Bruce E. Blough

N-substituted piperazines abused by humans mimic the molecular mechanism of 3,4-methylenedioxymethamphetamine (MDMA, or 'Ecstasy').

3,4-Methylenedioxymethamphetamine (MDMA, or ‘Ecstasy’) is an illicit drug that stimulates the release of serotonin (5-HT) and dopamine (DA) from neurons. Recent evidence reveals that drug users are ingesting piperazine analogs, like 1-benzylpiperazine (BZP, or ‘A2’) and 1-(m-trifluoromethylphenyl)piperazine (TFMPP, or ‘Molly’), to mimic psychoactive effects of MDMA. In the present study, we compared the neurochemistry of MDMA, BZP, and TFMPP in rats. The effects of MDMA, BZP, and TFMPP on transporter-mediated efflux of [3H]5-HT and [3H]MPP+ (DA transporter substrate) were determined in synaptosomes. The effects of drugs on extracellular levels of 5-HT and DA were examined using in vivo microdialysis in conscious rats. MDMA evoked transporter-mediated release of [3H]5-HT and [3H]MPP+. BZP released [3H]MPP+, whereas TFMPP was a selective releaser of [3H]5-HT. MDMA (1–3 mg/kg, i.v.) increased dialysate 5-HT and DA in a dose-related fashion, with actions on 5-HT being predominant. BZP (3–10 mg/kg, i.v.) elevated dialysate DA and 5-HT, while TFMPP (3–10 mg/kg, i.v.) elevated 5-HT. Administration of BZP plus TFMPP at a 1:1 ratio (BZP/TFMPP) produced parallel increases in dialysate 5-HT and DA; a 3 mg/kg dose of BZP/TFMPP mirrored the effects of MDMA. At a 10 mg/kg dose, BZP/TFMPP increased dialysate DA more than the summed effects of each drug alone, and some rats developed seizures. Our results show that BZP/TFMPP and MDMA share the ability to evoke monoamine release, but dangerous drug–drug synergism may occur when piperazines are coadministered at high doses.

Interaction of Amphetamines and Related Compounds at the Vesicular Monoamine Transporter

Amphetamine-type agents interact with the vesicular monoamine transporter type 2 (VMAT2), promoting the release of intravesicular neurotransmitter and an increase in cytoplasmic neurotransmitter. Some compounds, such as reserpine, “release” neurotransmitter by inhibiting the ability of VMAT2 to accumulate neurotransmitter in the vesicle, whereas other types of compounds can release neurotransmitter via a carrier-mediated exchange mechanism. The purpose of this study was to determine, for 42 mostly amphetamine-related compounds, their mode of interaction with the VMAT2. We used a crude vesicular fraction prepared from rat caudate to assay VMAT2 activity. Test compounds were assessed in several assays, including 1) inhibition of [3H]dihydrotetrabenazine binding, 2) inhibition of vesicular [3H]dopamine uptake, and 3) release of preloaded [3H]dopamine and [3H]tyramine. Several important findings derive from this comprehensive study. First, our work indicates that most agents are VMAT2 substrates. Second, our data strongly suggest that amphetamine-type agents deplete vesicular neurotransmitter via a carrier-mediated exchange mechanism rather than via a weak base effect, although this conclusion needs to be confirmed via direct measurement of vesicular pH. Third, our data fail to reveal differential VMAT2 interactions among agents that do and do not produce long-term 5-hydroxytryptamine depletion. Fourth, the data reported revealed the presence of two pools of [3H]amine within the vesicle, one pool that is free and one pool that is tightly associated with the ATP/protein complex that helps store amine. Finally, the VMAT2 assays we have developed should prove useful for guiding the synthesis and evaluation of novel VMAT2 agents as possible treatment agents for addictive disorders.

Use of intracranial self‐stimulation to evaluate abuse‐related and abuse‐limiting effects of monoamine releasers in rats

Monoamine releasers constitute a class of drugs that promote the release of dopamine (DA), serotonin (5‐HT) and/or norepinephrine. Although some drugs in this class are well‐known drugs of abuse (amphetamine, methamphetamine), others are thought to have reduced (3,4‐methylenedioxy‐N‐methylamphetamine [MDMA]) or no (fenfluramine) abuse potential. The purpose of this study was to further elucidate the role of dopamine versus serotonin selectivity on expression of abuse‐related effects produced by monoamine releasers in an assay of intracranial self‐stimulation (ICSS) in rats.

In Vivo Effects of Amphetamine Analogs Reveal Evidence for Serotonergic Inhibition of Mesolimbic Dopamine Transmission in the Rat

Evidence suggests that elevations in extracellular serotonin (5-HT) in the brain can diminish stimulant effects of dopamine (DA). To assess this proposal, we evaluated the pharmacology of amphetamine analogs (m-fluoroamphetamine, p-fluoroamphetamine, m-methylamphetamine, p-methylamphetamine), which display similar in vitro potency as DA releasers (EC50 = 24–52 nM) but differ in potency as 5-HT releasers (EC50 = 53–1937 nM). In vivo microdialysis was used to assess the effects of drugs on extracellular DA and 5-HT in rat nucleus accumbens, while simultaneously measuring ambulation (i.e., forward locomotion) and stereotypy (i.e., repetitive movements). Rats received two intravenous injections of drug, 1 mg/kg at time 0 followed by 3 mg/kg 60 min later. All analogs produced dose-related increases in dialysate DA and 5-HT, but the effects on DA did not agree with in vitro predictions. Maximal elevation of dialysate DA ranged from 5- to 14-fold above baseline and varied inversely with 5-HT response, which ranged from 6- to 24-fold above baseline. All analogs increased ambulation and stereotypy, but drugs causing greater 5-HT release (e.g., p-methylamphetamine) were associated with significantly less forward locomotion. The magnitude of ambulation was positively correlated with extracellular DA (p < 0.001) and less so with the ratio of DA release to 5-HT release (i.e., percentage DA increase divided by percentage 5-HT increase) (p < 0.029). Collectively, our findings are consistent with the hypothesis that 5-HT release dampens stimulant effects of amphetamine-type drugs, but further studies are required to address the precise mechanisms underlying this phenomenon.

Monoamine Releasers with Varying Selectivity for Dopamine/Norepinephrine versus Serotonin Release as Candidate “Agonist” Medications for Cocaine Dependence: Studies in Assays of Cocaine Discrimination and Cocaine Self-Administration in Rhesus Monkeys

Monoamine releasers constitute one class of drugs under investigation as candidate medications for the treatment of cocaine abuse. Promising preclinical and clinical results have been obtained with amphetamine, which has high selectivity for releasing dopamine/norepinephrine versus serotonin. However, use of amphetamine as a pharmacotherapy is complicated by its high abuse potential. Recent preclinical studies suggest that nonselective monoamine releasers or serotonin-selective releasers have lower abuse liability and may warrant evaluation as alternatives to amphetamine. To address this issue, the present study evaluated the effects of five monoamine releasers in assays of cocaine discrimination and cocaine self-administration in rhesus monkeys. The releasers varied along a continuum from dopamine/norepinephrine-selective to serotonin-selective [m-fluoroamphetamine (PAL-353), methamphetamine, m-methylamphetamine (PAL-314), 1-napthyl-2-aminopropane (PAL-287), fenfluramine]. In drug discrimination studies, rhesus monkeys were trained to discriminate saline from cocaine (0.4 mg/kg i.m.) in a two-key, food-reinforced drug discrimination procedure. Substitution for cocaine was positively associated with selectivity for dopamine/norepinephrine versus serotonin release. In drug self-administration studies, rhesus monkeys responded for cocaine (0.01 and 0.032 mg/kg/injection) and food (1-g pellets) under a second-order fixed-ratio 2 (variable-ratio 16:S) schedule. In general, monoamine releasers produced dose-dependent and sustained decreases in cocaine self-administration. However, the dopamine/norepinephrine-selective releasers decreased cocaine self-administration with minimal effects on food-maintained responding, whereas the more serotonin-selective releasers produced nonselective reductions in both cocaine- and food-maintained responding. These results are consistent with the conclusion that dopamine/norepinephrine-selective releasers retain cocaine-like abuse-related effects but may also be capable of producing relatively selective reductions in the reinforcing effects of cocaine.

Effects of “Legal X” Piperazine Analogs on Dopamine and Serotonin Release in Rat Brain

Abstract: 3,4‐Methylenedioxymethamphetamine (MDMA) is a popular illicit drug that evokes transporter‐mediated release of serotonin (5‐HT) and dopamine (DA) from nerve cells. Recently, drug users have ingested combinations of the piperazine analogs, 1‐benzylpiperazine (BZP) and 1‐(m‐trifluoromethylphenyl)piperazine (TFMPP), in an attempt to mimic the subjective effects of MDMA. In the present study, we compared neurochemical effects of MDMA, BZP, and TFMPP in rat brain. The ability of MDMA, BZP, and TFMPP to stimulate efflux of [3H]5‐HT and [3H]MPP+ (a DA transporter substrate) was determined in vitro using release assays in synaptosomes. The ability of these drugs to increase extracellular 5‐HT and DA in vivo was assessed using intracranial microdialysis in nucleus accumbens. MDMA stimulated transporter‐mediated release of 5‐HT (EC50= 58 nM) and MPP+ (EC50= 119 nM). BZP was a selective releaser of MPP+ (EC50= 175 nM), whereas TFMPP was a selective releaser of 5‐HT (EC50= 121 nM). MDMA injections (1 and 3 mg/kg, i.v.) increased dialysate 5‐HT and DA in a dose‐related manner, but actions on 5‐HT were predominant. BZP (3 and 10 mg/kg, i.v.) elevated dialysate DA and 5‐HT, while TFMPP (3 and 10 mg/kg, i.v.) elevated only 5‐HT. The coadministration of BZP plus TFMPP (BZP/TFMPP) produced marked elevations in extracellular 5‐HT and DA that mirrored the effects of MDMA. At the high dose of BZP/TFMPP (10 mg/kg, i.v.), the rise in dialysate DA exceeded the summed effects of the drugs alone. Our results support the hypothesis that the BZP/TFMPP combination mimics the neurochemical mechanism of MDMA, providing a basis for recreational use of these agents. Additionally, the findings suggest possible drug‐drug synergism when piperazine drugs are coadministered at high doses.

Synthesis and biological evaluation of bupropion analogues as potential pharmacotherapies for smoking cessation.

Bupropion (2a) analogues were synthesized and tested for their ability to inhibit monoamine uptake and to antagonize the effects of human alpha3beta4*, alpha4beta2, alpha4beta4, and alpha1* nAChRs. The analogues were evaluated for their ability to block nicotine-induced effects in four tests in mice. Nine analogues showed increased monoamine uptake inhibition. Similar to 2a, all but one analogue show inhibition of nAChR function selective for human alpha3beta4*-nAChR. Nine analogues have higher affinity at alpha3beta4*-nAChRs than 2a. Four analogues also had higher affinity for alpha4beta2 nAChR. Analogues 2r, 2m, and 2n with AD(50) values of 0.014, 0.015, and 0.028 mg/kg were 87, 81, and 43 times more potent than 2a in blocking nicotine-induced antinociception in the tail-flick test. Analogue 2x with IC(50) values of 31 and 180 nM for DA and NE, respectively, and with IC(50) of 0.62 and 9.8 microm for antagonism of alpha3beta4 and alpha4beta2 nAChRs had the best overall in vitro profile relative to 2a.

Dual dopamine/serotonin releasers as potential medications for stimulante and alcohol addictions

We have advocated the idea of agonist therapy for treating cocaine addiction. This strategy involves administration of stimulant-like medications (eg, monoamine releasers) to alleviate withdrawal symptoms and prefent relapse. A major limitation, of this approach is that many candidate medicines possess significant abuse potential because of activation of mesolimbic dopamine (DA) neurons in central nervous system reward circuits. Previous data suggest that serotonin (5-HT) neurons can provide an inhibitory influence over mesolimbic DA neurons. Thus, it might be predicted that the balance between DA and 5-HT transmission is important to consider when developing medications with reduced stimulant side effects. In this article, we discuss several issues related to the development of dual DA/5-HT releasers for the treatment of substance use disorders. First, we discuss evidence supporting the existence of a dual deficit in DA and 5-HT function during withdrawal from chronic cocaine or alcohol abuse. Then we summarize studies that have tested the hypothesis that 5-HT neurons can dampen the effects mediated by mesolimbic DA. For example, it has been shown that pharmacological manipulations that increase extracellular 5-HT attenuate stimulant effects produced by DA release, such as locomotor stimulation and self-administration behavior. Finally, we discuss our recently published data about PAL-287 (naphthylisopropylamine), a novel non-amphetamine DA-/5-HT-releasing agent that suppresses cocaine self-administration but lacks positive reinforcing properties. It is conclude that DA/5-HT releasers might be useful therapeutic adjuncts for the treatment of cocaine and alcohol addiction, obesity, and even attention deficit disorder and depression.

Behavioral, biological, and chemical perspectives on atypical agents targeting the dopamine transporter ☆

BACKGROUND Treatment of stimulant-use disorders remains a formidable challenge, and the dopamine transporter (DAT) remains a potential target for antagonist or agonist-like substitution therapies. METHODS This review focuses on DAT ligands, such as benztropine, GBR 12909, modafinil, and DAT substrates derived from phenethylamine or cathinone that have atypical DAT-inhibitor effects, either in vitro or in vivo. The compounds are described from a molecular mechanistic, behavioral, and medicinal-chemical perspective. RESULTS Possible mechanisms for atypicality at the molecular level can be deduced from the conformational cycle for substrate translocation. For each conformation, a crystal structure of a bacterial homolog is available, with a possible role of cholesterol, which is also present in the crystal of Drosophila DAT. Although there is a direct relationship between behavioral potencies of most DAT inhibitors and their DAT affinities, a number of compounds bind to the DAT and inhibit dopamine uptake but do not share cocaine-like effects. Such atypical behavior, depending on the compound, may be related to slow DAT association, combined sigma-receptor actions, or bias for cytosol-facing DAT. Some structures are sterically small enough to serve as DAT substrates but large enough to also inhibit transport. Such compounds may display partial DA releasing effects, and may be combined with release or uptake inhibition at other monoamine transporters. CONCLUSIONS Mechanisms of atypical DAT inhibitors may serve as targets for the development of treatments for stimulant abuse. These mechanisms are novel and their further exploration may produce compounds with unique therapeutic potential as treatments for stimulant abuse.

Comparative effects of substituted amphetamines (PMA, MDMA, and METH) on monoamines in rat caudate: a microdialysis study.

Abstract: Paramethoxyamphetamine (PMA) is a methoxylated phenethylamine derivative that has been used illicitly in Australia since 1994. PMA is also becoming popular at rave parties in the United States. PMA raised concern when a series of fatalities resulted after its use in South Australia, where it was marketed as “ecstasy,” which is the colloquial name for MDMA. In the present study, we evaluated the comparative neurotoxicity of substituted amphetamines in rats. Extracellular levels of dopamine (DA), 3,4‐dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), serotonin (5‐HT), and 5‐hydroxyindoleacetic acid (5‐HIAA) were assayed in the caudate of freely moving rats using microdialysis and HPLC‐EC. Dialysates were assayed every 20 minutes for 4 hours after an intraperitoneal (i.p.) injection of PMA (2.5, 5, 10, 20 mg/kg), MDMA (10 and 20 mg/kg), or METH (2.5 mg/kg). METH produced a significant increase in extracellular DA (700%), and significant decreases in extracellular DOPAC and HVA (30% and 50%), with no detectable changes in either 5‐HT or 5‐HIAA. MDMA produced significant increases in DA (700% at 10 mg/kg and 950% at 20 mg/kg) and decreases in DOPAC (15% for both 10 and 20 mg/kg), and HVA (50% at 10 mg/kg and 35% at 20 mg/kg). MDMA also increased 5‐HT (350% at 10, and 575% at 20 mg/kg), and decreased 5‐HIAA to 60% for both dose levels. PMA produced no detectable increases in DA at dose levels of 2.5, 5, or 10 mg/kg, but significantly increased DA (975%) at a dose of 20 mg/kg. However, PMA significantly decreased DOPAC at all dose levels (75% at 2.5; 40% at 5; 30% at 10; 10% at 20 mg/kg), with comparable decreases in HVA at all dose levels. PMA also produced significant increases in 5‐HT at 10 and 20 mg/kg (350% for both dose levels), with no detectable changes in 5‐HT at 2.5 or 5 mg/kg. All dose levels of PMA significantly decreased 5‐HIAA (50 to 70%). These data suggest that PMA, like MDMA and METH, is capable of producing dopaminergic and serotonergic neurotoxicity.