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.

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 dopamine transporter (DAT) mediates reuptake of dopamine from the synaptic cleft into the presynaptic terminus and plays a critical role in maintaining the normal function of dopaminergic neurons. DAT is the major target of widely abused psychostimulant drugs, including cocaine and amphetamine. DAT also figures into disease states, and it is a target for therapeutic drugs. It is known that cathinone and methcathinone, β-keto analogs of amphetamine and methamphetamine, respectively, produce pharmacological actions similar to amphetamine. Cathinone and methcathinone analogs are recently gaining in popularity on the clandestine market (e.g. ‘bath salts’). Cathinone and methcathinone analogs as well as their amphetamine and methamphetamine counterparts were synthesized and examined at the hDAT expressed in Xenopus oocytes. One of the two major constituents of ‘bath salts’ (i.e., mephedrone) produced an electrophysiological signature similar to the dopamine releasing agent S(+)-amphetamine while the other major constituent (i.e., MDPV) produced an electrophysiological signature similar to the dopamine re-uptake inhibitor cocaine.

Class of 2014 Abstract / Specific Aims: To compare trends in user demographics, clinical effects, and clinical outcomes associated with the use of synthetic cannabinoids and cathinones before and after signing into law the Synthetic Drug Abuse Prevention Act of 2012 on July 9, 2012. Methods: Reports generated by the National Poison and Drug Information Center’s Toxic Exposure Surveillance System were used to isolate calls regarding patients who reportedly used either synthetic cannabinoids or synthetic cathinones from July 2011 to March 2013. Clinical effects, clinical outcomes, and demographic information of patients associated with these calls from July 9, 2011 to July 8, 2012, were compared to that of patients associated with calls from July 9, 2012 to July 8, 2013. Main Results: Pending Conclusion: Pending

O surgimento de novas substâncias psicoativas (NPS-New Psychoactive Substances) levantou muitas questões no contexto da aplicação da lei e políticas públicas de drogas. De acordo com o Escritório das Nações Unidas sobre Drogas e Crime (UNODC- United Nations Office on Drugs and Crime) como uma alternativa às drogas proibidas. Esses novos compostos foram projetados e formulados para escapar à legislação de controle de drogas, criando um fenômeno que se tornou um problema internacional. No Brasil, essas substâncias são controladas e penalmente puníveis, no pela Lei 11.343/2006, também conhecida como Lei de Drogas. Este trabalho traz estudos relativos às catinonas sintéticas com metodologia in silico para investigar mecanismos de detecção e tendência de atuação no organismo humano. No estudo relacionado à detecção utilizamos a reação dessas drogas com o isotiocianato de fluoresceína (FITC Fluorescein isothiocyanate). Para essa proposta foram feitos estudos de viabilidade de métodos de cálculo, análise conformacional do FITC, avaliação energética da reação com as catinonas e os espectros de emissão. Em relação à viabilidade dos métodos de cálculo temos que a otimização prévia dos compostos envolvidos com o semi-empírico PM6 e posterior refinamento com o método B3LYP/6-31G** foram adequados para os cálculos. A avaliação energética mostrou que a reação é favorável para anfetaminas, aminoácidos e catinonas, e os menores valores foram encontrados no último caso. Nos estudos de emissão obtivemos resultados semelhantes ao perfil energético; no entanto, observamos que os espectros são únicos, representando uma baixa probabilidade de falsos positivos. Avaliações de docking mostraram que as catinonas têm mais afinidade com o receptor dopaminérgico do que suas anfetaminas homólogas, confirmando dados experimentais relatados na literatura. Por fim, os estudos realizados neste trabalho demonstraram a importância e a capacidade dos métodos in silico que apresentam grau potencial na área e que podem ser amplamente utilizados em investigações com diferentes propósitos no campo forense. / The emergence of new psychoactive substances (NPSs) has raised many issues in the context of law enforcement and public drug policies. According to the United Nations Office on Drugs and Crime (UNODC), NPS were created as an alternative to forbidden drugs. These new compounds were designed and formulated to escape the drug control legislation, creating a phenomenon that has become an international problem. In Brazil, these substances are controlled and punishable by Law 11,343 / 2006, also known as the Drug Law. This work presents studies on synthetic cathinones with in silico methodology to investigate mechanisms of detection and tendency of action in the human organism. In the detection-related study, we used the reaction of these drugs with fluorescein isothiocyanate (FITC). For this proposal were made studies regarding to the viability of the calculation methods, FITC conformational analysis, energetic evaluation of the reaction with the cathinones and the emission spectra. In relation to the viability of the calculation methods we have that the previous optimization of the compounds involved with the semi-empirical PM6 and subsequent refinement with the B3LYP / 6-31G ** method were adequate for the calculations. The energetic evaluation showed that the reaction is favorable for amphetamines, amino acids and cathinones, and the lowest values were found in the last case. In the emission studies we obtained similar results to the energy profile; however, we observed that the spectra are unique representing a low probability of false positive. Docking evaluations have shown that cathinones have more affinity to the dopaminergic receptor than their homologous amphetamines, confirming experimental data reported in the literature. Finally, the studies carried out in this work demonstrated the importance and the capacity of the in silico methods that present with potential grade in the area and that can be widely used in investigations with different purposes in the forensic field.

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.

O surgimento de novas substâncias psicoativas (NPS-New Psychoactive Substances) levantou muitas questões no contexto da aplicação da lei e políticas públicas de drogas. De acordo com o Escritório das Nações Unidas sobre Drogas e Crime (UNODC- United Nations Office on Drugs and Crime) como uma alternativa às drogas proibidas. Esses novos compostos foram projetados e formulados para escapar à legislação de controle de drogas, criando um fenômeno que se tornou um problema internacional. No Brasil, essas substâncias são controladas e penalmente puníveis, no pela Lei 11.343/2006, também conhecida como Lei de Drogas. Este trabalho traz estudos relativos às catinonas sintéticas com metodologia in silico para investigar mecanismos de detecção e tendência de atuação no organismo humano. No estudo relacionado à detecção utilizamos a reação dessas drogas com o isotiocianato de fluoresceína (FITC Fluorescein isothiocyanate). Para essa proposta foram feitos estudos de viabilidade de métodos de cálculo, análise conformacional do FITC, avaliação energética da reação com as catinonas e os espectros de emissão. Em relação à viabilidade dos métodos de cálculo temos que a otimização prévia dos compostos envolvidos com o semi-empírico PM6 e posterior refinamento com o método B3LYP/6-31G** foram adequados para os cálculos. A avaliação energética mostrou que a reação é favorável para anfetaminas, aminoácidos e catinonas, e os menores valores foram encontrados no último caso. Nos estudos de emissão obtivemos resultados semelhantes ao perfil energético; no entanto, observamos que os espectros são únicos, representando uma baixa probabilidade de falsos positivos. Avaliações de docking mostraram que as catinonas têm mais afinidade com o receptor dopaminérgico do que suas anfetaminas homólogas, confirmando dados experimentais relatados na literatura. Por fim, os estudos realizados neste trabalho demonstraram a importância e a capacidade dos métodos in silico que apresentam grau potencial na área e que podem ser amplamente utilizados em investigações com diferentes propósitos no campo forense. / The emergence of new psychoactive substances (NPSs) has raised many issues in the context of law enforcement and public drug policies. According to the United Nations Office on Drugs and Crime (UNODC), NPS were created as an alternative to forbidden drugs. These new compounds were designed and formulated to escape the drug control legislation, creating a phenomenon that has become an international problem. In Brazil, these substances are controlled and punishable by Law 11,343 / 2006, also known as the Drug Law. This work presents studies on synthetic cathinones with in silico methodology to investigate mechanisms of detection and tendency of action in the human organism. In the detection-related study, we used the reaction of these drugs with fluorescein isothiocyanate (FITC). For this proposal were made studies regarding to the viability of the calculation methods, FITC conformational analysis, energetic evaluation of the reaction with the cathinones and the emission spectra. In relation to the viability of the calculation methods we have that the previous optimization of the compounds involved with the semi-empirical PM6 and subsequent refinement with the B3LYP / 6-31G ** method were adequate for the calculations. The energetic evaluation showed that the reaction is favorable for amphetamines, amino acids and cathinones, and the lowest values were found in the last case. In the emission studies we obtained similar results to the energy profile; however, we observed that the spectra are unique representing a low probability of false positive. Docking evaluations have shown that cathinones have more affinity to the dopaminergic receptor than their homologous amphetamines, confirming experimental data reported in the literature. Finally, the studies carried out in this work demonstrated the importance and the capacity of the in silico methods that present with potential grade in the area and that can be widely used in investigations with different purposes in the forensic field.

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.

Synthetic cathinones are stimulant drugs of abuse that act at monoamine transporters e.g. the dopamine transporter (DAT) as releasing agents or as reuptake inhibitors. More than >150 new synthetic cathinones have emerged on the clandestine market and have attracted considerable attention from the medical and law enforcement communities. threo-Methylphenidate (tMP) is an FDA approved drug for the treatment of ADHD and narcolepsy, which also acts as a DAT reuptake inhibitor and is widely abused. tMP and synthetic cathinones share some structural similarities and extensive structure-activity relationship (SAR) studies on tMP have been conducted. However, much less is known about the SAR of synthetic cathinones, and the available MP literature might assist in understanding it. The main focus of this research was to compare SAR between methylphenidate-cathinone hybrids and available methylphenidate SAR in order to identify some guiding principles that might allow
us to predict their abuse potential and to identify which cathinones should be
targeted for more extensive evaluation. In the present study, we evaluated eight 2-benzoylpiperidine analogs and a descarbonyl analog to determine if tMP SAR can be applied to cathinone SAR. We conducted molecular modeling and docking studies and predicted the order of potency to be tMP > 2-benzoylpiperidine > 2-benzylpiperidine based on the number of hydrogen bonds. The synthesized analogs were evaluated in a competition assay using live-cell imaging against APP+ in HEK293 cells stably expressing hDAT. All compounds were found to be DAT reuptake inhibitors and, as the modeling studies predicted, the order of potency in our functional studies was also found to be tMP > 2-benzoylpiperidine > 2- benzylpiperidine. A significant correlation was obtained between the potency of the benzoylpiperidines and tMP binding data (r = 0.91) suggesting that the SAR of tMP analogs might be applicable to the synthetic cathinones as DAT reuptake inhibitors.