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.

Novel psychoactive substances (NPS) are a novel problem of the drug scene. NPS mimic effects of the "classic" illicit drugs, but since they have a different chemical structure, they are usually not covered by legislative control. The exact nature of the aforementioned effects depends merely on the description by users, with a proper scientific assessment still absent. Aim of this study is to evaluate effects and addictive potential of naphyrone (a derivative of cathinone) in Wistar rats. High concentration of naphyrone in the brain tissue discovered by pharmacokinetic analysis proved its high blood-brain barrier permeability. Brain level of naphyrone peaked at approximately 30 min after the treatment, nearly at the same time as in the serum. Since naphyrone administration significantly rises body temperature and increases overall locomotion, its stimulant effect is prominently apparent. Our study failed to prove any effect of naphyrone on sensorimotor gating. Tendency to produce conditioned place preference was observed but was not significant. My thesis reports on initial and novel findings about impact of naphyrone administration on physiological parameters of the animal model.

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.

Background: The rapid diffusion of Novel Psychoactive Substances (NPS) constitutes an important challenge in terms of public health and a novelty in clinical settings, where these compounds may lead to erratic symptoms, unascertained effects and multi-intoxication scenarios, especially in emergency situations. The number of NPS available on the illicit drug market is astonishing: official reports suggest the appearance of a new drug every week. NPS may be enlisted in many different families such as synthetic phenethylamines, tryptamines, cathinones, piperazines, ketamine-like compounds, cannabimimetics and other plant-derived, medical products and derivatives. Therefore, healthcare services and professionals are often called to face this unknown 'galaxy' where NPS users seem to perceive traditional services 'unfitting' for their needs, requiring an attention which is quite different from known classic drug abusers. In this context, the Recreational Drugs European Network (ReDNet), a research project funded the European Commission and led by the University of Hertfordshire, aimed to explore the NPS galaxy and develop information tools for vulnerable individuals and professionals working with them. This initiative reported specific Technical Folders on new drugs and disseminated the collected information through innovative communication technologies (e.g. multimedia tools, social networking and mobile phone services) internationally. Aim and objectives: The aim of this work is to evaluate and contribute to expand the knowledge of health professionals on NPS. The key objectives are: 1) to assess the level of knowledge on NPS amongst a sample of Italian healthcare professionals; 2) to evaluate the effectiveness of dissemination tools developed by ReDNet, including an SMS-Email/mobile service (SMAIL); 3) to understand the clinical impact of NPS by providing four Technical Folders and collecting two clinical cases on NPS. Methodology: According to the objectives, the methodological approach has been articulated in the following three phases. Phase 1: investigating knowledge and preferred channels of information via an online survey among health professionals in Italy. This first Italian study on NPS awareness had been online from February to July 2011, recruiting participants from Departments of Addiction, Psychiatry and other services. Phase 2: evaluating the ReDNet initiative. An evaluation questionnaire was designed and disseminated online to assess the various resources provided by ReDNet project; it had been online from April to July 2013, targeting professionals registered to ReDNet services. This phase also investigated the SMAIL service, a mobile application that was the latest technological tool developed by ReDNet team. Phase 3: promoting evidence based work in clinical practice through the preparation of four Technical Folders and two case reports. Technical Folders followed the methodology optimised during the ReDNet experience, organising NPS data under specific headings, measured for the need of health professionals. Case reports were collected in a Dual Diagnosis Unit in Italy ('Casa di Cura Parco dei Tigli'); assessed patients revealed for the first time the use of NPS; clinical interviews were conducted to collect a full anamnesis while for the first time psychopathological characteristics were measured in NPS abusers, using a psychometric instrument (MMPI-2). Results: In Phase 1 Italian services, in particular interviewees (n=243) from Departments of Psychiatry and Addiction, showed a strong interest for the subject but a poor understanding of NPS: 26.7% of respondents did not know if their patients ever used NPS; at the same time they considered this phenomenon as very relevant to their profession (e.g. psychomotor agitation [75.7%], errors in the assessment [75.7%], management of the clients [72%]); in addition less of a quarter of them had reliable information on new substances. Interviewees also reported the need for easily accessible channels of information to expand their expertise in the field (including emails [70%] and dedicated websites [51.9%]). The ReDNet initiative (Phase 2) reached professionals (n=270) from European countries and various other regions; they appreciated the website above all (48.5%), which provided access to other information (in form of academic papers, news, technical folders, etc.). The integration of technological-based and classic educational resources was used to self-educate professionals (52.6%) and supply information for research (33.7%) with up-to-date and 3 reliable information; in the same Phase the SMAIL service was analysed in its first 557 searches: in the pilot period 122 professionals used SMS inquiries (95%), asking information on NPS while highlighting the increasing number of NPS available on the market. Technical folders (Phase 3) described two new phenethylamines (Bromo-dragonfly and 25I-NBOMe), a novel ethno drug (Kratom) and a new synthetic cathinone (alpha-PVP) whose severe effects were also described in one of the clinical cases. The first case report (Alice) involved a clubber who used mephedrone and other NPS with a severe worsening of her psychiatric disturbances; the second one (Marvin) described a patient who was referred by a psychiatric service and revealed himself as a 'psychonaut' with an intense abuse of alpha-PVP. Conclusions: The exploration of the NPS galaxy is a new challenge for healthcare professionals. In this study, Italian services seemed to be unprepared to face the emergency and requested rapid access to reliable information; the ReDNet project provided both technology-based and traditional resources to expand knowledge on NPS, making professionals more aware of emerging issues and helping especially clinicians working in the field (e.g. via SMAIL service and Technical Folders). Overall, it can be observed that effective information services on NPS targeted at professionals initiatives should include an online interface integrating up-to-date information, describing NPS through specific Technical Folders and disseminating scientific literature; the use of technological tools, including mobile applications, is an important strategy to support health professionals in their activity. Finally, more 'visual' guidelines, possibly in the form of a 'map' of these heterogeneous compounds, could be a useful framework to describe NPS to physicians and other professionals who are often unprepared and unconfident to face such an expanding galaxy.

In recent years, the abuse of synthetic cathinones or ‘bath salts’ has become a major public health concern. Although these compounds were initially sold legally and labeled “not for human consumption”, the ‘bath salts’ are psychostimulants, with similar structures and pharmacologic mechanisms to cocaine, the amphetamines, and 3,4 methylendioxymethamphetamine (MDMA, Molly, or Ecstasy). The reported use of these substances by women of child-bearing age highlights the necessity of studies seeking to delineate risks of prenatal exposure. Three popular drugs of this type are methylone, mephedrone, and 3, 4-methylenedioxypyrovalerone (MDPV). Unfortunately, there is currently no information available on the teratogenicity of these compounds, or of the extent to which they cross the placenta. As such, the purpose of this study was to examine the pharmacokinetic profile of the ‘bath salts’ in a pregnancy model. Pregnant mice (E17.5 gestation) were injected intraperitoneally with a cocktail of 5mg/kg methylone, 10mg/kg mephedrone, and 3mg/kg (MDPV) dissolved in sterile saline. Maternal brain, maternal plasma, placenta, and fetal brain were collected at 30s, 1min, 5min, 10min, 15min, 30min, 1h, 2h, 4h, and 8h following injection. Methylone, mephedrone, and MDPV were extracted from tissue by solid phase extraction, and concentrations were determined using a previously validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. Interestingly, all 3 cathinones reached measurable concentrations in the placenta, as well as the fetal brain; in fact, for MDPV, the maximal concentration (Cmax) was highest in fetal brain, while mephedrone's highest Cmax value was achieved in placenta. Additionally, the total drug exposure for all 3 compounds (as represented by area under the curve, AUC) was higher in fetal matrices (placenta and fetal brain) than in maternal matrices (maternal brain and plasma), and the half-lives for the drugs were longer. Given the extensive presence of methylone, mephedrone, and MDPV in the fetal brain following prenatal exposure, fetal risk is definitely a concern. As there are currently no prenatal studies available on the teratogenicity of these agents, pregnant patients should be informed about the potential risks that these substances may have.

Synthetic cathinones and related agents represent an international drug abuse problem, and at the same time an important class of clinically useful compounds. Structure-activity relationship studies are needed to elucidate molecular features underlying the pharmacology of these agents. Illicit methcathinone (i.e., MCAT), the prototype of the synthetic cathinone class, exists as a racemic mixture. Though the differences in potency and target selectivity between the positional and optical isomers of synthetic cathinones and related agents have been demonstrated to have important implications for abuse and therapeutic potential, the two MCAT isomers have never been directly compared at their molecular targets: the monoamine transporters (MATs). Additionally, previous studies have found that the carbonyl oxygen atom can be replaced with a methoxy group, but this results in two chiral centers (i.e., four possible optical isomers for synthesis and evaluation). Here, the individual isomers of MCAT, their racemate, and achiral MCAT analogs were prepared where necessary, and examined in vitro and in silico at the MATs. All agents were active as substrates, with a rank order of potency suggesting that α-position chirality, in either configuration, is favored but not required, with the S(-) configuration slightly preferred. Either chiral center removal approach resulted in a reduction in potency, suggesting both favorable interactions with the α-methyl, and limited bulk tolerance. To further investigate this possibility, docking studies were conducted using homology models of the MATs. Common binding modes were identified that were similar to the binding mode of S(+)amphetamine co-crystallized at drosophila DAT. Taken together, these studies supported our conclusions, as steric hindrance was observed in the α-methyl region of the proposed binding site for the R(+)MCAT isomer. Inclusion of the original synthetic cathinones among Schedule I controlled substances has driven the clandestine development of a second generation of agents, resulting in an array of new synthetic cathinones diverse in structure and effect.Pyrrolidinophenones are a major constituent of second-generation bath salts. Little is known about their structure-activity relationships. Here, we have synthesized and examined a series of aryl-substituted pyrrolidinophenone analogs, as well as an achiral pyrrolidinophenone analog, utilizing novel synthetic chemistry and an innovative cell-based epifluorescence Ca2+ imaging technique. Herein, we evaluated the neurochemical properties of these novel compounds at the dopamine transporter (DAT), considered to exert a major role in actions of drugs of abuse. For future structure-activity relationship studies, additional analogs of synthetic cathinone-related agents were produced using novel synthetic approaches, including analogs and isomers of known amphetamine drugs of abuse. Finally, though much has been learned about the role of the dopamine and serotonin transporters in the mechanisms of action of synthetic cathinones, the role of the norepinephrine transporter is poorly understood. Homology models of the human norephinephrine transporter were built and docking studies conducted to inform the study of MAT ligand selectivity, activity, and binding. In conclusion, these studies represent progress towards the establishment of comprehensive structure-activity relationships for synthetic cathinones and related agents. Particular emphasis was placed on the SAR of the phenylalkylamine α-carbon in the synthetic cathinone context, and the role of the norepinephrine transporter in their activity.