A molecular receptor for mephedrone, an amphetamine-like NPS, was developed using host-guest chemistry and pharmacophoric design. The in-field detection of new psychoactive substances (NPS) is an area that has garnered considerable attention in the last few years. With the continuously expanding number of NPS on the market, traditional detection mechanisms lack the selectivity needed. In this project a new methodology has been developed for the design of host molecules for use in in-field detection, based on biomimetic design. To understand what a sensory molecular needs to be selective against, GC-MS and HPLC analysis were employed to identify and quantify thirteen aminoindane internet samples. It was found that the composition of internet samples varies greatly in terms of concentration of active ingredient, with a range of 17-95 % w/w of active ingredient identified. It was also found that caffeine was the most common cutting agent with a range of 27.7-30.2 % w/w identified. This highlights the need for both selectivity and sensitivity in detection mechanisms. Using the principles of biomimetic design, a methodology for the treatment of protein-ligand interactions was developed. Protein-ligand binding data collected from the Protein Databank was analysed for mephedrone related structures and common cutting agents, identified through aminoindane internet sample analysis and literature sources. From this work a three-point pharmacophoric model was developed, upon which two host molecules were considered, macrocyclic calixarenes and acyclic anthraquinones. Both contained the three binding interactions deduced from the pharmacophore design; two p-stacking interactions and one hydrogen bond acceptor. The final host molecule taken forward for testing was 1,8-dibenzylthiourea anthracene (Probe 1). The binding affinity of Probe 1 to mephedrone was tested using 1H-NMR. An estimated association constant of 104 M-1 was calculated, with a 1:1 binding stoichiometry. Along with ESI-MS and DFT calculations, it was found that mephedrone binds to Probe 1 in a concerted fashion with a three-point binding geometry, with two hydrogen bonds and one p-stacking interaction. A modest optical response using fluorescence spectroscopy was also observed between mephedrone and Probe 1 at high molar concentrations. A more pronounced response was observed upon addition of high molar concentrations of flephedrone. 1H-NMR showed that Probe 1 selectively bound mephedrone over methamphetamine as well as the four most common cutting agents identified from literature: lidocaine, caffeine, paracetamol and benzocaine, which have been shown to cause false positives in previous studies. Probe 1 showed significant selectivity for the β-ketoamine arrangement. This is supported by the systematic analysis of mephedrone, methamphetamine, mephedrone precursor and flephedrone. This is the first time this has been achieved using host-guest chemistry. A protocol was developed to successfully detect mephedrone via Probe 1 using NMR spectroscopy in a simulated street sample containing two of the most common cutting agents, benzocaine and caffeine. To further aid future design of small host molecules a methodology for the in silico analysis of small molecule host-guest binding using metadynamics was explored. Solvent interactions with the host and guest molecules were observed, highlighting the importance of solvent choice in binding studies. Metadynamics shows potential to be used in further work for improving the approach in which host molecules are designed in future.

Objective: Unusual clinical course Background: "Bath salts" is the street name for a group of recently identified and increasingly abused stimulant synthetic cathinones that are associated with multiple systemic effects. We present a case of a patient who developed reversible dilated cardiomyopathy secondary to their use. Case Report: A 27 year old male with no past medical history was brought to emergency department with agitation. He had been inhaling and intravenously injecting "bath salts", containing a mephedrone/Methylenedioxypyrovalerone (MDPV) combination. On presentation, he was tachycardic, hypotensive and febrile. His initial labs showed an elevated white count, creatinine and creatinine phosphokinase levels. His erythrocyte sedimentation rate; C-reactive protein; urinalysis; urine drug screen; Human Immunodeficiency Virus, hepatitis, coxsackie, and influenza serology were normal. EKG showed sinus tachycardia. An echocardiogram was done which showed dilated cardiomyopathy with an ejection fraction (EF) of 15-20% and global hypokinesia. A left heart catheterization was done and was negative for coronary artery disease. At a 20 week follow up, he had stopped abusing bath salts and was asymptomatic. A repeat echocardiogram showed an EF of 52%. Cocnlusions: Bath salts (MDPV, mephedrone) are synthetic cathinones with amphetamine/cocaine like properties with potential cardiotoxic effects. Cardiovascular manifestations reported include tachycardia, hypertension, myocardial infarction, arrhythmias and cardiac arrest. "Bath salts" can also cause severe reversible dilated cardiomyopathy. Prior to diagnosis, other causes of cardiomyopathy including ischemic, infectious, familial, immunological, metabolic and cytotoxic may need to be ruled out; as was done in our patient.

Objective: Unusual clinical course Background: "Bath salts" is the street name for a group of recently identified and increasingly abused stimulant synthetic cathinones that are associated with multiple systemic effects. We present a case of a patient who developed reversible dilated cardiomyopathy secondary to their use. Case Report: A 27 year old male with no past medical history was brought to emergency department with agitation. He had been inhaling and intravenously injecting "bath salts", containing a mephedrone/Methylenedioxypyrovalerone (MDPV) combination. On presentation, he was tachycardic, hypotensive and febrile. His initial labs showed an elevated white count, creatinine and creatinine phosphokinase levels. His erythrocyte sedimentation rate; C-reactive protein; urinalysis; urine drug screen; Human Immunodeficiency Virus, hepatitis, coxsackie, and influenza serology were normal. EKG showed sinus tachycardia. An echocardiogram was done which showed dilated cardiomyopathy with an ejection fraction (EF) of 15-20% and global hypokinesia. A left heart catheterization was done and was negative for coronary artery disease. At a 20 week follow up, he had stopped abusing bath salts and was asymptomatic. A repeat echocardiogram showed an EF of 52%. Cocnlusions: Bath salts (MDPV, mephedrone) are synthetic cathinones with amphetamine/cocaine like properties with potential cardiotoxic effects. Cardiovascular manifestations reported include tachycardia, hypertension, myocardial infarction, arrhythmias and cardiac arrest. "Bath salts" can also cause severe reversible dilated cardiomyopathy. Prior to diagnosis, other causes of cardiomyopathy including ischemic, infectious, familial, immunological, metabolic and cytotoxic may need to be ruled out; as was done in our patient.

Synthetic cathinones are an emerging class of novel psychoactive substances whose rising rates of abuse have made them a significant public health issue. Recently, synthetic cathinones have emerged as popular drugs of abuse in the United States and Europe. Illicit drug dealers have synthesized stable analogues of cathinones and marketed them via the Internet as “legal high” alternatives to commonly abused psychostimulants. Some adverse effects of synthetic cathinone intoxication include depression, anxiety, myocardial infarction, cardiac dysrhythmias, pulmonary edema, renal failure, stroke and death. Two synthetic cathinones that have gained popularity over the past decade are 4-methylmethcathinone (mephedrone, MEPH) and 3,4-methylenedioxypyrovalerone (MDPV). Analogous to other amphetamines and cathinones, MEPH is a chiral molecule with two enantiomers, R-MEPH and S-MEPH. Pharmacological differences can exist between enantiomers. Given the enantiomeric specificity that exists between the enantiomers of MEPH, the overall goal of this dissertation was to investigate potential therapeutic effects of S-MEPH and to determine the reinforcing effects and potential abuse liability of each enantiomer of MEPH. To date, there are no approved medications for psychostimulant abuse despite high rates of relapse even with treatment. The lack of pharmacotherapies suggests that new approaches for psychostimulant abuse remain to be identified. Further exploration into the stereochemistry of MEPH will characterize a new approach to manage psychostimulant abuse during acute withdrawal that causes anxiety and relapse. The first experimental chapter of this thesis (Chapter 2) evaluated the effects of S-MEPH on cocaine and MDPV withdrawal-induced anxiety- and depression-like behavior in the elevated plus maze (EPM) and forced swim test (FST), respectively. EPM and FST were used to test the hypothesis that S-MEPH reduces anxiety-like and depression-like behaviors in rats withdrawn from a 10-day chronic binge cocaine paradigm (10 mg/kg, 3x/day in 1-hr intervals) or binge MDPV paradigm (1 mg/kg, 3x/day in 1-hr intervals). Control animals received saline injections. Both cocaine and MDPV induced heightened anxiety-like behavior on the EPM compared to saline controls. Rats withdrawn from chronic cocaine exposure received treatment with 10 mg/kg S-MEPH (COC SM 10). S-MEPH increased the percentage of time spent on the open arms compared to treatment with saline (COC SAL). Similar efficacy was observed for 10 mg/kg of S-MEPH where rats withdrawn from chronic MDPV exposure and treated with S-MEPH during the drug-free period (MDPV SM 10) increased the percentage of time spent on the open arms compared to treatment with saline (MDPV SAL). Treatment with a higher dose of S-MEPH (30 mg/kg) also produced an increase in time spent on the open arms for rats withdrawn from either cocaine (COC SM 30) or MDPV (MDPV SM 30). However, treatment with 30 mg/kg S-MEPH by itself (SAL S-MEPH) caused a slight, but significant, decrease in time spent on the open arms compared to saline controls (SAL SAL). Cocaine and MDPV withdrawn rats spent more time in the immobile state, indicative of depression-like behavior in the FST compared to saline controls. Subsequent treatment with 10 mg/kg S-MEPH (COC SM 10), following withdrawal from chronic cocaine exposure, reduced the time spent in the immobile state compared to treatment with saline (COC SAL). Similar efficacy was observed for 10 mg/kg of S-MEPH where rats withdrawn from chronic MDPV exposure and treated with S-MEPH during the drug-free period (MDPV SM 10) decreased the time spent in the immobile state compared to treatment with saline (MDPV SAL). Treatment with a higher dose of S-MEPH (30 mg/kg) also produced a decrease in immobile state for rats withdrawn from either cocaine (COC SM 30) or MDPV (MDPV SM 30). However, treatment with 30 mg/kg S-MEPH by itself (SAL S-MEPH) caused a reduction in time spent in the immobile compared to saline controls (SAL SAL). The present data suggest S-MEPH has therapeutic potential during early withdrawal from psychostimulant abuse and may be a possible structural and pharmacological template to develop maintenance therapy for psychostimulant abuse. Although dopamine (DA) and serotonin (5-HT) release is the primary mechanism of action of S-MEPH, in vitro studies assessed the receptor binding and activity of S-MEPH to elucidate a possible secondary mechanism that could contribute to the clinical effects of S-MEPH. Indeed, standard antidepressants (e.g. fluoxetine) have a dual mechanism of action, and not only increase 5-HT levels, but also block 5-HT2C receptors that help to prevent the initial onset of anxiety reported by patients. Binding and functional assays were performed at the National Institute of Mental Health Psychoactive Drug Screening Program (PDSP). For radioligand binding, S-MEPH was screened at 10 μM for binding to a battery of receptors including a range of 5-HT, DA, sigma, kappa opioid, adrenergic, muscarinic, and nicotinic receptor subtypes. It was found that S-MEPH binds to 5-HT2 receptors (2A, 2B, 2C) but showed negligible binding for dopaminergic, adrenergic and nicotinic receptors. Functional assays revealed S-MEPH has no agonist activity. These results suggest that S-MEPH may be a possible structural and pharmacological template to develop a maintenance therapy for addicts with acute anxiety and depression during early withdrawal by enhancing the release of 5-HT and/or through 5-HT2 receptor interactions. Chapter 3 investigated whether S-MEPH can reinstate drug-seeking behavior in rats with a history of cocaine exposure. Using the drug intravenous self-administration (IVSA) model, rats were trained to self-administer cocaine (0.375 mg/kg/infusion) under FR-1 schedule of reinforcement during daily 2-hour sessions for 12 days. Following acquisition of cocaine self-administration, rats were subject to daily 2-hour extinction sessions. After meeting extinction criteria (less than 15 active lever presses), rats underwent drug-primed reinstatement, where non-contingent injections of R-MEPH (10 mg/kg), S-MEPH (10, 20, 30 mg/kg), or cocaine (10 mg/kg) were administered prior to the reinstatement session. S-MEPH (10, 20, 30 mg/kg) prior to the reinstatement session was not efficacious in producing drug-seeking behavior. R-MEPH-prime injection reinstated cocaine-seeking behavior. Rats reinstated responding after cocaine-prime injection. After observing differences in priming injections with R-MEPH and S-MEPH during reinstatement to cocaine-seeking (Chapter 3), we further examined the reinforcing effects and potential for abuse of S-MEPH in Chapter 4. Fixed-ratio 1 (FR-1) (acquisition) and progressive-ratio (motivation) schedules of reinforcement were used to determine the rewarding properties and motivational incentive for R-MEPH, S-MEPH as well as racemic MEPH. Rats were trained to self-administer MEPH (0.5 mg/kg/infusion) under a fixed ratio (FR-1) schedule in daily 2-hour sessions for 14 days. After acquisition, rats underwent dose substitution to examine effects of R-MEPH (0.5 mg/kg/infusion) and S-MEPH (0.25, 0.5, 2. mg/kg/ infusion). Dose-substitution studies revealed the lowest (0.25 mg/kg/ infusion) and middle (0.5 mg/kg/ infusion) dose of S-MEPH significantly increased the number of reinforcers earned, while the highest (2.0 mg/kg/ infusion) dose decreased the number of reinforcers earned during the two hour sessions compared to acquisition of MEPH. Following the observations that rats readily acquire S-MEPH when a history of MEPH administration is already established, in a separate cohort of rats, acquisition of S-MEPH self-administration was studied using three doses of S-MEPH (0.25, 0.5, 2.0 mg/kg/infusion) and R-MEPH (0.5 mg/kg/infusion) on a fixed-ratio 1 (FR-1) and progressive-ratio schedule of reinforcement using drug naïve rats. In this cohort, 50-kHz ultrasonic vocalizations (USVs) were recorded during FR-1 sessions to assess potential stereospecific differences in drug-associated positive affect. Rats were trained to self-administer R-MEPH (0.5 mg/kg/ infusion) or S-MEPH (0.25, 0.5, 2.0 mg/kg/ infusion) for 10 days followed by progressive-ratio for 10 days to determine motivation for drug taking. Rats readily self-administer both enantiomers of MEPH under a FR-1 schedule. Equivalent doses of R-MEPH and S-MEPH did not significantly differ in number of infusions and active lever presses. Self-administration of R-MEPH elicited greater rates of 50-kHz USVs compared to S-MEPH. Progressive-ratio studies determined that R-MEPH- trained rats had significantly higher breakpoints than that of S-MEPH- trained rats, indicating an increase in motivation to work for reinforcer. These data suggest that escalation of S-MEPH intake occurred when a previous history of MEPH was established; rats exposed to MEPH are compensating with escalating their intake of S-MEPH to reach the previous euphoric state during acquisition of MEPH. These findings indicate that rats readily acquire both enantiomers of MEPH, which signify an abuse liability for these drugs. However, these data suggest that R-MEPH, rather than the S-MEPH, is responsible for the rewarding and reinforcing effects seen with the racemic form of MEPH, due to the lack of motivation to work for the S-enantiomer. In conclusion, during early withdrawal from chronic cocaine or MDPV, rats demonstrated heightened anxiety-like behavior on the EPM and increased depression-like behavior in FST. Binding and functional assays indicate affinity for the 5-HT2 receptors (2A, 2B, 2C) and no agonist activity of S-MEPH. Results show R-MEPH reinstates cocaine-seeking behavior and S-MEPH does not reinstate cocaine-seeking. Furthermore, R-MEPH and S-MEPH both have reinforcing effects, with R-MEPH having significantly greater reinforcing strength compared to S-MEPH. These studies identified unique differences in the behavioral profile of MEPH enantiomers. This data not only expands the body of literature on the stereospecific effects of MEPH, but also developing the pharmacological profile of MDPV in the context of dependence and addiction. Continued research is needed profiling MEPH and MDPV to develop efficacious pharmacotherapeutics for treating psychostimulant abuse to reduce relapse rates. / Pharmacology

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

In the last few years, there has been continued concern about synthetic drug abuse in both the United States and worldwide. Small adjustments in drug compound structure often allow synthetic drug makers to manufacture a legal product that can produce the same highs as illegal counterparts. Unfortunately, this is happening faster than the government can outlaw the drug compounds, and a wide variety of synthetics are now appearing on the street. This study evaluated the effects on the human serotonin transporter of six different 4-para substituted methcathinone compounds. Using a Xenopus oocyte model, the efficacy of each MCAT analogue at hSERT was calculated by applying the Hill equation to the oocyte data. This study suggests that volume, size, and steric bulk of the compound may generally influence efficacy at hSERT in a direct manner, but that other factors, like lipophilicity, may also play an important role in potency at the transporter.

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.

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.