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 EPM and FST were used to test the hypothesis that S-MEPH reduces anxiety-like and depression-like behaviors in rats withdrawn from a 10-day chronic binge cocaine paradigm (10 mg/kg, 3x/day in 1-hr intervals) or binge MDPV paradigm (1 mg/kg, 3x/day in 1-hr intervals).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 Rats withdrawn from chronic cocaine exposure received treatment with 10 mg/kg S-MEPH (COC SM 10).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

Psychostimulant abuse is a major public health concern yet no FDA-approved medications exist. Synthetic cathinones (“bath salts”) are a class of psychostimulants that have emerged relatively recently worldwide. One synthetic cathinone, MDPV (3,4-methylenedioxypyrovalerone) is mechanistically similar to cocaine but is over ten times more potent, possesses high abuse potential, and is relatively understudied. Recent studies have revealed involvement of inflammatory proteins called chemokines in the rewarding effects of MDPV and the mechanistically similar drug, cocaine. We and others have shown that the chemokine-receptor ligand pair CXCL12-CXCR4 is recruited in the rewarding effects of cocaine and MDPV. Humans and animal models of cocaine addiction have dysregulated CXCL12 and the commercially-available CXCR4 antagonist, AMD3100, can reverse cocaine use and relapse in preclinical models of addiction. Specifically, AMD3100 reduces self-administration and reinstatement to cocaine-seeking with concomitant alterations in CXCL12 gene expression in the midbrain. Here, I employ several complementary methods to demonstrate that AMD3100 also reverses MDPV-elicited behaviors. I demonstrate that (i) AMD3100 reverses MDPV-induced hyperlocomotion, conditioned place preference (preclinical model of drug reward), self-administration and reinstatement to MDPV-seeking behavior; (ii) AMD3100 can rescue MDPV-induced deficits in measures of anxiety and recognition memory shortly after a binge; and (iii) repeated MDPV exposure upregulates CXCL12 gene expression in the nucleus accumbens with concomitant downregulation of dendrite morphometrics and a related synapse scaffolding protein gene expression. These findings implicate CXCR4-CXCL12 signaling in the modulation of MDPV-elicited behaviors, suggesting that AMD3100 is a viable therapeutic option for the effects of this synthetic cathinone. / Psychology

Aptamers are short oligonucleotide sequences (DNA or RNA) capable of high affinity and specific binding to a molecule or a family of molecules. Aptamers are lower in cost and exhibit higher reproducibility when compared to antibodies and thus are well-suited for recognition and detection of small molecular targets such as drugs of abuse and small medical biomarkers. While aptamers have been extensively utilized for development of small molecule sensors, several limitations prevent measurements of complex or real-world samples. This dissertation describes methods, technologies, and assays that were developed with the goal of producing and/or improving aptamer-based sensors for target detection in complex samples. Aptamer engineering is detailed as an important facet of maximizing aptamer-sensor sensitivity and specificity, along with adaptation to various read-out mechanisms for improved selectivity. In chapter 3, an aptamer vii sensor for cocaine is developed based on binding between the fluorophore ATMND to the cocaine aptamer which results in quenching (i.e., ‘turn-off’) of the fluorescence of ATMND. Cocaine binding results in displacement of the ATMND and recovery of the fluorescence signal. Detection of cocaine is demonstrated with an engineered cocaine aptamer with higher affinity for cocaine, permitting over a 50-fold increase in sensitivity over other aptamer-based sensors. The method can be used in dilute biological fluids (e.g., saliva) with a single step reaction (seconds) and robust signal output. In chapter 4, a new adenosine specific aptamer is identified through rational engineering of a previously reported ATP-binding aptamer. The new adenosine aptamer is utilized to develop an electrochemical sensor for detection of adenosine in undiluted serum. The method displays 40-fold higher sensitivity in undiluted serum measurements over previously reported aptamer-based sensors for adenosine but also demonstrates specificity for adenosine over ATP, ADP and AMP that has not been previously reported. In chapter 5, a nuclease-guided truncation method is developed to yield optimal structure-switching aptamer sequences for the emergent illicit drug methylenedioxypyrovalerone (MDPV) and medical biomarkers ATP and deoxycorticosterone 21-glucoside (DOG). The method intelligently removes unessential nucleotides, producing truncated aptamer sequences with structure-switching functionality. This technique will be immediately useful for simple and low-cost development of aptamer-based electrochemical sensors.

MDPV är en drog som fått stort genomslag i massmedier och Norrköping är en av de städer där användandet av drogen har ökat. Det här arbetet syftar till att studera hur personer som missbrukar MDPV beskrivs i de lokala tidningarna Norrköpings Tidningar och Folkbladet i Norrköping. 56 utvalda artiklar skrivna i dessa tidningar analyserades genom en inledande kvantifiering och en kvalitativ textanalys. Empirin är sedan kopplad till socialkonstruktionism som är vår teoretiska ansats, till begreppen stigma och stämpling samt till tidigare forskning kring MDPV, narkotikamissbruk, media och sociala problem i media. Materialet delades sedan in i tre teman; Avvikare, Offer för omständigheterna och Samhällsproblem. Dessa teman representerar olika sätt som MDPV-missbrukare beskrivs på i tidningarna. I temat Avvikare beskrivs missbrukarna opersonligt och med egenskaper kopplade till drogen och brottslighet, medan de i temat Offer för omständigheterna beskrivs mer personligt genom att missbrukarna själva och anhöriga uttalar sig. I temat Samhällsproblem beskrivs missbrukarna som anledningen till professionellas försämrade arbetsmiljö och till socialnämndens budgetunderskott. Studien visar också att MDPV-missbrukarna beskrivs olika beroende på vem som uttalar sig i tidningsartikeln, vilket tyder på att det är viktigt att alla involverade får komma till tals.

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.

Two deaths involving 3, 4-methylenedioxypyrovalerone (MDPV) are reported. MDPV is a synthetic cathinone stimulant found in "bath salts" with neurological and cardiovascular toxicity. Biological specimens were analyzed for MDPV by GC/MS and LC/MS. A White man was found dead with signs of nausea and vomiting after repeatedly abusing bath salts during a weekend binge. Femoral venous blood and urine had MDPV concentrations of 39 ng/mL and 760 ng/mL. The second fatality was a White man with a history of drug and bath salt abuse found dead at a scene in total disarray after exhibiting fits of anger and psychotic behavior. Femoral venous blood and urine had MDPV concentrations of 130 ng/mL and 3800 ng/mL. The blood and urine MDPV concentrations are within the reported recreational concentration ranges (blood 24-241 ng/mL and urine 34-3900 ng/mL). Both decedents' deaths were attributed to relevant natural causes in a setting of MDPV abuse.

Two deaths involving 3, 4-methylenedioxypyrovalerone (MDPV) are reported. MDPV is a synthetic cathinone stimulant found in "bath salts" with neurological and cardiovascular toxicity. Biological specimens were analyzed for MDPV by GC/MS and LC/MS. A White man was found dead with signs of nausea and vomiting after repeatedly abusing bath salts during a weekend binge. Femoral venous blood and urine had MDPV concentrations of 39 ng/mL and 760 ng/mL. The second fatality was a White man with a history of drug and bath salt abuse found dead at a scene in total disarray after exhibiting fits of anger and psychotic behavior. Femoral venous blood and urine had MDPV concentrations of 130 ng/mL and 3800 ng/mL. The blood and urine MDPV concentrations are within the reported recreational concentration ranges (blood 24-241 ng/mL and urine 34-3900 ng/mL). Both decedents' deaths were attributed to relevant natural causes in a setting of MDPV abuse.

No description available.