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Previous research has demonstrated therapeutic potential for VMAT2 inhibitors in rat models of methamphetamine use disorder. Here, we report on the neurochemical and behavioral effects of 1-(2-methoxyphenethyl)-4-phenethypiperazine (JPC-141), a novel analog of lobelane. JPC-141 potently inhibited (Ki = 52 nM) [ 3 H]dopamine uptake by VMAT2 in striatal vesicles with 50 to 250-fold greater selectivity for VMAT2 over dopamine, norepinephrine and serotonin plasmalemma transporters. Also, JPC-141 was 57-fold more selective for inhibiting VMAT2 over [ 3 H]dofetilide binding to hERG channels expressed by HEK293, suggesting relatively low potential for cardiotoxicity. When administered in vivo to rats, JPC-141 prevented the METH-induced reduction in striatal dopamine content when given either prior to or after a high dose of METH, suggesting a reduction in METH-induced dopaminergic neurotoxicity. In behavioral assays, JPC-141 decreased METH-stimulated locomotor activity in METH-sensitized rats at doses of JPC-141 which did not alter locomotor activity in the saline control group. Moreover, JPC-141 specifically decreased iv METH self-administration at doses that had no effect on food-maintained responding. These findings support the further development of VMAT2 inhibitors as pharmacotherapies for individuals with methamphetamine use disorder., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Despite escalating methamphetamine use and high relapse rates, pharmacotherapeutics for methamphetamine use disorders are not available. Our iterative drug discovery program had found that R - N -(1,2-dihydroxypropyl)-2,6- cis -di-(4-methoxyphenethyl)piperidine hydrochloride (GZ-793A), a selective vesicular monoamine transporter-2 (VMAT2) inhibitor, specifically decreased methamphetamine's behavioral effects. However, GZ-793A inhibited human-ether-a-go-go-related gene (hERG) channels, suggesting cardiotoxicity and prohibiting clinical development. The current study determined if replacement of GZ-793A's piperidine ring with a phenylalkyl group to yield S -3-(4-methoxyphenyl)- N -(1-phenylpropan-2-yl)propan-1-amine (GZ-11608) diminished hERG interaction while retaining pharmacological efficacy. VMAT2 inhibition, target selectivity, and mechanism of GZ-11608-induced inhibition of methamphetamine-evoked vesicular dopamine release were determined. We used GZ-11608 doses that decreased methamphetamine-sensitized activity to evaluate the potential exacerbation of methamphetamine-induced dopaminergic neurotoxicity. GZ-11608-induced decreases in methamphetamine reinforcement and abuse liability were determined using self-administration, reinstatement, and substitution assays. Results show that GZ-11608 exhibited high affinity (K i = 25 nM) and selectivity (92-1180-fold) for VMAT2 over nicotinic receptors, dopamine transporter, and hERG, suggesting low side-effects. GZ-11608 (EC 50 = 620 nM) released vesicular dopamine 25-fold less potently than it inhibited VMAT2 dopamine uptake. GZ-11608 competitively inhibited methamphetamine-evoked vesicular dopamine release (Schild regression slope = 0.9 ± 0.13). GZ-11608 decreased methamphetamine sensitization without altering striatal dopamine content or exacerbating methamphetamine-induced dopamine depletion, revealing efficacy without neurotoxicity. GZ-11608 exhibited linear pharmacokinetics and rapid brain penetration. GZ-11608 decreased methamphetamine self-administration, and this effect was not surmounted by increasing methamphetamine unit doses. GZ-11608 reduced cue- and methamphetamine-induced reinstatement, suggesting potential to prevent relapse. GZ-11608 neither served as a reinforcer nor substituted for methamphetamine, suggesting low abuse liability. Thus, GZ-11608, a potent and selective VMAT2 inhibitor, shows promise as a therapeutic for methamphetamine use disorder. SIGNIFICANCE STATEMENT: GZ-11608 is a potent and selective vesicular monoamine transporter-2 inhibitor that decreases methamphetamine-induced dopamine release from isolated synaptic vesicles from brain dopaminergic neurons. Results from behavioral studies show that GZ-11608 specifically decreases methamphetamine-sensitized locomotor activity, methamphetamine self-administration, and reinstatement of methamphetamine-seeking behavior, without exhibiting abuse liability. Tolerance does not develop to the efficacy of GZ-11608 to decrease the behavioral effects of methamphetamine. In conclusion, GZ-11608 is an outstanding lead in our search for a therapeutic to treat methamphetamine use disorder., (Copyright © 2019 by The American Society for Pharmacology and Experimental Therapeutics.)

Despite increased methamphetamine use worldwide, pharmacotherapies are not available to treat methamphetamine use disorder. The vesicular monoamine transporter-2 (VMAT2) is an important pharmacological target for discovery of treatments for methamphetamine use disorder. VMAT2 inhibition by the natural product, lobeline, reduced methamphetamine-evoked dopamine release, methamphetamine-induced hyperlocomotion, and methamphetamine self-administration in rats. Compared to lobeline, lobelane exhibited improved affinity and selectivity for VMAT2 over nicotinic acetylcholine receptors. Lobelane inhibited neurochemical and behavioral effects of methamphetamine, but tolerance developed to its behavioral efficacy in reducing methamphetamine self-administration, preventing further development. The lobelane analog, R-N-(1,2-dihydroxypropyl)-2,6-cis-di-(4-methoxyphenethyl)piperidine hydrochloride (GZ-793A), potently and selectively inhibited VMAT2 function and reduced neurochemical and behavioral effects of methamphetamine. However, GZ-793A exhibited potential to induce ventricular arrhythmias interacting with human-ether-a-go-go (hERG) channels. Herein, a new lead, R-3-(4-methoxyphenyl)-N-(1-phenylpropan-2-yl)propan-1-amine (GZ-11610), from the novel scaffold (N-alkyl(1-methyl-2-phenylethyl)amine) was evaluated as a VMAT2 inhibitor and potential therapeutic for methamphetamine use disorder. GZ-11610 was 290-fold selective for VMAT2 over dopamine transporters, suggesting that it may lack abuse liability. GZ-11610 was 640- to 3500-fold selective for VMAT2 over serotonin transporters and nicotinic acetylcholine receptors. GZ-11610 exhibited > 1000-fold selectivity for VMAT2 over hERG, representing a robust improvement relative to our previous VMAT2 inhibitors. GZ-11610 (3-30 mg/kg, s.c. or 56-300 mg/kg, oral) reduced methamphetamine-induced hyperactivity in methamphetamine-sensitized rats. Thus, GZ-11610 is a potent and selective inhibitor of VMAT2, may have low abuse liability and low cardiotoxicity, and after oral administration is effective and specific in inhibiting the locomotor stimulant effects of methamphetamine, suggesting further investigation as a potential therapeutic for methamphetamine use disorder.

A small library of fluoroethoxy-1,4-diphenethyl piperidine and fluoroethoxy-1,4-diphenethyl piperazine derivatives were designed, synthesized and evaluated for their ability to inhibit [ 3 H]dopamine (DA) uptake at the vesicular monoamine transporter-2 (VMAT2) and dopamine transporter (DAT), [ 3 H]serotonin (5-HT) uptake at the serotonin transporter (SERT), and [ 3 H]dofetilide binding at the human-ether-a-go-go-related gene (hERG) channel. The majority of the compounds exhibited potent inhibition of [ 3 H]DA uptake at VMAT2, Ki changes in the nanomolar range (K i  = 0.014-0.073 µM). Compound 15d exhibited the highest affinity (K i  = 0.014 µM) at VMAT2, and had 160-, 5-, and 60-fold greater selectivity for VMAT2 vs. DAT, SERT and hERG, respectively. Compound 15b exhibited the greatest selectivity (>60-fold) for VMAT2 relative to all the other targets evaluated, and 15b had high affinity for VMAT2 (K i  = 0.073 µM). Compound 15b was considered the lead compound from this analog series due to its high affinity and selectivity for VMAT2., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Lobeline and lobelane inhibit the behavioral and neurochemical effects of methamphetamine via an interaction with the vesicular monoamine transporter-2 (VMAT2). However, lobeline has high affinity for nicotinic receptors, and tolerance develops to the behavioral effects of lobelane. A water-soluble analog of lobelane, R-N-(1,2-dihydroxypropyl)-2,6-cis-di-(4-methoxyphenethyl)piperidine hydrochloride (GZ-793A), also interacts selectively with VMAT2 to inhibit the effects of methamphetamine, but does not produce behavioral tolerance. The current study further evaluated the mechanism underlying the GZ-793A-mediated inhibition of the neurochemical effects of methamphetamine. In contrast to lobeline, GZ-793A does not interact with the agonist recognition site on α4β2 * and α7 * nicotinic receptors. GZ-793A (0.3-100µM) inhibited methamphetamine (5µM)-evoked fractional dopamine release from rat striatal slices, and did not evoke dopamine release in the absence of methamphetamine. Furthermore, GZ-793A (1-100µM) inhibited neither nicotine (30µM)-evoked nor electrical field-stimulation-evoked (100Hz/1min) fractional dopamine release. Unfortunately, GZ-793A inhibited [ 3 H]dofetilide binding to human-ether-a-go-go related gene channels expressed on human embryonic kidney cells, and further, prolonged action potentials in rabbit cardiac Purkinje fibers, suggesting the potential for GZ-793A to induce ventricular arrhythmias. Thus, GZ-793A selectively inhibits the neurochemical effects of methamphetamine and lacks nicotinic receptor interactions; however, development as a pharmacotherapy for methamphetamine use disorders will not be pursued due to its potential cardiac liabilities., (Copyright © 2016. Published by Elsevier B.V.)

A small library of 1,4-diphenethylpiperazine analogs was synthesized and evaluated for inhibition of [(3)H]dihydrotetrabenazine binding and [(3)H]dopamine uptake at the vesicular monoamine transporter-2 (VMAT2). Results from these studies identified three novel molecules, 6b, 6e and 9a (Ki=35nM, 48nM and 37nM, respectively) that exhibit similar potency for inhibition of VMAT2 function compared with lobelane (Ki=45nM), and importantly, have enhanced water-solubility when compared to the previously reported 1,4-diphenethylpiperidine analogs. These 1,4-diphenethylpiperazine analogs constitute promising new leads in the discovery of potential pharmacotherapeutics for treatment of methamphetamine use disorders., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

A series of 1,4-diphenalkylpiperidine analogs were synthesized and evaluated for their affinity and inhibitory potency at the [(3)H]dihydrotetrabenazine (DTBZ) binding site and [(3)H]dopamine (DA) uptake site on the vesicular monoamine transporter-2 (VMAT2). Results revealed that translocation of the phenethyl side chains of lobelane from C2 and C6 to C1 and C4 around the central piperidine ring slightly reduces affinity and inhibitory potency at VMAT2 with respect to lobelane. However, methoxy and fluoro-substitution of either phenyl ring of these 1,4-diphenethyl analogs afforded VMAT2 inhibition comparable or higher (5-fold) affinity at the DTBZ binding and DA uptake sites relative to lobelane, whereas replacement of the 4-phenethyl moiety in these analogs with a 4-phenmethyl moiety markedly reduced affinity for the DTBZ binding and DA uptake sites by 3- and 5-fold, respectively. Among the twenty five 1,4-diphenethylpiperidine analogs evaluated, compounds containing a 4-(2-methoxyphenethyl) moiety exhibited the most potent inhibition of DTBZ binding and vesicular DA uptake. From this subgroup, analogs 8h, 8j and 8m exhibited Ki values of 9.3nM, 13nM and 13nM, respectively, for inhibition of [(3)H]DA uptake by VMAT2, and represent some of the most potent inhibitors of VMAT2 function reported thus far., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

A series of lobelane and GZ-793A analogues that incorporate aromatic 4-hydroxy and 4-(2-fluoroethoxy) substituents were synthesized and evaluated for inhibition of [(3)H]dopamine (DA) uptake at the vesicular monoamine transporter-2 (VMAT2) and the dopamine transporter (DAT), and [(3)H]serotonin uptake at the serotonin transporter (SERT). Most of these compounds exhibited potent inhibition of DA uptake at VMAT2 in the nanomolar range (Ki=30-70nM). The two most potent analogues, 7 and 14, both exhibited a Ki value of 31nM for inhibition of VMAT2. The lobelane analogue 14, incorporating 4-(2-fluoroethoxy) and 4-hydroxy aromatic substituents, exhibited 96- and 335-fold greater selectivity for VMAT2 versus DAT and SERT, respectively, in comparison to lobelane. Thus, lobelane analogues bearing hydroxyl and fluoroethoxy moieties retain the high affinity for VMAT2 of the parent compound, while enhancing selectivity for VMAT2 versus the plasmalemma transporters., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

We have previously shown that quinolyl moieties are attractive structural replacements for the phenyl groups in lobelane. These quinolyl analogues had improved water-solubility over lobelane and retained the potent vesicular monoamine transporter-2 (VMAT-2) inhibitory properties of the parent compound, with quinlobelane (4) exhibiting potent inhibition of uptake at VMAT-2 (Ki=51nM). However, the VMAT-2 inhibitory properties of quinolyl analogues of norlobelane, which is equipotent with lobeline as an inhibitor of [(3)H]dopamine (DA) uptake at VMAT-2, have not been reported. In the current communication, we describe the synthesis of some novel des-methyl quinolyl analogues of lobelane that exhibit greater affinity (Ki=178-647nM) for the dihydrotetrabenazine binding site located on VMAT-2 compared with lobelane (Ki=970nM), norlobelane (Ki=2310nM) and quinlobelane (Ki=2640nM). The most potent compounds, 14 and 15, also exhibited inhibition of [(3)H]DA uptake at VMAT-2 (Ki=42nM) which was comparable to both lobelane (Ki=45nM) and norlobelane (Ki=43nM). Results reveal that binding affinity at VMAT-2 serves as an accurate predictor of inhibition of the function of VMAT-2 for the majority of these analogues. These novel analogues are under consideration for further development as treatments for methamphetamine abuse., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Environmental factors influence a variety of health-related outcomes. In general, being raised in an environment possessing social, sensory, and motor enrichment reduces the rewarding effects of various drugs, thus protecting against abuse vulnerability. However, in the case of methamphetamine (METH), which acts at the vesicular monoamine transporter 2 (VMAT2) to enhance dopamine release from the cytosol, previous evidence suggests that METH reward may not be altered by environmental enrichment. This study examined the influence of an enriched environment on measures of METH reward, METH seeking, and VMAT2 function. Rats were raised from weaning to adulthood in either an enriched environment (presence of social cohorts and novel objects) or an isolated environment (no cohorts or novel objects). Rats in these two conditions were subsequently tested for their acquisition of conditioned place preference (CPP), METH self-administration, maintenance of self-administration at various unit doses of METH (0.001-0.5mg/kg/infusion), and cue-induced reinstatement. VMAT2 function in striatum from these two groups also was assessed. No significant environment effects were found in CPP or METH self-administration, which paralleled a lack of effect in VMAT2 function between groups. However, cue-induced reinstatement was reduced by environmental enrichment. Together, these results suggest that environmental enrichment does not alter VMAT2 function involved in METH reward. However, the enrichment-induced decrease in cue-induced reinstatement indicates that enrichment may have a beneficial effect against relapse following a period of extinction via a neural mechanism other than striatal VMAT2 function., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Methamphetamine abuse escalates, but no approved therapeutics are available to treat addicted individuals. Methamphetamine increases extracellular dopamine in reward-relevant pathways by interacting at vesicular monoamine transporter-2 (VMAT2) to inhibit dopamine uptake and promote dopamine release from synaptic vesicles, increasing cytosolic dopamine available for reverse transport by the dopamine transporter (DAT). VMAT2 is the target of our iterative drug discovery efforts to identify pharmacotherapeutics for methamphetamine addiction. Lobeline, the major alkaloid in Lobelia inflata, potently inhibited VMAT2, methamphetamine-evoked striatal dopamine release, and methamphetamine self-administration in rats but exhibited high affinity for nicotinic acetylcholine receptors (nAChRs). Defunctionalized, unsaturated lobeline analog, meso-transdiene (MTD), exhibited lobeline-like in vitro pharmacology, lacked nAChR affinity, but exhibited high affinity for DAT, suggesting potential abuse liability. The 2,4-dicholorophenyl MTD analog, UKMH-106, exhibited selectivity for VMAT2 over DAT, inhibited methamphetamine-evoked dopamine release, but required a difficult synthetic approach. Lobelane, a saturated, defunctionalized lobeline analog, inhibited the neurochemical and behavioral effects of methamphetamine; tolerance developed to the lobelane-induced decrease in methamphetamine self-administration. Improved drug-likeness was afforded by the incorporation of a chiral N-1,2-dihydroxypropyl moiety into lobelane to afford GZ-793A, which inhibited the neurochemical and behavioral effects of methamphetamine, without tolerance. From a series of 2,5-disubstituted pyrrolidine analogs, AV-2-192 emerged as a lead, exhibiting high affinity for VMAT2 and inhibiting methamphetamine-evoked dopamine release. Current results support the hypothesis that potent, selective VMAT2 inhibitors provide the requisite preclinical behavioral profile for evaluation as pharmacotherapeutics for methamphetamine abuse and emphasize selectivity for VMAT2 relative to DAT as a criterion for reducing abuse liability of the therapeutic., (© 2014 Elsevier Inc. All rights reserved.)

Despite the high prevalence of use of methamphetamine (METH), there is no FDA-approved pharmacological treatment available currently for METH addiction. The vesicular monoamine transporter (VMAT2) has been proposed as a novel target to treat METH abuse. GZ-793A, a lobelane analog and selective VMAT2 inhibitor, has been shown previously to decrease METH self-administration specifically when administered via the subcutaneous route in rats. Since oral administration is the preferred clinical route, the present experiments determined if oral administration of GZ-793A would decrease specifically METH self-administration. Experiments 1 and 2 assessed the dose-effect functions of oral administration of GZ-793A (30-240 mg/kg) on intravenous METH self-administration and food-maintained responding, respectively. Experiments 3 and 4 assessed the time-course (20-180 min pretreatment) of oral administration of GZ-793A on METH self-administration and food-maintained responding, respectively. Oral administration of GZ-793A dose-dependently decreased METH self-administration, with the highest dose (240 mg/kg) producing an 85% decrease compared to control baseline. The decrease in METH self-administration produced by GZ-793A (120 mg/kg) lasted at least 180 min. In contrast, GZ-793A failed to alter food-maintained responding at any of the doses or pretreatment intervals tested. The oral effectiveness and the specificity of GZ-793A to decrease methamphetamine self-administration support the feasibility of developing VMAT2 inhibitors as treatments for METH abuse., (© 2013.)

Lobelane analogs that incorporate a central piperidine or pyrrolidine moiety have previously been reported by our group as potent inhibitors of VMAT2 function. Further central ring size reduction of the piperidine moiety in lobelane to a four-membered heterocyclic ring has been carried out in the current study to afford novel cis-and trans-azetidine analogs. These azetidine analogs (15a-15c and 22a-22c) potently inhibited [(3)H]dopamine (DA) uptake into isolated synaptic vesicles (Ki⩽66nM). The cis-4-methoxy analog 22b was the most potent inhibitor (Ki=24nM), and was twofold more potent that either lobelane (2a, Ki=45nM) or norlobelane (2b, Ki=43nM). The trans-methylenedioxy analog, 15c (Ki=31nM), was equipotent with the cis-analog, 22b, in this assay. Thus, cis- and trans-azetidine analogs 22b and 15c represent potential leads in the discovery of new clinical candidates for the treatment of methamphetamine abuse., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

(R)-3-[2,6-cis-Di(4-methoxyphenethyl)piperidin-1-yl]propane-1,2-diol (GZ-793A) inhibits methamphetamine-evoked dopamine release from striatal slices and methamphetamine self-administration in rats. GZ-793A potently and selectively inhibits dopamine uptake at the vesicular monoamine transporter-2 (VMAT2). This study determined GZ-793A's ability to evoke [³H]dopamine release and inhibit methamphetamine-evoked [³H]dopamine release from isolated striatal synaptic vesicles. Results show GZ-793A concentration-dependent [³H]dopamine release; nonlinear regression revealed a two-site model of interaction with VMAT2 (High- and Low-EC₅₀ = 15.5 nM and 29.3 μM, respectively). Tetrabenazine and reserpine completely inhibited GZ-793A-evoked [³H]dopamine release, however, only at the High-affinity site. Low concentrations of GZ-793A that interact with the extravesicular dopamine uptake site and the High-affinity intravesicular DA release site also inhibited methamphetamine-evoked [³H]dopamine release from synaptic vesicles. A rightward shift in the methamphetamine concentration-response was evident with increasing concentrations of GZ-793A, and the Schild regression slope was 0.49 ± 0.08, consistent with surmountable allosteric inhibition. These results support a hypothetical model of GZ-793A interaction at more than one site on the VMAT2 protein, which explains its potent inhibition of dopamine uptake, dopamine release via a High-affinity tetrabenazine- and reserpine-sensitive site, dopamine release via a Low-affinity tetrabenazine- and reserpine-insensitive site, and a low-affinity interaction with the dihydrotetrabenazine binding site on VMAT2. GZ-793A inhibition of the effects of methamphetamine supports its potential as a therapeutic agent for the treatment of methamphetamine abuse., (© 2013 International Society for Neurochemistry.)

Vesicular monoamine transporter-2 (VMAT2) inhibitors reduce methamphetamine (METH) reward in rats. The current study determined the effects of VMAT2 inhibitors lobeline (LOB; 1 or 3 mg/kg) and N-(1,2R-dihydroxylpropyl)-2,6-cis-di(4-methoxyphenethyl)piperidine hydrochloride (GZ-793A; 15 or 30 mg/kg) on METH-induced (0.5 mg/kg, SC) changes in extracellular dopamine (DA) and its metabolite dihydroxyphenylacetic acid (DOPAC) in the reward-relevant nucleus accumbens (NAc) shell using in vivo microdialysis. The effect of GZ-793A (15 mg/kg) on DA synthesis in tissue also was investigated in NAc, striatum, medial prefrontal cortex and orbitofrontal cortex. In NAc shell, METH produced a time-dependent increase in extracellular DA and decrease in DOPAC. Neither LOB nor GZ-793A alone altered extracellular DA; however, both drugs increased extracellular DOPAC. In combination with METH, LOB did not alter the effects of METH on DA; however, GZ-793A, which has greater selectivity than LOB for inhibiting VMAT2, reduced the duration of the METH-induced increase in extracellular DA. Both LOB and GZ-793A enhanced the duration of the METH-induced decrease in extracellular DOPAC. METH also increased tissue DA synthesis in NAc and striatum, whereas GZ-793A decreased synthesis; no effect of METH or GZ-793A on DA synthesis was found in medial prefrontal cortex or orbitofrontal cortex. These results suggest that selective inhibition of VMAT2 produces a time-dependent decrease in DA release in NAc shell as a result of alterations in tyrosine hydroxylase activity, which may play a role in the ability of GZ-793A to decrease METH reward., (© 2013 International Society for Neurochemistry.)

Central heterocyclic ring size reduction from piperidinyl to pyrrolidinyl in the vesicular monoamine transporter-2 (VMAT2) inhibitor GZ-793A and its analogs resulted in novel N-propane-1,2(R)-diol analogs 11a-i. These compounds were evaluated for their affinity for the dihydrotetrabenazine (DTBZ) binding site on VMAT2 and for their ability to inhibit vesicular dopamine (DA) uptake. The 4-difluoromethoxyphenethyl analog 11f was the most potent inhibitor of [(3)H]-DTBZ binding (Ki=560 nM), with 15-fold greater affinity for this site than GZ-793A (Ki=8.29 μM). Analog 11f also showed similar potency of inhibition of [(3)H]-DA uptake into vesicles (Ki=45 nM) compared to that for GZ-793A (Ki=29 nM). Thus, 11f represents a new water-soluble inhibitor of VMAT function., (Copyright © 2013. Published by Elsevier Ltd.)

Rationale: The vesicular monoamine transporter 2 (VMAT2) has been identified as a potential target for the treatment of methamphetamine (METH) abuse. GZ-793A is a potent and selective VMAT2 inhibitor that has been shown to block the primary and conditioned reinforcing effects of METH, while demonstrating no abuse liability when given alone., Objectives: The aim of the current study was to determine if GZ-793A attenuates METH- or cue-induced reinstatement of METH-seeking after a period of extinction. The effect of acute GZ-793A on locomotor activity also was assessed., Methods: After a period of extinction, rats were administered GZ-793A (15 mg/kg, s.c.) 15 min prior to a priming injection of METH or re-exposure to cues associated with METH infusions. GZ-793A also was administered 20 min prior to an injection of METH (0.5 mg/kg, s.c.) or saline to determine its effect on locomotor behavior., Results: Pretreatment with GZ-793A (15 mg/kg) decreased cue-induced reinstatement, without demonstrating any response suppressive effects when administered in the absence of reinstating stimuli. GZ-793A also decreased methamphetamine-induced reinstatement; however, response suppressant effects of GZ-793A were obtained when the compound was presented alone. In this latter experiment, GZ-793A may have reduced responding for the conditioned reinforcing effects of the contingently available cues rather than having nonspecific effects on baseline responding. GZ-793A had no effect on locomotor activity when administered alone or with METH., Conclusions: GZ-793A and related VMAT2 inhibitors may be promising leads for reducing the risk of relapse to METH use following exposure to drug-associated cues.

Rationale: Previous research suggests that the vesicular monoamine transporter-2 (VMAT2) is a novel target for the treatment of methamphetamine (METH) abuse., Objective: The effects GZ-793A, a novel, selective, and potent lobelane analog, on the rewarding effects of METH, cocaine, and palatable food in rats were determined., Method: GZ-793A (3-30 mg/kg, s.c.) was administered 20 min prior to each session in which the groups of rats pressed a lever for infusions of METH (0.03 mg/kg/infusion), cocaine (0.3 mg/kg/infusion), or food pellets. Tolerance to repeated GZ-793A (15 mg/kg, s.c. for 7 days) on METH self-administration and food-maintained responding was determined. The ability of increasing doses of METH (0.001-0.56 mg/kg, i.v.) to surmount inhibition produced by GZ-793A (15 mg/kg, s.c.) was determined. Self-administration of GZ-793A (0.01-0.3 mg/kg/infusion, i.v.) was tested as a substitute for METH infusion. GZ-793A (15 mg/kg, s.c.) was administered 20 min prior to METH or saline conditioning in a place preference test., Results: GZ-793A specifically decreased METH self-administration, without the development of tolerance. Increasing the unit dose of METH did not surmount the inhibition produced by GZ-793A on METH self-administration. GZ-793A did not serve as a substitute for self-administered METH. GZ-793A blocked METH-induced conditioned place preference (CPP) and did not induce CPP alone., Conclusions: These results indicate that VMAT2 is a viable target for pharmacological inhibition of METH reward and that GZ-793A represents a new lead in the discovery of a treatment for METH abuse.

Lobelane, a chemically defunctionalized saturated analog of lobeline, has increased selectivity for the vesicular monoamine transporter 2 (VMAT2) compared with the parent compound. Lobelane inhibits methamphetamine-evoked dopamine (DA) release and decreases methamphetamine self-administration. Unfortunately, tolerance develops to the ability of lobelane to decrease these behavioral effects of methamphetamine. Lobelane has low water solubility, which is problematic for drug development. The aim of the current study was to determine the pharmacological effect of replacement of the N-methyl moiety with a chiral N-1,2-dihydroxypropyl (N-1,2-diol) moiety, which enhances water solubility, altering the configuration of the N-1,2-diol moiety and incorporating phenyl ring substituents into the analogs. To determine VMAT2 selectivity, structure-activity relationships also were generated for inhibition of DA and serotonin transporters. Analogs with the highest potency for inhibiting DA uptake at VMAT2 and at least 10-fold selectivity were evaluated further for ability to inhibit methamphetamine-evoked DA release from superfused striatal slices. (R)-3-[2,6-cis-di(4-methoxyphenethyl)piperidin-1-yl]propane-1,2-diol (GZ-793A), the (R)-4-methoxyphenyl-N-1,2-diol analog, and (R)-3-[2,6-cis-di(1-naphthylethyl)piperidin-1-yl]propane-1,2-diol (GZ-794A), the (R)-1-naphthyl-N-1,2-diol analog, exhibited the highest potency (K(i) ∼30 nM) inhibiting VMAT2, and both analogs inhibited methamphetamine-evoked endogenous DA release (IC(50) = 10.6 and 0.4 μM, respectively). Thus, the pharmacophore for VMAT2 inhibition accommodates the N-1,2-diol moiety, which improves drug-likeness and enhances the potential for the development of these clinical candidates as treatments for methamphetamine abuse.

Lobeline, a nicotinic receptor antagonist and neurotransmitter transporter inhibitor, is a candidate pharmacotherapy for methamphetamine abuse. meso-Transdiene (MTD), a lobeline analog, lacks nicotinic receptor affinity, retains affinity for vesicular monoamine transporter 2 (VMAT2), and, surprisingly, has enhanced affinity for dopamine (DA) and serotonin transporters [DA transporter (DAT) and serotonin transporter (SERT), respectively]. In the current study, MTD was evaluated for its ability to decrease methamphetamine self-administration in rats relative to food-maintained responding. MTD specifically decreased methamphetamine self-administration, extending our previous work. Classical structure-activity relationships revealed that more conformationally restricted MTD analogs enhanced VMAT2 selectivity and drug likeness, whereas affinity at the dihydrotetrabenazine binding and DA uptake sites on VMAT2 was not altered. Generally, MTD analogs exhibited 50- to 1000-fold lower affinity for DAT and were equipotent or had 10-fold higher affinity for SERT, compared with MTD. Representative analogs from the series potently and competitively inhibited [(3)H]DA uptake at VMAT2. (3Z,5Z)-3,5-bis(2,4-dichlorobenzylidene)-1-methylpiperidine (UKMH-106), the 3Z,5Z-2,4-dichlorophenyl MTD analog, had improved selectivity for VMAT2 over DAT and importantly inhibited methamphetamine-evoked DA release from striatal slices. In contrast, (3Z,5E)-3,5-bis(2,4-dichlorobenzylidene)-1-methylpiperidine (UKMH-105), the 3Z,5E-geometrical isomer, inhibited DA uptake at VMAT2, but did not inhibit methamphetamine-evoked DA release. Taken together, these results suggest that these geometrical isomers interact at alternate sites on VMAT2, which are associated with distinct pharmacophores. Thus, structural modification of the MTD molecule resulted in analogs exhibiting improved drug likeness and improved selectivity for VMAT2, as well as the ability to decrease methamphetamine-evoked DA release, supporting the further evaluation of these analogs as treatments for methamphetamine abuse.

Lobeline attenuates the behavioral effects of methamphetamine via inhibition of the vesicular monoamine transporter (VMAT2). To increase selectivity for VMAT2, chemically defunctionalized lobeline analogs, including lobelane, were designed to eliminate nicotinic acetylcholine receptor affinity. The current study evaluated the ability of lobelane analogs to inhibit [³H]dihydrotetrabenazine (DTBZ) binding to VMAT2 and [³H]dopamine (DA) uptake into isolated synaptic vesicles and determined the mechanism of inhibition. Introduction of aromatic substituents in lobelane maintained analog affinity for the [³H]DTBZ binding site on VMAT2 and inhibitory potency in the [³H]DA uptake assay assessing VMAT2 function. The most potent (K(i) = 13-16 nM) analogs in the series included para-methoxyphenyl nor-lobelane (GZ-252B), para-methoxyphenyl lobelane (GZ-252C), and 2,4-dichlorphenyl lobelane (GZ-260C). Affinity of the analogs for the [³H]DTBZ binding site did not correlate with inhibitory potency in the [³H]DA uptake assay. It is noteworthy that the N-benzylindole-, biphenyl-, and indole-bearing meso-analogs 2,6-bis[2-(1-benzyl-1H-indole-3-yl)ethyl]-1-methylpiperidine hemifumarate (AV-1-292C), 2,6-bis(2-(biphenyl-4-yl)ethyl)piperidine hydrochloride (GZ-272B), and 2,6-bis[2-(1H-indole-3-yl)ethyl]-1-methylpiperidine monofumarate (AV-1-294), respectively] inhibited VMAT2 function (K(i) = 73, 127, and 2130 nM, respectively), yet had little to no affinity for the [³H]DTBZ binding site. These results suggest that the analogs interact at an alternate site to DTBZ on VMAT2. Kinetic analyses of [³H]DA uptake revealed a competitive mechanism for 2,6-bis(2-(4-methoxyphenyl)ethyl)piperidine hydrochloride (GZ-252B), 2,6-bis(2-(4-methoxyphenyl)ethyl)-1-methylpiperidine hydrochloride (GZ-252C), 2,6-bis(2-(2,4-dichlorophenyl)ethyl)piperidine hydrochloride (GZ-260C), and GZ-272B. Similar to methamphetamine, these analogs released [³H]DA from the vesicles, but with higher potency. In contrast to methamphetamine, these analogs had higher potency (>100-fold) at VMAT2 than DAT, predicting low abuse liability. Thus, modification of the lobelane molecule affords potent, selective inhibitors of VMAT2 function and reveals two distinct pharmacological targets on VMAT2.

The vesicular monoamine transporter-2 (VMAT2) is considered as a new target for the development of novel therapeutics to treat psychostimulant abuse. Current information on the structure, function and role of VMAT2 in psychostimulant abuse are presented. Lobeline, the major alkaloidal constituent of Lobelia inflata, interacts with nicotinic receptors and with VMAT2. Numerous studies have shown that lobeline inhibits both the neurochemical and behavioral effects of amphetamine in rodents, and behavioral studies demonstrate that lobeline has potential as a pharmacotherapy for psychostimulant abuse. Systematic structural modification of the lobeline molecule is described with the aim of improving selectivity and affinity for VMAT2 over neuronal nicotinic acetylcholine receptors and other neurotransmitter transporters. This has led to the discovery of more potent and selective ligands for VMAT2. In addition, a computational neural network analysis of the affinity of these lobeline analogs for VMAT2 has been carried out, which provides computational models that have predictive value in the rational design of VMAT2 ligands and is also useful in identifying drug candidates from virtual libraries for subsequent synthesis and evaluation.

Both lobeline and lobelane attenuate methamphetamine self-administration in rats by decreasing methamphetamine-induced dopamine release via interaction with vesicular monoamine transporter-2 (VMAT2). A novel derivative of nor-lobelane, cis-2,5-di-(2-phenethyl)-pyrrolidine hydrochloride (UKCP-110), and its trans-isomers, (2R,5R)-trans-di-(2-phenethyl)-pyrrolidine hydrochloride (UKCP-111) and (2S,5S)-trans-di-(2-phenethyl)-pyrrolidine hydrochloride (UKCP-112), were evaluated for inhibition of [(3)H]dihydrotetrabenazine binding and [(3)H]dopamine uptake by using a rat synaptic vesicle preparation to assess VMAT2 interaction. Compounds were evaluated for inhibition of [(3)H]nicotine and [(3)H]methyllycaconitine binding to assess interaction with the major nicotinic receptor subtypes. In addition, compounds were evaluated for inhibition of methamphetamine-evoked endogenous dopamine release by using striatal slices. The most promising compound, UKCP-110, was evaluated for its ability to decrease methamphetamine self-administration and methamphetamine discriminative stimulus cues and for its effect on food-maintained operant responding. UKCP-110, UKCP-111, and UKCP-112 inhibited [(3)H]dihydrotetrabenazine binding (K(i) = 2.66 ± 0.37, 1.05 ± 0.10, and 3.80 ± 0.31 μM, respectively) and had high potency inhibiting [(3)H]dopamine uptake (K(i) = 0.028 ± 0.001, 0.046 ± 0.008, 0.043 ± 0.004 μM, respectively), but lacked affinity at nicotinic receptors. Although the trans-isomers did not alter methamphetamine-evoked dopamine release, UKCP-110 inhibited (IC(50) = 1.8 ± 0.2 μM; I(max) = 67.18 ± 6.11 μM) methamphetamine-evoked dopamine release. At high concentrations, UKCP-110 also increased extracellular dihydroxyphenylacetic acid. It is noteworthy that UKCP-110 decreased the number of methamphetamine self-infusions, while having no effect on food-reinforced behavior or the methamphetamine stimulus cue. Thus, UKCP-110 represents a new lead in the development of novel pharmacotherapies for the treatment of methamphetamine abuse.

Replacing the phenyl groups in the structure of the VMAT2 inhibitor, lobelane with either pyridyl, quinolyl or indolyl groups affords novel analogues with improved water solubility. The synthetic methodologies reported herein also underscore the paucity of hydrogenation methods that offer selectivity in the synthesis of the different classes of heteroaromatic lobelane analogues. The quinolyl group was the only replacement for the phenyl group in lobelane that retained VMAT2 inhibition., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Lobeline is currently being evaluated in clinical trials as a methamphetamine abuse treatment. Lobeline interacts with nicotinic receptor subtypes, dopamine transporters (DATs), and vesicular monoamine transporters (VMAT2s). Methamphetamine inhibits VMAT2 and promotes dopamine (DA) release from synaptic vesicles, resulting ultimately in increased extracellular DA. The present study generated structure-activity relationships by defunctionalizing the lobeline molecule and determining effects on [(3)H]dihydrotetrabenazine binding, inhibition of [(3)H]DA uptake into striatal synaptic vesicles and synaptosomes, the mechanism of VMAT2 inhibition, and inhibition of methamphetamine-evoked DA release. Compared with lobeline, the analogs exhibited greater potency inhibiting DA transporter (DAT) function. Saturated analogs, lobelane and nor-lobelane, exhibited high potency (K(i) = 45 nM) inhibiting vesicular [(3)H]DA uptake, and lobelane competitively inhibited VMAT2 function. Lobeline and lobelane exhibited 67- and 35-fold greater potency, respectively, in inhibiting VMAT2 function compared to DAT function. Lobelane potently decreased (IC(50) = 0.65 microM; I(max) = 73%) methamphetamine-evoked DA overflow, and with a greater maximal effect compared with lobeline (IC(50) = 0.42 microM, I(max) = 56.1%). These results provide support for VMAT2 as a target for inhibition of methamphetamine effects. Both trans-isomers and demethylated analogs of lobelane had reduced or unaltered potency inhibiting VMAT2 function and lower maximal inhibition of methamphetamine-evoked DA release compared with lobelane. Thus, defunctionalization, cis-stereochemistry of the side chains, and presence of the piperidino N-methyl are structural features that afford greatest inhibition of methamphetamine-evoked DA release and enhancement of selectivity for VMAT2. The current results reveal that lobelane, a selective VMAT2 inhibitor, inhibits methamphetamine-evoked DA release and is a promising lead for the development of a pharmacotherapeutic for methamphetamine abuse.

Ring size reduction of the central piperidine ring of lobelane yielded pyrrolidine analogues that showed marked inconsistencies in their ability to bind to the dihydrotetrabenazine (DTBZ) binding site on the vesicular monoamine transporter-2 (VMAT2) and their ability to inhibit VMAT2 function. The structure-activity relationships indicate that structural modification within the pyrrolidine series resulted in analogues that interact with two different sites, i.e., the DTBZ binding site and an alternative site on VMAT2 to inhibit transporter function.

A series of lobelane homologues has been synthesized and evaluated for their [(3)H]DTBZ binding affinity at the vesicular monoamine transporter-2 (VMAT2). The structure-activity relationships (SAR) indicate that for retention of binding affinity at VMAT2, the lengths of the methylene linkers should be no shorter than one methylene unit at C-6 of the piperidine ring, and no shorter than two methylene units at C-2 of the piperidine ring. These results indicate that the intramolecular distances between the piperidine ring and two phenyl rings in lobelane analogues are an important criterion for retention of high affinity at VMAT2.

Back-propagation artificial neural networks (ANNs) were trained on a dataset of 104 VMAT2 ligands with experimentally measured log(1/K(i)) values. A set of related descriptors, including topological, geometrical, GETAWAY, aromaticity, and WHIM descriptors, was selected to build nonlinear quantitative structure-activity relationships. A partial least squares (PLS) regression model was also developed for comparison. The nonlinearity of the relationship between molecular descriptors and VMAT2 ligand activity was demonstrated. The obtained neural network model outperformed the PLS model in both the fitting and predictive ability. ANN analysis indicated that the computed activities were in excellent agreement with the experimentally observed values (r(2)=0.91, rmsd=0.225; predictive q(2)=0.82, loormsd=0.316). The generated models were further tested by use of an external prediction set of 15 molecules. The nonlinear ANN model has r(2)=0.93 and root-mean-square errors of 0.282 compared with the experimentally measured activity of the test set. The stability test of the model with regard to data division was found to be positive, indicating that the generated model is predictive. The modeling study also reflected the important role of atomic distribution in the molecules, size, and steric structure of the molecules when they interact with the target, VMAT2. The developed models are expected to be useful in the rational design of new chemical entities as ligands of VMAT2 and for directing synthesis of new molecules in the future.

In the central nervous system, vesicular monoamine transporter 2 (VMAT2) is the only transporter that moves cytoplasmic dopamine (DA) into synaptic vesicles for storage and subsequent exocytotic release. Pharmacologically enhancing DA sequestration by VMAT2, and thus preventing the oxidation of DA in the cytoplasm, may be a strategy for treating diseases such as Parkinson's disease. VMAT2 may also be a novel target for the development of treatments for psychostimulant abuse. This review summarizes the possible role of VMAT2 as a therapeutic target, VMAT2 ligands reported in the literature, and the structure-activity relationship of these ligands, including tetrabenazine analogs, ketanserin analogs, lobeline analogs, and 3-amine-2-phenylpropene analogs. The molecular structure of VMAT2 and its relevance to ligand binding are briefly discussed.