Neurochemical and Cardiovascular Effects of 4-Chloro Ring-Substituted Synthetic Cathinones in Rats.

Synthetic cathinones are a class of new psychoactive substances that display psychomotor stimulant properties, and novel cathinone analogs continue to emerge in illicit drug markets worldwide. The aim of the present study was to characterize the pharmacology of 4-chloro ring-substituted cathinones that are appearing in illicit drug markets compared with the effects of 4-methylmethcathinone (mephedrone). Synaptosomes were prepared from rat caudate for dopamine transporter (DAT) assays or from whole brain minus caudate and cerebellum for norepinephrine transporter (NET) and serotonin transporter (SERT) assays. Findings from transporter uptake inhibition and release assays showed that mephedrone and 4-chloromethcathinone (4-CMC) function as substrates at DAT, NET, and SERT, with similar potency at all three transporters. In contrast, 4-chloro-α-pyrrolidinopropiophenone (4-CαPPP) was an uptake inhibitor at DAT and NET, with similar potency at each site, but had little activity at SERT. 4-Chloroethcathinone (4-CEC) was a low-potency uptake inhibitor at DAT and NET but a substrate at SERT. In rats implanted with telemetry transmitters, mephedrone and 4-CMC increased blood pressure, heart rate, and locomotor activity to a similar extent. 4-CEC and 4-CαPPP were less potent at increasing blood pressure and had modest stimulatory effects on heart rate and activity. 4-CMC also transiently decreased temperature at the highest dose tested. All three 4-chloro ring-substituted cathinones are biologically active, but only 4-CMC has potency comparable to mephedrone. Collectively, our findings suggest that 4-CMC and other 4-chloro cathinones may have abuse potential and adverse effects in humans that are analogous to those associated with mephedrone. SIGNIFICANCE STATEMENT: The 4-chloro ring-substituted cathinones all produced significant cardiovascular stimulation, with 4-chloromethcathinone (4-CMC) showing potency similar to mephedrone. All of the drugs are likely to be abused given their effects at the dopamine transporter, particularly 4-CMC.

Amphetamine-like Neurochemical and Cardiovascular Effects of α-Ethylphenethylamine Analogs Found in Dietary Supplements.

Dietary supplements often contain additives not listed on the label, including α-ethyl homologs of amphetamine such as N,α-diethylphenethylamine (DEPEA). Here, we examined the neurochemical and cardiovascular effects of α-ethylphenethylamine (AEPEA), N-methyl-α-ethylphenethylamine (MEPEA), and DEPEA as compared with the effects of amphetamine. All drugs were tested in vitro using uptake inhibition and release assays for monoamine transporters. As expected, amphetamine acted as a potent and efficacious releasing agent at dopamine transporters (DAT) and norepinephrine transporters (NET) in vitro. AEPEA and MEPEA were also releasers at catecholamine transporters, with greater potency at NET than DAT. DEPEA displayed fully efficacious release at NET but weak partial release at DAT (i.e., 40% of maximal effect). In freely moving, conscious male rats fitted with biotelemetry transmitters for physiologic monitoring, amphetamine (0.1-3.0 mg/kg, s.c.) produced robust dose-related increases in blood pressure (BP), heart rate (HR), and motor activity. AEPEA (1-10 mg/kg, s.c.) produced significant increases in BP but not HR or activity, whereas DEPEA and MEPEA (1-10 mg/kg, s.c.) increased BP, HR, and activity. In general, the phenethylamine analogs were approximately 10-fold less potent than amphetamine. Our results show that α-ethylphenethylamine analogs are biologically active. Although less potent than amphetamine, they produce cardiovascular effects that could pose risks to humans. Given that MEPEA and DEPEA increased locomotor activity, these substances may also have significant abuse potential. SIGNIFICANCE STATEMENT: The α-ethyl homologs of amphetamine have significant cardiovascular, behavioral, and neurochemical effects in rats. Given that these compounds are often not listed on the ingredient labels of dietary supplements, these compounds could pose a risk to humans using these products.

The Supplement Adulterant ß-Methylphenethylamine Increases Blood Pressure by Acting at Peripheral Norepinephrine Transporters.

ß-Methylphenethylamine [(BMPEA), 2-phenylpropan-1-amine] is a structural isomer of amphetamine (1-phenylpropan-2-amine) that has been identified in preworkout and weight loss supplements, yet little information is available about its pharmacology. Here, the neurochemical and cardiovascular effects of BMPEA and its analogs, N-methyl-2-phenylpropan-1-amine (MPPA) and N,N-dimethyl-2-phenylpropan-1-amine (DMPPA), were compared with structurally related amphetamines. As expected, amphetamine and methamphetamine were potent substrate-type releasing agents at dopamine transporters (DATs) and norepinephrine transporters (NETs) in rat brain synaptosomes. BMPEA and MPPA were also substrates at DATs and NETs, but they were at least 10-fold less potent than amphetamine. DMPPA was a weak substrate only at NETs. Importantly, the releasing actions of BMPEA and MPPA were more potent at NETs than DATs. Amphetamine produced significant dose-related increases in blood pressure (BP), heart rate (HR), and locomotor activity in conscious rats fitted with surgically implanted biotelemetry transmitters. BMPEA, MPPA, and DMPPA produced increases in BP that were similar to the effects of amphetamine, but the compounds failed to substantially affect HR or activity. The hypertensive effect of BMPEA was reversed by the α-adrenergic antagonist prazosin but not the ganglionic blocker chlorisondamine. Radioligand binding at various G protein-coupled receptors did not identify nontransporter sites of action that could account for cardiovascular effects of BMPEA or its analogs. Our results show that BMPEA, MPPA, and DMPPA are biologically active. The compounds are unlikely to be abused due to weak effects at DATs, but they could produce adverse cardiovascular effects via substrate activity at peripheral NET sites.

Binding Mode Selection Determines the Action of Ecstasy Homologs at Monoamine Transporters.

Determining the structural elements that define substrates and inhibitors at the monoamine transporters is critical to elucidating the mechanisms underlying these disparate functions. In this study, we addressed this question directly by generating a series of N-substituted 3,4-methylenedioxyamphetamine analogs that differ only in the number of methyl substituents on the terminal amine group. Starting with 3,4-methylenedioxy-N-methylamphetamine, 3,4-methylenedioxy-N,N-dimethylamphetamine (MDDMA) and 3,4-methylenedioxy-N,N,N-trimethylamphetamine (MDTMA) were prepared. We evaluated the functional activities of the compounds at all three monoamine transporters in native brain tissue and cells expressing the transporters. In addition, we used ligand docking to generate models of the respective protein-ligand complexes, which allowed us to relate the experimental findings to available structural information. Our results suggest that the 3,4-methylenedioxyamphetamine analogs bind at the monoamine transporter orthosteric binding site by adopting one of two mutually exclusive binding modes. 3,4-methylenedioxyamphetamine and 3,4-methylenedioxy-N-methylamphetamine adopt a high-affinity binding mode consistent with a transportable substrate, whereas MDDMA and MDTMA adopt a low-affinity binding mode consistent with an inhibitor, in which the ligand orientation is inverted. Importantly, MDDMA can alternate between both binding modes, whereas MDTMA exclusively binds to the low-affinity mode. Our experimental results are consistent with the idea that the initial orientation of bound ligands is critical for subsequent interactions that lead to transporter conformational changes and substrate translocation.

Studies of the biogenic amine transporters 15. Identification of novel allosteric dopamine transporter ligands with nanomolar potency.

Novel allosteric modulators of the dopamine transporter (DAT) have been identified. We have shown previously that SRI-9804 [N-(diphenylmethyl)-2-phenyl-4-quinazolinamine], SRI-20040 [N-(2,2-diphenylethyl)-2-phenyl-4-quinazolinamine], and SRI-20041 [N-(3,3-diphenylpropyl)-2-phenyl-4-quinazolinamine] partially inhibit [(125)I]RTI-55 ([(125)I]3ß-(4'-iodophenyl)tropan-2ß-carboxylic acid methyl ester) binding and [(3)H]dopamine ([(3)H]DA) uptake, slow the dissociation rate of [(125)I]RTI-55 from the DAT, and allosterically modulate d-amphetamine-induced, DAT-mediated DA release. We synthesized and evaluated the activity of >500 analogs of these ligands and report here on 36 selected compounds. Using synaptosomes prepared from rat caudate, we conducted [(3)H]DA uptake inhibition assays, DAT binding assays with [(3)H]WIN35428 ([(3)H]2ß-carbomethoxy-3ß-(4-fluorophenyl)tropane), and DAT-mediated release assays with either [(3)H]MPP(+) ([(3)H]1-methyl-4-phenylpyridinium) or [(3)H]DA. We observed three groups of [(3)H]DA uptake inhibitors: 1) full-efficacy agents with a one-site fit, 2) full-efficacy agents with a two-site fit, and 3) partial-efficacy agents with a one-site fit-the focus of further studies. These agents partially inhibited DA, serotonin, and norepinephrine uptake, yet were much less potent at inhibiting [(3)H]WIN35428 binding to the DAT. For example, SRI-29574 [N-(2,2-diphenylethyl)-2-(imidazo[1,2-a]pyridin-6-yl)quinazolin-4-amine] partially inhibited DAT uptake, with an IC50 = 2.3 ± 0.4 nM, without affecting binding to the DAT. These agents did not alter DAT-mediated release of [(3)H]MPP(+) in the absence or presence of 100 nM d-amphetamine. SRI-29574 had no significant effect on the d-amphetamine EC50 or Emax value for DAT-mediated release of [(3)H]MPP(+). These studies demonstrate the existence of potent DAT ligands that partially block [(3)H]DA uptake, without affecting DAT binding or d-amphetamine-induced [(3)H]MPP(+) release. These compounds may prove to be useful probes of biogenic amine transporter function as well as novel therapeutics.

Ethylenedioxy homologs of N-methyl-(3,4-methylenedioxyphenyl)-2-aminopropane (MDMA) and its corresponding cathinone analog methylenedioxymethcathinone: Interactions with transporters for serotonin, dopamine, and norepinephrine.

N-Methyl-(3,4-methylenedioxyphenyl)-2-aminopropane (MDMA; 'Ecstasy'; 1) and its ß-keto analog methylone (MDMC; 2) are popular drugs of abuse. Little is known about their ring-expanded ethylenedioxy homologs. Here, we prepared N-methyl-(3,4-ethylenedioxyphenyl)-2-aminopropane (EDMA; 3), both of its optical isomers, and ß-keto EDMA (i.e., EDMC; 4) to examine their effects at transporters for serotonin (SERT), dopamine (DAT), and norepinephrine (NET). In general, ring-expansion of the methylenedioxy group led to a several-fold reduction in potency at all three transporters. With respect to EDMA (3), S(+)3 was 6-fold, 50-fold, and 8-fold more potent than its R(-) enantiomer at SERT, DAT, and NET, respectively. Overall, in the absence of a ß-carbonyl group, the ethylenedioxy (i.e., 1,4-dioxane) substituent seems better accommodated at SERT than at DAT and NET.

Alpha-ethyltryptamines as dual dopamine-serotonin releasers.

The dopamine (DA), serotonin (5-HT), and norepinephrine (NE) transporter releasing activity and serotonin-2A (5-HT2A) receptor agonist activity of a series of substituted tryptamines are reported. Three compounds, 7b, (+)-7d and 7f, were found to be potent dual DA/5-HT releasers and were >10-fold less potent as NE releasers. Additionally, these compounds had different activity profiles at the 5-HT2A receptor. The unique combination of dual DA/5-HT releasing activity and 5-HT2A receptor activity suggests that these compounds could represent a new class of neurotransmitter releasers with therapeutic potential.

Comparative Pharmacological Effects of Lisuride and Lysergic Acid Diethylamide Revisited.

Lisuride is a non-psychedelic serotonin (5-HT) 2A receptor (5-HT2A) agonist and analogue of the psychedelic lysergic acid diethylamide (LSD). Lisuride also acts as an agonist at the serotonin 1A receptor (5-HT1A), a property known to counter psychedelic effects. Here, we tested whether lisuride lacks psychedelic activity due to a dual mechanism: (1) partial agonism at 5-HT2A and (2) potent agonism at 5-HT1A. The in vitro effects of lisuride, LSD, and related analogues on 5-HT2A signaling were characterized by using miniGαq and ß-arrestin 2 recruitment assays. The 5-HT1A- and 5-HT2A-mediated effects of lisuride and LSD were also compared in male C57BL/6J mice. The in vitro results confirmed that LSD is an agonist at 5-HT2A, with high efficacy and potency for recruiting miniGαq and ß-arrestin 2. By contrast, lisuride displayed partial efficacy for both functional end points (6-52% of 5-HT or LSD Emax) and antagonized the effects of LSD. The mouse experiments demonstrated that LSD induces head twitch responses (HTRs)(ED50 = 0.039 mg/kg), while lisuride suppresses HTRs (ED50 = 0.006 mg/kg). Lisuride also produced potent hypothermia and hypolocomotion (ED50 = 0.008-0.023 mg/kg) that was blocked by the 5-HT1A antagonist WAY100635 (3 mg/kg). Blockade of 5-HT1A prior to lisuride restored basal HTRs, but it failed to increase HTRs above baseline levels. HTRs induced by LSD were blocked by lisuride (0.03 mg/kg) or the 5-HT1A agonist 8-OH-DPAT (1 mg/kg). Overall, our findings show that lisuride is an ultrapotent 5-HT1A agonist in C57BL/6J mice, limiting its use as a 5-HT2A ligand in mouse studies examining acute drug effects. Results also indicate that the 5-HT2A partial agonist-antagonist activity of lisuride explains its lack of psychedelic effects.

Studies of the biogenic amine transporters. 14. Identification of low-efficacy "partial" substrates for the biogenic amine transporters.

Several compounds have been identified that display low-efficacy, "partial substrate" activity. Here, we tested the hypothesis that the mechanism of this effect is a slower rate of induced neurotransmitter efflux than that produced by full substrates. Biogenic amine transporter release assays were carried out in rat brain synaptosomes and followed published procedures. [(3)H]1-methyl-4-phenylpyridinium (MPP(+)) was used to assess release from dopamine (DA) and norepinephrine nerve terminals, whereas [(3)H]5-hydroxytryptamine (5-HT) was used to assess release from 5-HT nerve terminals. A detailed time-course evaluation of DA transporter (DAT)-mediated efflux was conducted by measuring the efflux of [(3)H]MPP(+) after the addition of various test compounds. In vivo microdialysis experiments compared the effects of the full substrates [(±)-1-(2-naphthyl)propan-2-amine (PAL-287) and (S)-N-methyl-1-(2-naphthyl)propan-2-amine (PAL-1046)], to that of a partial DAT/5-HT transporter substrate [(S)-N-ethyl-1-(2-naphthyl)propan-2-amine (PAL-1045)] on extracellular DA and 5-HT in the nucleus accumbens of the rat. The in vitro release assays demonstrated that partial substrate activity occurs at all three transporters. In the DAT efflux experiments, D-amphetamine (full substrate) promoted a fast efflux (K1 = 0.24 min(-1)) and a slow efflux (K2 = 0.008 min(-1)). For the partial DAT substrates, K1 = ∼0.04 min(-1), and K2 approximated zero. The in vivo microdialysis experiments showed that the partial substrate (PAL-1045) was much less effective in elevating extracellular DA and 5-HT than the comparator full substrates. We conclude that low-efficacy partial DAT substrates promote efflux at a slower rate than full substrates, and "partiality" reflects the ultra-slow K2 constant, which functionally limits the ability of these compounds to increase extracellular DA. We speculate that partial biogenic amine transporter substrates bind to the transporter but are less effective in inducing conformational changes required for reverse transport activity.

Structure-activity relationships for 5F-MDMB-PICA and its 5F-pentylindole analogs to induce cannabinoid-like effects in mice.

Synthetic cannabinoid receptor agonists (SCRAs) are an evolving class of new psychoactive substances found on recreational drug markets worldwide. The indole-containing compound, 5F-MDMB-PICA, is a popular SCRA associated with serious medical consequences, including overdose and hospitalizations. In vitro studies reveal that 5F-MDMB-PICA is a potent agonist at cannabinoid type 1 receptors (CB1), but little information exists regarding in vivo pharmacology of the drug. To this end, we examined the in vitro and in vivo cannabinoid-like effects produced by 5F-MDMB-PICA and related 5F-pentylindole analogs with differing composition of the head group moiety (i.e., 5F-NNEI, 5F-SDB-006, 5F-CUMYL-PICA, 5F-MMB-PICA). In mouse brain membranes, 5F-MDMB-PICA and its analogs inhibited binding to [3H]rimonabant-labeled CB1 and displayed agonist actions in [35S]GTPγS functional assays. 5F-MDMB-PICA exhibited the highest CB1 affinity (Ki = 1.24 nM) and functional potency (EC50 = 1.46 nM), but head group composition markedly influenced activity in both assays. For example, the 3,3-dimethylbutanoate (5F-MDMB-PICA) and cumyl (5F-CUMYL-PICA) head groups engendered high CB1 affinity and potency, whereas a benzyl (5F-SDB-006) head group did not. In C57BL/6J mice, all 5F-pentylindole SCRAs produced dose- and time-dependent hypothermia, catalepsy, and analgesia that were reversed by rimonabant, indicating CB1 involvement. In vitro Ki and EC50 values were positively correlated with in vivo ED50 potency estimates. Our findings demonstrate that 5F-MDMB-PICA is a potent SCRA, and subtle alterations to head group composition can have profound influence on pharmacological effects at CB1. Importantly, measures of CB1 binding and efficacy in mouse brain tissue seem to accurately predict in vivo drug potency in this species.

Structure-Activity Relationships for Psilocybin, Baeocystin, Aeruginascin, and Related Analogues to Produce Pharmacological Effects in Mice.

4-Phosphoryloxy-N,N-dimethyltryptamine (psilocybin) is a naturally occurring tertiary amine found in many mushroom species. Psilocybin is a prodrug for 4-hydroxy-N,N-dimethyltryptamine (psilocin), which induces psychedelic effects via agonist activity at the serotonin (5-HT) 2A receptor (5-HT2A). Several other 4-position ring-substituted tryptamines are present in psilocybin-containing mushrooms, including the secondary amine 4-phosphoryloxy-N-methyltryptamine (baeocystin) and the quaternary ammonium 4-phosphoryloxy-N,N,N-trimethyltryptamine (aeruginascin), but these compounds are not well studied. Here, we investigated the structure-activity relationships for psilocybin, baeocystin, and aeruginascin, as compared to their 4-acetoxy and 4-hydroxy analogues, using in vitro and in vivo methods. Broad receptor screening using radioligand binding assays in transfected cells revealed that secondary and tertiary tryptamines with either 4-acetoxy or 4-hydroxy substitutions display nanomolar affinity for most human 5-HT receptor subtypes tested, including the 5-HT2A and the serotonin 1A receptor (5-HT1A). The same compounds displayed affinity for 5-HT2A and 5-HT1A in mouse brain tissue in vitro and exhibited agonist efficacy in assays examining 5-HT2A-mediated calcium mobilization and ß-arrestin 2 recruitment. In mouse experiments, only the tertiary amines psilocin, psilocybin, and 4-acetoxy-N,N-dimethyltryptamine (psilacetin) induced head twitch responses (ED50 0.11-0.29 mg/kg) indicative of psychedelic-like activity. Head twitches were blocked by 5-HT2A antagonist pretreatment, supporting 5-HT2A involvement. Both secondary and tertiary amines decreased body temperature and locomotor activity at higher doses, the effects of which were blocked by 5-HT1A antagonist pretreatment. Across all assays, the pharmacological effects of 4-acetoxy and 4-hydroxy compounds were similar, and these compounds were more potent than their 4-phosphoryloxy counterparts. Importantly, psilacetin appears to be a prodrug for psilocin that displays substantial serotonin receptor activities of its own.

Evidence for noncompetitive modulation of substrate-induced serotonin release.

Prior work indicated that serotonin transporter (SERT) inhibitors competitively inhibit substrate-induced [(3)H]5-HT release, producing rightward shifts in the substrate-dose response curve and increasing the EC(50) value without altering the E(max). We hypothesized that this finding would not generalize across a number of SERT inhibitors and substrates, and that the functional dissociation constant (Ke) of a given SERT inhibitor would not be the same for all tested substrates. To test this hypothesis, we utilized a well-characterized [(3)H]5-HT release assay that measures the ability of a SERT substrate to release preloaded [(3)H]5-HT from rat brain synaptosomes. Dose-response curves were generated for six substrates (PAL-287 [naphthylisopropylamine], (+)-fenfluramine, (+)-norfenfluramine, mCPP [meta-chlorophenylpiperazine], (±)-MDMA, 5-HT) in the absence and presence of a fixed concentration of three SERT inhibitors (indatraline, BW723C86, EG-1-149 [4-(2-(benzhydryloxy)ethyl)-1-(4-bromobenzyl)piperidine oxalate]). Consistent with simple competitive inhibition, all SERT inhibitors increased the EC(50) value of all substrates. However, in many cases a SERT inhibitor decreased the E(max) value as well, indicating that in the presence of the SERT inhibitor the substrate became a partial releaser. Moreover, the Ke values of a given SERT inhibitor differed among the six SERT substrates, indicating that each inhibitor/substrate combination had a unique interaction with the transporter. Viewed collectively, these findings suggest that it may be possible to design SERT inhibitors that differentially regulate SERT function.

The psychoactive aminoalkylbenzofuran derivatives, 5-APB and 6-APB, mimic the effects of 3,4-methylenedioxyamphetamine (MDA) on monoamine transmission in male rats.

RATIONALE: The nonmedical use of new psychoactive substances (NPS) is a worldwide public health concern. The so-called "benzofury" compounds, 5-(2-aminopropyl)benzofuran (5-APB) and 6-(2-aminopropyl)benzofuran (6-APB), are NPS with stimulant-like properties in human users. These substances are known to interact with monoamine transporters and 5-HT receptors in transfected cells, but less is known about their effects in animal models. METHODS: Here, we used in vitro monoamine transporter assays in rat brain synaptosomes to characterize the effects of 5-APB and 6-APB, together with their N-methyl derivatives 5-MAPB and 6-MAPB, in comparison with 3,4-methylenedioxyamphetamine (MDA) and 3,4-methylenedioxymethamphetamine (MDMA). In vivo neurochemical and behavioral effects of 5-APB (0.3 and 1.0 mg/kg, i.v.) and 6-APB (0.3 and 1.0 mg/kg, i.v.) were assessed in comparison with MDA (1.0 and 3.0 mg/kg, i.v.) using microdialysis sampling in the nucleus accumbens of conscious male rats. RESULTS: All four benzofuran derivatives were substrate-type releasers at dopamine transporters (DAT), norepinephrine transporters (NET), and serotonin transporters (SERT) with nanomolar potencies, similar to the profile of effects produced by MDA and MDMA. However, the benzofurans were at least threefold more potent than MDA and MDMA at evoking transporter-mediated release. Like MDA, both benzofurans induced dose-related elevations in extracellular dopamine and serotonin in the brain, but benzofurans were more potent than MDA. The benzofuran derivatives also induced profound behavioral activation characterized by forward locomotion which lasted for at least 2 h post-injection. CONCLUSIONS: Overall, benzofurans are more potent than MDA in vitro and in vivo, producing sustained stimulant-like effects in rats. These data suggest that benzofuran-type compounds may have abuse liability and could pose risks for adverse effects, especially if used in conjunction with abused drugs or medications which enhance monoamine transmission in the brain.

Studies of the biogenic amine transporters. 13. Identification of "agonist" and "antagonist" allosteric modulators of amphetamine-induced dopamine release.

Recent studies identified novel allosteric modulators of the dopamine (DA) transporter (DAT). N-(Diphenylmethyl)-2-phenyl-4-quinazolinamine (SoRI-9804), N-(2,2-diphenylethyl)-2-phenyl-4-quinazolinamine (SoRI-20040), and N-(3,3-diphenylpropyl)-2-phenyl-4-quinazolinamine (SoRI-20041) partially inhibited [(125)I]3beta-(4'-iodophenyl)tropan-2beta-carboxylic acid methyl ester (RTI-55) binding, slowed the dissociation rate of [(125)I]RTI-55 from the DAT, and partially inhibited [(3)H]dopamine uptake. In the present study, we report that SoRI-9804 and SoRI-20040, at doses that do not alter release, partially inhibited d-amphetamine-induced DAT-mediated release of [(3)H]1-methyl-4-phenylpyridinium (MPP(+))or[(3)H]dopamine from striatal synaptosomes ("DAT-mediated DA release") in a dose-dependent manner. SoRI-20041, which does not alter DAT-mediated DA release measured with [(3)H]DA, reversed the effect of SoRI-20040. SoRI-20040 and SoRI-9804 also partially inhibited DAT-mediated DA release induced by DA or (+/-)-3,4-methylenedioxyamphetamine, demonstrating that the observed partial inhibition is not specific for a particular DAT substrate. SoRI-9804 and SoRI-20040 did not attenuate D-amphetamine-induced release of [(3)H]5-hydroxytryptamine from serotonergic, or [(3)H]MPP(+) from noradrenergic, nerve terminals. Kinetic experiments demonstrated that SoRI-9804, in contrast to cocaine, slowed D-amphetamine-induced release of [(3)H]MPP(+) from dopaminergic nerve terminals without altering the apparent rate constants. The two major findings of this study are 1) the identification of both "agonist" (SoRI-9804 and SoRI-20040) and "antagonist" (SoRI-20041) allosteric modulators of D-amphetamine-induced DAT-mediated DA release and 2) [(3)H]DA uptake and d-amphetamine-induced DAT-mediated efflux can be separately modulated. Such agents may have therapeutic potential for the treatment of stimulant addiction, Parkinson's disease, and other psychiatric disorders.

Evidence for the involvement of dopamine transporters in behavioral stimulant effects of modafinil.

Modafinil is prescribed for numerous medical conditions, but the drug's mechanism of action is unclear. Here, we examined the interaction of modafinil with receptors and transporters in vitro and compared pharmacological effects of the drug with those produced by indirect dopamine (DA) agonists 1-[2-[bis(4-fluorophenyl)methoxy]ethyl]-4-(3-phenylpropyl)piperazine (GBR12909) and (+)-methamphetamine (METH). Modafinil was screened at various receptors and transporters using binding assays. Transporter-mediated uptake and release were examined in rat brain synaptosomes. Effects of modafinil on motor activity and neurochemistry were determined in rats undergoing in vivo microdialysis in nucleus accumbens. Of the receptors and transporters assayed, modafinil displayed measurable potency only at DA transporters (DAT), inhibiting [(3)H]DA uptake, with an IC(50) value of 4.0 microM. Accordingly, modafinil pretreatment (10 microM) antagonized METH-induced release of the DAT substrate [(3)H]1-methyl-4-phenylpyridinium. Intravenous modafinil (20 and 60 mg/kg) produced dose-dependent increases in motor activity and extracellular DA, without affecting serotonin (5-HT). Analogous results were observed for GBR12909 (1 and 3 mg/kg), whereas METH (0.3 and 1 mg/kg) increased DA and 5-HT. Locomotor effects of all drugs were positively correlated with dialysate DA (P < 0.001). Interestingly, modafinil pretreatment reduced METH-induced ambulation and DA release. Our data show that modafinil interacts with DAT sites in rat brain, a property shared with agonist medications under investigation for treating cocaine dependence. Nondopaminergic mechanisms may also contribute to the pharmacology of modafinil. Finally, the results suggest that modafinil should be tested as an adjunct for treating METH addiction.

The dopamine, serotonin and norepinephrine releasing activities of a series of methcathinone analogs in male rat brain synaptosomes.

RATIONALE: Novel synthetic "bath salt" cathinones continue to appear on the street as abused and addictive drugs. The range of subjective experiences produced by different cathinones suggests that some compounds have primarily dopaminergic activity (possible stimulants) while others have primarily serotonergic activity (possible empathogenics). An understanding of the structure activity relationships (SARs) of these compounds will help in assessing the likely behavioral effects of future novel structures, and to define potential therapeutic strategies to reverse any reinforcing effects. OBJECTIVES: A series of methcathinone analogs was systematically studied for their activity at the dopamine and serotonin transporters. Compound structures varied at the aromatic group, either by substituent or by replacement of the phenyl ring with a naphthalene or indole ring. METHODS: A novel, high-yielding synthesis of methcathinone hydrochlorides was developed which avoids isolation of the unstable free bases. Neurotransmitter transporter release activity was determined in rat brain synaptosomes as previously reported. Compounds were also screened for activity at the norepinephrine transporter. RESULTS: Twenty-eight methcathinone analogs were analyzed and fully characterized in dopamine and serotonin transporter release assays. Compounds substituted at the 2-position (ortho) were primarily dopaminergic. Compounds substituted at the 3-position (meta) were found to be much less dopaminergic, with some substituents favoring serotonergic activity. Compounds substituted at the 4-position (para) were found to be far more serotonergic, as were disubstituted compounds and other large aromatic groups. One exception was the fluoro-substituted analogs which seem to favor the dopamine transporter. CONCLUSIONS: The dopaminergic to serotonergic ratio can be manipulated by choice of substituent and location on the aromatic ring. It is therefore likely possible to tweak the subjective and reinforcing effects of these compounds by adjusting their structure. Certain substituents like a fluoro group tend to favor the dopamine transporter, while others like a trifluoromethyl group favor the serotonin transporter.

The synthetic cathinones, butylone and pentylone, are stimulants that act as dopamine transporter blockers but 5-HT transporter substrates.

RATIONALE: Synthetic cathinones continue to emerge in recreational drug markets worldwide. 1-(1,3-Benzodioxol-5-yl)-2-(methylamino)butan-1-one (butylone) and 1-(1,3-benzodioxol-5-yl)-2-(methylamino)pentan-1-one (pentylone) are derivatives of the cathinone compound, 1-(1,3-benzodioxol-5-yl)-2-(methylamino)propan-1-one (methylone), that are being detected in drug products and human casework. OBJECTIVES: The purpose of the present study was to examine the neuropharmacology of butylone and pentylone using in vitro and in vivo methods. METHODS: In vitro uptake and release assays were carried out in rat brain synaptosomes and in cells expressing human dopamine transporters (DAT) and 5-HT transporters (SERT). In vivo microdialysis was performed in the nucleus accumbens of conscious rats to assess drug-induced changes in neurochemistry. RESULTS: Butylone and pentylone were efficacious uptake blockers at DAT and SERT, though pentylone was more DAT-selective. Both drugs acted as transporter substrates that evoked release of [3H]5-HT at SERT, while neither evoked release at DAT. Consistent with the release data, butylone and pentylone induced substrate-associated inward currents at SERT but not DAT. Administration of butylone or pentylone to rats (1 and 3 mg/kg, i.v.) increased extracellular monoamines and motor activity, but pentylone had weaker effects on 5-HT and stronger effects on motor stimulation. CONCLUSIONS: Our data demonstrate that increasing the α-carbon chain length of methylone creates "hybrid" transporter compounds which act as DAT blockers but SERT substrates. Nevertheless, butylone and pentylone elevate extracellular dopamine and stimulate motor activity, suggesting both drugs possess significant risk for abuse.

Studies of the biogenic amine transporters. 12. Identification of novel partial inhibitors of amphetamine-induced dopamine release.

Previous studies identified partial inhibitors and allosteric modulators of 5-hydroxytryptamine ([5-amino-3-(3,4-dichlorophenyl)-1,2-dihydropyrido[3,4-b]pyrazin-7-yl]carbamic acid ethyl ester [SoRI-6238], 4-(2-[bis(4-fluorophenyl)methoxy]ethyl)-1-(2-trifluoromethyl-benzyl)-piperidine [TB-1-099]) and dopamine transporters N-(diphenylmethyl)-2-phenyl-4-quinazolinamine, [SoRI-9804]). We report here the identification of three novel allosteric modulators of the dopamine transporter [N-(2,2-diphenylethyl)-2-phenyl-4-quinazolinamine [SoRI-20040], N-(3,3-diphenylpropyl)-2-phenyl-4-quinazolinamine [SoRI-20041], and [4-amino-6-[(diphenylmethyl)amino]-5-nitro-2-pyridinyl]carbamic acid ethyl ester [SoRI-2827]]. Membranes were prepared from human embryonic kidney cells expressing the cloned human dopamine transporter (hDAT). [(125)I]3beta-(4'-Iodophenyl)tropan-2beta-carboxylic acid methyl ester ([(125)I]RTI-55) binding and other assays followed published procedures. SoRI-20040, SoRI-20041, and SoRI-2827 partially inhibited [(125)I]RTI-55 binding, with EC(50) values ranging from approximately 1.4 to 3 microM and E(max) values decreasing as the [(125)I]RTI-55 concentrations increased. All three compounds decreased the [(125)I]RTI-55 B(max) value and increased the apparent K(d) value in a manner well described by a sigmoid dose-response curve. In dissociation rate experiments, SoRI-20040 (10 microM) and SoRI-20041 (10 microM), but not SoRI-2827 (10 microM), slowed the dissociation of [(125)I]RTI-55 from hDAT by approximately 30%. Using rat brain synaptosomes, all three agents partially inhibited [(3)H]dopamine uptake, with EC(50) values ranging from 1.8 to 3.1 microM and decreased the V(max) value in a dose-dependent manner. SoRI-9804 and SoRI-20040 partially inhibited amphetamine-induced dopamine transporter-mediated release of [(3)H]1-methyl-4-phenylpyridinium ion from rat caudate synaptosomes in a dose-dependent manner. Viewed collectively, we report several compounds that allosterically modulate hDAT binding and function, and we identify novel partial inhibitors of amphetamine-induced dopamine release.

Herkinorin analogues with differential beta-arrestin-2 interactions.

Salvinorin A is a psychoactive natural product that has been found to be a potent and selective kappa opioid receptor agonist in vitro and in vivo. The activity of salvinorin A is unusual compared to other opioids such as morphine in that it mediates potent kappa opioid receptor signaling yet leads to less receptor downregulation than observed with other kappa agonists. Our initial chemical modifications of salvinorin A have yielded one analogue, herkinorin ( 1c), with high affinity at the microOR. We recently reported that 1c does not promote the recruitment of beta-arrestin-2 to the microOR or receptor internalization. Here we describe three new derivatives of 1c ( 3c, 3f, and 3i) with similar properties and one, benzamide 7b, that promotes recruitment of beta-arrestin-2 to the microOR and receptor internalization. When the important role micro opioid receptor regulation plays in determining physiological responsiveness to opioid narcotics is considered, micro opioids derived from salvinorin A may offer a unique template for the development of functionally selective mu opioid receptor-ligands with the ability to produce analgesia while limiting adverse side effects.

Evidence for a mu-delta opioid receptor complex in CHO cells co-expressing mu and delta opioid peptide receptors.

Based on non-competitive binding interactions we suggested that mu and delta receptors associate as a mu/delta receptor complex in rat brain. We hypothesized that the same non-competitive binding interactions observed in rat brain will be seen in CHO cells that co-express mu and delta receptors, but not in cells that express just mu or delta receptors. We used CHO cells expressing the cloned human mu receptor, cloned human delta receptor, or cloned mouse delta/human mu ("dimer cell"). Cell membranes were prepared from intact cells pretreated with 100nM SUPERFIT. [(3)H][d-Ala(2),d-Leu(5)]enkephalin binding assays followed published procedures. SUPERFIT, a delta-selective irreversible ligand, decreased [(3)H][d-Ala(2),d-Leu(5)]enkephalin binding to delta receptors by approximately 75% and to mu receptors by approximately 50% in dimer cells. SUPERFIT treatment did not decrease [(3)H][d-Ala(2),d-Leu(5)]enkephalin binding to mu cells. The IC(50) values observed in SUPERFIT-treated dimer cells were: [d-Pen(2),d-Pen(5)]enkephalin (1820nM) and morphine (171nM). Saturation binding experiments with SUPERFIT-treated dimer cells showed that [d-Pen(2),d-Pen(5)]enkephalin (5000nM) was a competitive inhibitor. In contrast, morphine (1000nM) lowered the B(max) from 1944fmol/mg to 1276fmol/mg protein (35% decrease). Both [d-Pen(2),d-Pen(5)]enkephalin and morphine competitively inhibited [(3)H][d-Ala(2),d-Leu(5)]enkephalin binding to SUPERFIT-treated mu cells. The results indicate that the mu-delta opioid receptor complex defined on the basis of non-competitive binding interactions in rat brain over 20 years ago likely occurs as a consequence of the formation of mu-delta heterodimers. SUPERFIT-treated dimer cells may provide a useful model to study the properties of mu-delta heterodimers.