 mg/kg). The present demonstration that cocaine-induced locomotor activation is enhanced by prior MEPH exposure suggests that MEPH cross-sensitizes to cocaine and increases cocaine-evoked locomotor activity. Interestingly, MEPH cross-sensitization was not bidirectional and did not extend to METH, suggesting that the phenomenon is sensitive to specific psychostimulants. Similar to other cathinone and amphetamine-related compounds, MEPH has a chiral center at its alpha carbon, and exists stably as two enantiomers. To further explore enantiomer-specific MEPH neuropharmacology, individual MEPH enantiomers R-MEPH and S-MEPH were examined for their behavioral and neurochemical effects (detailed in Chapter 4). We analyzed both enantiomers in rat brain synaptosome neurotransmitter release assays and investigated each MEPH enantiomer for their acute ambulatory activity and repetitive movements, ability to produce behavioral sensitization, and rewarding properties. Both enantiomers displayed similar potency as substrates (i.e. releasers) at dopamine transporters, but R-MEPH was much less potent than S-MEPH at serotonin (5-HT) transporters. Locomotor activity was evaluated after acute administration of each enantiomer, with R-MEPH producing greater repetitive movements than S-MEPH across multiple doses. Pretreatment with the 5-HT2C antagonist SB242084 significantly increased S-MEPH locomotor activity, indicating 5-HT receptor activation is involved in suppressing S-MEPH locomotor activation. In repeated drug administration paradigms, R-MEPH, but not S-MEPH, produced sensitization of repetitive movements. R-MEPH also produced a conditioned place preference whereas S-MEPH produced no place preference at the doses tested. Lastly, R-MEPH and S-MEPH produced biphasic profiles in an assay of intracranial self-stimulation (ICSS), but R-MEPH produced greater ICSS facilitation than S-MEPH. Our data were the first to demonstrate stereospecific effects of MEPH enantiomers and suggests that the predominant dopaminergic actions of R-MEPH (i.e. the lack of serotonergic actions) render this stereoisomer more stimulant-like when compared to S-MEPH. Following the increased clinical presence of MDPV over MEPH in the United States, and reports from abusers detailing intense cravings to re-dose during drug administration sessions, our studies shifted focus onto the neuropharmacology of MDPV. The first investigation of MDPV evaluated the effects of non-contingent MDPV administration on the glutamate system (detailed in Chapter 5). To date, all pharmacological studies on MDPV have focused on monoaminegic systems, leaving a critical void in the literature. The glutamate system has been extensively studied with psychostimulants with similar monoamine mechanisms to MDPV, and glutamatergic dysregulation is an underlying component in behavioral sensitization and relapse to drug seeking. Two important regulators of glutamate homeostasis are the enzyme glutamate carboxypeptidase II (GCPII) and the glutamate transporter subtype 1 (GLT-1), which contribute to the synthesis and extrasynaptic reuptake of glutamate, respectively. Ceftriaxone (CEF), a beta-lactam activator at the glutamate transporter subtype 1 (GLT-1), has shown preclinical promise in attenuating the rewarding and reinforcing properties of cocaine. We provide the first investigation of the effects of MDPV on GLT-1 and GCPII expression in the reward center, and the role of GLT-1 in MDPV behavior. MDPV effects on GLT-1 and GCPII expression at multiple withdrawal time points following MDPV or saline administration in a 7-day variable-dose paradigm via Western blot. Compared to saline controls, MDPV-treated rats had decreased expression of GLT-1 from Withdrawal Day 2 to Withdrawal Day 10 in the nucleus accumbens, while no changes in GLT-1 expression were observed in the prefrontal cortex. GCPII expression was decreased in MDPV treated rats compared to saline controls at Withdrawal Day 0 in the nucleus accumbens, as well as Withdrawal Day 0 to 10 in the prefrontal cortex. The effects of repeated CEF treatment on acute MDPV locomotor activity was also evaluated across multiple doses of MDPV, and no differences were observed. To evaluate behavioral sensitization, MDPV or saline was administered to rats in a 7-day variable-dose paradigm. Rats in the CEF group received CEF (200 mg/kg IP) for four days prior to MDPV treatment, and received CEF 30 minutes prior to each MDPV injection. Following 10 days of MDPV abstinence, a challenge dose (0.5 mg/kg MDPV) was administered and locomotor activity was recorded. Sensitization of repetitive movements was observed with repeated administration of MDPV, and this sensitization was attenuated in rats pretreated with CEF. MDPV’s reward was evaluated using a 4-day conditioned place preference model. MDPV (2.0 mg/kg IP) produced significant place preference compared to saline, and this effect was attenuated with pretreatment with CEF. These data indicate that repeated MDPV exposure decreases GLT-1 and GCPII expression in the mesolimbic reward center, and that pharmacological activation of GLT-1 may be a viable target for developing therapeutics to attenuate the rewarding effects MDPV. To further expand on the role of GLT-1 in MDPV abuse liability, CEF and the cysteine-glutamate antiporter (xCT) substrate N-acetylcysteine (NAcetyl) were evaluated in operant intravenous self-administration (IVSA) models, including fixed-ratio 1 (FR-1) self-administration and reinstatement to drug seeking (detailed in Chapter 6). The first experiment assessed CEF and NAcetyl treatment when administered after MDPV IVSA had cease (i.e. during extinction procedures). Rats were trained to self-administer MDPV (0.056 mg/kg/infusion) in daily 2 hours sessions for 14 days, during which ultrasonic vocalizations (USVs) were recorded. Following acquisition of MDPV self-administration, rats were pretreated daily with either saline, CEF (200 mg/kg) or NAcetyl (100 mg/kg) 30 minutes prior to extinction procedures for 10 days. One day after extinction, rats underwent cue-induced reinstatement procedures in the absence of CEF/NAcetyl, followed 24 hours later by a cue+MDPV-primed reinstatement procedures, where a non-contingent MDPV injection (0.5 mg/kg) was administered immediately prior to the reinstatement session. Neither CEF nor NAcetyl altered the rate of extinction of MDPV drug seeking, nor did either treatment attenuate cue- or cue+MDPV-primed reinstatement. After observing no differences in treatment with CEF or NAcetyl during extinction of MDPV drug seeking, our second experiment explored CEF and NAcetyl against the acquisition of MDPV self-administration, as well as the effects of CEF and NAcetyl administered throughout acquisition on reinstatement. Rats were treated with either saline, CEF (200 mg/kg) or NAcetyl (100 mg/kg) daily for 10 days prior to the start of acquisition of MDPV IVSA. Rats continued saline/CEF/NAcetyl daily treatment 30 minutes prior to acquisition of MDPV self-administration for 14 days. After acquisition, rats underwent 10 days of extinction procedures in a drug-free state and reinstatement procedures identical to the first experiment. Pretreatment with CEF, but not NAcetyl, resulted in significantly less active lever presses and reinforcers throughout acquisition, as well as an increase in latency of active lever pressing (i.e. an increase in time spent between reinforcers) during the early load-up phase across the second week of acquisition. Neither treatment was efficacious in attenuating cue- or cue+MDPV-primed relapse to MDPV seeking. Further characterization of the rewarding and reinforcing properties of MDPV were performed during cocaine self-administration by quantifying positive affective ultrasonic vocalizations (USVs) in rats self-administering MDPV versus cocaine. After rats acquired IVSA, rats self-administering MDPV (0.056 mg/kg/infusion) produced a greater calling rate and slower decay of 50 kHz calls per infusion, compared to cocaine (0.56 mg/kg/infusion). Latency to active lever pressing was lower in MDPV rats compared to cocaine, indicating that rats self-administering MDPV wait a smaller amount of time between doses than cocaine. In summary, the experiments described in this dissertation aimed to highlight various aspects of the neuropharmacology of MEPH and MDPV; two pharmacologically distinct synthetic cathinones that are both commonly abused and serve as a pharmacological template for the development of second generation synthetic cathinones. MEPH produces locomotor behaviors similar to that of pharmacologically similar psychostimulants, as well as bi-phasic cross-sensitization with cocaine. Locomotor and reward behaviors observed with MEPH administration are stereospecific, with the R-enantiomer of MEPH possessing the more dopaminergic and stimulant like profile. Repeated MDPV administration and withdrawal induces depletions in GLT-1 and GCPII in the reward center, and pharmacologically targeting GLT-1 with CEF attenuates MDPV sensitization, reward, and reinforcement. Despite evaluating CEF and NAcetyl in multiple paradigms of administration, neither compound was found to be efficacious in attenuating relapse to MDPV seeking. MDPV self-administration produces a greater positive affective status, compared to cocaine, throughout the latter parts of acquisition of IVSA. These studies have identified crucial differences in the behavioral profile and neuroadaptations expressed during and after MDPV versus cocaine. In conclusion, our studies have expanded the neuropharmacology knowledge base of these two synthetic cathinones, MEPH and MDPV, and provide a strong foundation for future investigations into the neuropharmacology of this constantly-evolving class of drugs. The stereoselectivity of MEPH enantiomers towards the more rewarding R- enantiomer, compared to the S- enantiomer possessing a more serotonergic and less stimulant-like profile indicates that the change in steric orientation around the chiral carbon at MEPH is critically involved in dopaminergic and rewarding activity. This observation may be useful in the development of future pharmacotherapies aimed at targeting pathologies with a mixed monoaminergic substrate activity, similar to the cathinone bupropion. Additionally, our studies with MDPV have identified MDPV as a highly reinforcing and rewarding psychostimulant, with notable potency differences compared to cocaine. While our efforts to attenuate reinstatement of MDPV-seeking the promising compounds CEF and NAcetyl were unsuccessful, the lack of efficacy in these reinstatement studies continue to underlie the importance of investigating pharmacotherapies against MDPV reinstatement. The conclusions in this dissertation should be used as foundation for future studies investigating both MEPH and MDPV, as well as second-generation cathinones that continue to emerge as the problem of novel psychoactive substances evolves and persists. / Pharmacology

Synthetic cathinones represent threatening and high abuse-potential designer drugs. These are analogs of cathinone (the b-keto analog of amphetamine (AMPH)) a naturally occurring stimulant found in the plant Catha Edulis. Methcathinone (MCAT) was the first synthetic analog of cathinone to be identified in 1987 by Glennon and co-workers and it exerted its action primarily through the dopamine transporter (DAT). Most central stimulants exert their action via monomaine transporters by causing either the release (e.g. cathinone analogs such as MCAT) or by preventing the reuptake (e.g. cocaine) of the neurotransmitter dopamine (DA) thus increasing the extracellular synaptosomal concentration of this neurotransmitter. In 2010, a new class of designer cathinone-like drugs called ‘bath salts’, initially a combination of methylenedioxypyrovalerone (MDPV), methylone (methylenedioxymethcathione, MDMC) and mephedrone (MEPH), soared to popularity. It caused extremely detrimental side effects; it was exceedingly popular for its recreational use and posed a threat to public health. At the time, their mechanisms of action were unknown. Our group identified that MDPV produced actions distinct from other cathinone analogs (i.e., it was identified as the first cathinone-like compound to act as a reuptake inhibitor at the dopamine transporter (DAT)). These findings suggested that not all cathinone-like compounds act uniformly and this insinuated that unique structural features on the cathinone scaffold might contribute to different effects at the transporter level. The overall goal of this project was to study the mechanisms of action of synthetic cathinones (including ‘bath salts’) at the monoamine transporters. We investigated the contribution of each of various structural features on the cathinone scaffold (i.e, the terminal amine, a and b positions, and the phenyl ring). We also constructed homology models of the human dopamine and serotonin transporters (hDAT and hSERT respectively) to help explain differences in selectivity between the neurochemical and behavioral aspects of DAT and SERT. Overall we found that structural features contributed to similar or distinctive mechanisms of action and also contributed to selectivity at monoamine transporters. Our studies provide information that can be useful to drug and health regulatory agencies to help prevent, treat, or curb the future abuse of such drugs.

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.

The synthetic cathinones, 3,4- methylenedioxypyrovalerone (MDPV), 4-methylmethcathinone (mephedrone), and 3,4- methylenedioxymethcathinone (methylone), gained worldwide notoriety as the psychoactive components of ‘bath salts;’ a marketing term used to circumvent federal drug laws and permit their legal sale. Previous studies have shown that these drugs share pharmacological characteristics with cocaine and the amphetamines, however, descriptions of their neurotoxic properties are limited. Moreover, while forensic analysis has revealed that the most frequently abused bath salts ‘brands’ contain binary and ternary mixtures of MDPV, mephedrone, and methylone, the majority of preclinical research has focused on explicating the individual effects of these drugs. Therefore, the present dissertation aimed to address this limitation and characterize the acute and chronic effects of combined synthetic cathinone exposure on dopaminergic tone in mesolimbic and nigrostriatal brain regions. To accomplish this, male Swiss-Webster mice were administered MDPV, mephedrone, and methylone, individually or concomitantly, 1 time or 7 times over the course of two weeks and the corresponding effects of each treatment on mesolimbic and nigrostriatal brain tissue levels of dopamine (DA) and DA metabolites were analyzed using a high performance liquid chromatography – electrochemical detection (HPLC-ECD) assay. Additionally, motor-stimulant activity was evaluated after both dosing regimens using locomotor activity assays, while immunoblot and immunostaining techniques were used to evaluate the chronic effects of co-synthetic cathinone exposure on tissue levels of tyrosine hydroxylase (TH), dopamine transporter (DAT), monoamine oxidase B (MAO-B), catechol-O-methyltransferase (COMT), and glial fibrillary acidic protein (GFAP). Results from these studies provide evidence of a significant pharmacological interaction among major bath salt constituents, MDPV, mephedrone, and methylone. This was observed acutely as enhanced DA responses and chronically as functional toxicity at the DA synapse. Furthermore, such interactions may contribute to the deleterious effects reported by bath salt users. Together, these findings have shown that the composition of bath salts preparations can significantly influence their psychostimulant and toxic effects, substantiating the importance of modeling bath salts as drug mixtures.