Dual DAT and sigma receptor inhibitors attenuate cocaine effects on nucleus accumbens dopamine dynamics in rats.

Psychostimulant use disorders (PSUD) are prevalent; however, no FDA-approved medications have been made available for treatment. Previous studies have shown that dual inhibitors of the dopamine transporter (DAT) and sigma receptors significantly reduce the behavioral/reinforcing effects of cocaine, which have been associated with stimulation of extracellular dopamine (DA) levels resulting from DAT inhibition. Here, we employ microdialysis and fast scan cyclic voltammetry (FSCV) procedures to investigate the effects of dual inhibitors of DAT and sigma receptors in combination with cocaine on nucleus accumbens shell (NAS) DA dynamics in naïve male Sprague Dawley rats. In microdialysis studies, administration of rimcazole (3, 10 mg/kg; i.p.) or its structural analog SH 3-24 (1, 3 mg/kg; i.p.), compounds that are dual inhibitors of DAT and sigma receptors, significantly reduced NAS DA efflux stimulated by increasing doses of cocaine (0.1, 0.3, 1.0 mg/kg; i.v.). Using the same experimental conditions, in FSCV tests, we show that rimcazole pretreatments attenuated cocaine-induced stimulation of evoked NAS DA release but produced no additional effect on DA clearance rate. Under the same conditions, JJC8-091, a modafinil analog and dual inhibitor of DAT and sigma receptors, similarly attenuated cocaine-induced stimulation of evoked NAS DA release but produced no additional effect on DA clearance rate. Our results provide the neurochemical groundwork towards understanding actions of dual inhibitors of DAT and sigma receptors on DA dynamics that likely mediate the behavioral effects of psychostimulants like cocaine.

Ventilatory Effects of Fentanyl, Heroin, and d-Methamphetamine, Alone and in Mixtures in Male Rats Breathing Normal Air .

The number of drug overdoses and deaths has increased significantly over the past decade and co-use of opioids and stimulants is associated with greater likelihood of overdose and decreased likelihood of accessing treatment, compared with use of opioids alone. Potential adverse effects of opioid/stimulant mixtures, particularly methamphetamine, are not well characterized. Two structurally different drugs with agonist properties at µ-opioid receptors (MOR), fentanyl and heroin, and d-methamphetamine, alone and in mixtures, were assessed for their effects on ventilation in rats breathing normal air. Whole-body phethysmography chambers were equipped with a tower and swivel allowing infusions to indwelling intravenous catheters. After a 45-minute habituation period, saline, fentanyl, heroin, or d-methamphetamine, alone and in mixtures, was administered. Five minutes later, the opioid receptor antagonist naloxone or vehicle was injected. Fentanyl (0.0032-0.1 mg/kg) and heroin (0.32-3.2 mg/kg) decreased ventilation [frequency (f) and tidal volume (VT)] in a dose-related manner whereas d-methamphetamine (0.1-3.2 mg/kg) increased f to >400% of control and decreased VT to <60% of control, overall increasing minute volume (product of f and VT) to >240% of control. When combined, d-methamphetamine (0.1-3.2 mg/kg) attenuated the ventilatory depressant effects of fentanyl (0.1 mg/kg) and heroin (3.2 mg/kg). d-Methamphetamine did not alter the potency of naloxone to reverse the ventilatory depressant effects of fentanyl or heroin. These studies demonstrate that d-methamphetamine can attenuate the ventilatory depressant effects of moderate doses of opioid receptor agonists while not altering the potency of naloxone to reverse opioid hypoventilation. SIGNIFICANCE STATEMENT: Co-use of opioids and stimulants is associated with greater likelihood of overdose and decreased likelihood of accessing treatment, compared with use of opioids alone. Potential adverse effects of opioid/stimulant mixtures are not well characterized. This study reports that 1) d-methamphetamine attenuates the ventilatory depressant effects of moderate doses of two structurally different opioid receptor agonists, fentanyl and heroin, and 2) d-methamphetamine does not alter potency or effectiveness of naloxone to reverse the ventilatory depressant effects of these opioid receptor agonists.

Comparison of the µ-opioid receptor antagonists methocinnamox (MCAM) and naloxone to reverse and prevent the ventilatory depressant effects of fentanyl, carfentanil, 3-methylfentanyl, and heroin in male rats.

The number of opioid overdose deaths has increased significantly over the past decade. The life-threatening effect of opioids is hypoventilation that can be reversed by the µ-opioid receptor (MOR) antagonist naloxone; however, because of the very short duration of action of naloxone, re-emergence of MOR agonist-induced hypoventilation can occur, requiring additional doses of naloxone. The MOR antagonist methocinnamox (MCAM) antagonizes hypoventilation by the non-morphinan fentanyl and the morphinan heroin in laboratory animals with an unusually long duration of action. Whole-body plethysmography was used to compare the potency and effectiveness of MCAM and naloxone for preventing and reversing hypoventilation by fentanyl, heroin, and the ultra-potent and longer-acting fentanyl analogs carfentanil and 3-methylfentanyl in male rats breathing normal air. Sessions comprised a 45-minute habituation period followed by intravenous (i.v.) administration of saline or an acute dose of MOR agonist. The rank order of potency to decrease ventilation was 3-methylfentanyl > carfentanil > fentanyl > heroin. MCAM (0.0001-0.1 mg/kg) and naloxone (0.0001-0.01 mg/kg) dose-dependently reversed hypoventilation by 3-methylfentanyl (0.01 mg/kg), carfentanil (0.01 mg/kg), fentanyl (0.1 mg/kg), or heroin (3.2 mg/kg). For prevention studies, MCAM, naloxone, or vehicle was administered i.v. 22, 46, or 70 hours prior to a MOR agonist. When administered 22 hours earlier, MCAM (0.1-1.0 mg/kg) but not naloxone (1.0 mg/kg) prevented hypoventilation by each MOR agonist. This study demonstrates the effectiveness of MCAM to reverse and prevent hypoventilation by MOR agonists including ultra-potent fentanyl analogs that have a long duration of action. Significance Statement The number of opioid overdose deaths increased over the past decade despite the availability of antagonists that can prevent and reverse the effects of opioids. This study demonstrates the effectiveness and long duration of action of the µ-opioid receptor (MOR) antagonist methocinnamox (MCAM) for reversing and preventing hypoventilation by MOR agonists including ultra-potent fentanyl analogs. These results provide support for the notion that MCAM has the potential to positively impact the ongoing opioid crisis by reversing and preventing opioid overdose.

A Sigma1 Receptor Agonist Alters Fluidity and Stability of Lipid Monolayers.

Interactions between the sigma1 receptor agonist PRE-084 and various lipid monolayers, including dipalmitoylphosphatidylcholine (DPPC), DPP-ethanolamine (DPPE), DPP-glycerol (DPPG), DPP-serine (DPPS), palmitoylsphingomyelin (PSM), and cholesterol (Ch), were investigated to elucidate the effects of PRE-084 on membrane fluidity and stability. Their interactions with sigma1 receptor agonists have potential implications for neuroprotection, antidepressant, analgesic, and cognitive enhancement effects. In this study, we observed that the presence of PRE-084 in the subphase led to increased fluidity in DPPC and DPPE monolayers, whereas decreasing fluidity was observed in DPPG, DPPS, and PSM monolayers. The interaction of PRE-084 with Ch monolayers was found to be distinct from its interaction with other lipids. Fluorescence microscopy images revealed changes in the size and shape of liquid-condensed domains in the presence of PRE-084, supporting the notion of altered membrane fluidity. Our findings provide new insights into the interaction of PRE-084 with lipid monolayers and its potential implications for biological and membrane science.

Pharmacokinetic and Pharmacodynamic Consequences of Cytochrome P450 3A Inhibition on Mitragynine Metabolism in Rats.

Mitragynine, an opioidergic alkaloid present in Mitragyna speciosa (kratom), is metabolized by cytochrome P450 3A (CYP3A) to 7-hydroxymitragynine, a more potent opioid receptor agonist. The extent to which conversion to 7-hydroxymitragynine mediates the in vivo effects of mitragynine is unclear. The current study examined how CYP3A inhibition (ketoconazole) modifies the pharmacokinetics of mitragynine in rat liver microsomes in vitro. The study further examined how ketoconazole modifies the discriminative stimulus and antinociceptive effects of mitragynine in rats. Ketoconazole [30 mg/kg, oral gavage (o.g.)] increased systemic exposure to mitragynine (13.3 mg/kg, o.g.) by 120% and 7-hydroxymitragynine exposure by 130%. The unexpected increase in exposure to 7-hydroxymitragynine suggested that ketoconazole inhibits metabolism of both mitragynine and 7-hydroxymitragynine, a finding confirmed in rat liver microsomes. In rats discriminating 3.2 mg/kg morphine from vehicle under a fixed-ratio schedule of food delivery, ketoconazole pretreatment increased the potency of both mitragynine (4.7-fold) and 7-hydroxymitragynine (9.7-fold). Ketoconazole did not affect morphine's potency. Ketoconazole increased the antinociceptive potency of 7-hydroxymitragynine by 4.1-fold. Mitragynine (up to 56 mg/kg, i.p.) lacked antinociceptive effects both in the presence and absence of ketoconazole. These results suggest that both mitragynine and 7-hydroxymitragynine are cleared via CYP3A and that 7-hydroxymitragynine is formed as a metabolite of mitragynine by other routes. These results have implications for kratom use in combination with numerous medications and citrus juices that inhibit CYP3A. SIGNIFICANCE STATEMENT: Mitragynine is an abundant kratom alkaloid that exhibits low efficacy at the µ-opioid receptor (MOR). Its metabolite, 7-hydroxymitragynine, is also an MOR agonist but with higher affinity and efficacy than mitragynine. Our results in rats demonstrate that cytochrome P450 3A (CYP3A) inhibition can increase the systematic exposure of both mitragynine and 7-hydroxymitragynine and their potency to produce MOR-mediated behavioral effects. These data highlight potential interactions between kratom and CYP3A inhibitors, which include numerous medications and citrus juices.

Interactive Effects of µ-Opioid and Adrenergic-α 2 Receptor Agonists in Rats: Pharmacological Investigation of the Primary Kratom Alkaloid Mitragynine and Its Metabolite 7-Hydroxymitragynine.

The primary kratom alkaloid mitragynine is proposed to act through multiple mechanisms, including actions at µ-opioid receptors (MORs) and adrenergic-α 2 receptors (Aα 2Rs), as well as conversion in vivo to a MOR agonist metabolite (i.e., 7-hydroxymitragynine). Aα 2R and MOR agonists can produce antinociceptive synergism. Here, contributions of both receptors to produce mitragynine-related effects were assessed by measuring receptor binding in cell membranes and, in rats, pharmacological behavioral effect antagonism studies. Mitragynine displayed binding affinity at both receptors, whereas 7-hydroxymitragynine only displayed MOR binding affinity. Compounds were tested for their capacity to decrease food-maintained responding and rectal temperature and to produce antinociception in a hotplate test. Prototypical MOR agonists and 7-hydroxymitragynine, but not mitragynine, produced antinociception. MOR agonist and 7-hydroxymitragynine rate-deceasing and antinociceptive effects were antagonized by the opioid antagonist naltrexone but not by the Aα 2R antagonist yohimbine. Hypothermia only resulted from reference Aα 2R agonists. The rate-deceasing and hypothermic effects of reference Aα 2R agonists were antagonized by yohimbine but not naltrexone. Neither naltrexone nor yohimbine antagonized the rate-decreasing effects of mitragynine. Mitragynine and 7-hydroxymitragynine increased the potency of the antinociceptive effects of Aα 2R but not MOR reference agonists. Only mitragynine produced hypothermic effects. Isobolographic analyses for the rate-decreasing effects of the reference Aα 2R and MOR agonists were also conducted. These results suggest mitragynine and 7-hydroxymitragynine may produce antinociceptive synergism with Aα 2R and MOR agonists. When combined with Aα 2R agonists, mitragynine could also produce hypothermic synergism. SIGNIFICANCE STATEMENT: Mitragynine is proposed to target the µ-opioid receptor (MOR) and adrenergic-α2 receptor (Aα2R) and to produce behavioral effects through conversion to its MOR agonist metabolite 7-hydroxymitragynine. Isobolographic analyses indicated supra-additivity in some dose ratio combinations. This study suggests mitragynine and 7-hydroxymitragynine may produce antinociceptive synergism with Aα2R and MOR agonists. When combined with Aα2R agonists, mitragynine could also produce hypothermic synergism.

The Lack of Contribution of 7-Hydroxymitragynine to the Antinociceptive Effects of Mitragynine in Mice: A Pharmacokinetic and Pharmacodynamic Study.

Kratom (Mitragyna speciosa), a Southeast Asian tree, has been used for centuries in pain relief and mitigation of opium withdrawal symptoms. Mitragynine (MTG), the major kratom alkaloid, is being investigated for its potential to provide analgesia without the deleterious effects associated with typical opioids. Concerns have been raised regarding the active metabolite of MTG, 7-hydroxymitragynine (7HMG), which has higher affinity and efficacy at µ-opioid receptors than MTG. Here we investigated the hotplate antinociception, pharmacokinetics, and tissue distribution of MTG and 7HMG at equianalgesic oral doses in male and female C57BL/6 mice to determine the extent to which 7HMG metabolized from MTG accounts for the antinociceptive effects of MTG and investigate any sex differences. The mechanism of action was examined by performing studies with the opioid receptor antagonist naltrexone. A population pharmacokinetic/pharmacodynamic model was developed to predict the behavioral effects after administration of various doses of MTG and 7HMG. When administered alone, 7HMG was 2.8-fold more potent than MTG to produce antinociception. At equivalent effective doses of MTG and 7HMG, there was a marked difference in the maximum brain concentration of 7HMG achieved, i.e., 11-fold lower as a metabolite of MTG. The brain concentration of 7HMG observed 4 hours post administration, producing an analgesic effect <10%, was still 1.5-fold higher than the maximum concentration of 7HMG as a metabolite of MTG. These results provide strong evidence that 7HMG has a negligible role in the antinociceptive effects of MTG in mice. SIGNIFICANCE STATEMENT: Mitragynine (MTG) is being investigated for its potential to aid in pain relief, opioid withdrawal syndrome, and opioid use disorder. The active metabolite of MTG, 7-hydroxymitragynine (7HMG), has been shown to have abuse potential and has been implicated in the opioid-like analgesic effect after MTG administration. The results of this study suggest a lack of involvement of 7HMG in the antinociceptive effects of MTG in mice.

The discriminative stimulus effects of baclofen and gamma hydroxybutyrate in C57BL/6J mice.

Baclofen and γ-hydroxybutyrate (GHB) exert γ-aminobutyric acid (GABA)B receptor agonism and have therapeutic utility but possess different pharmacological activities. We examined whether separate groups of mice could be trained to discriminate either baclofen or GHB, and the contribution of GABAB receptors to discriminative stimulus effects. Male C57BL/6J mice were trained to discriminate either baclofen (3.2 mg/kg, intraperitoneal) or GHB (178 mg/kg, intraperitoneal) from saline under a fixed-ratio 10 schedule. The GABAB antagonist 3-aminopropyl(diethoxymethyl)phosphinic acid (CGP 35348) was used to pharmacologically assess GABAB receptor involvement. The selectivity of the resulting discriminations was assessed with the opioid agonist morphine and the benzodiazepine midazolam. In baclofen-trained mice, both baclofen and GHB were readily discriminated. Baclofen produced a maximum of 86% baclofen-appropriate responding. CGP 35348 (320 mg/kg, i.p.) produced a 4.7-fold rightward shift in the dose-effect function. GHB produced a maximum of 85.8% baclofen-appropriate responding. In GHB-trained mice, both GHB and baclofen were readily discriminated. In GHB-trained mice, GHB produced a maximum of 85.3% drug-appropriate responding; CGP 35348 (320 mg/kg, i.p.) produced a 1.8-fold rightward shift in the GHB discrimination dose-effect function. Baclofen produced up to 70.0% GHB-appropriate responding. CGP 35348 (320 mg/kg, i.p.) significantly antagonized baclofen discrimination and baclofen produced up to 37% GHB-appropriate responding up to doses that disrupted operant responding. Morphine did not produce substitution for either baclofen or GHB. Midazolam produced partial substitution for both. GHB and baclofen discrimination assays in mice provide a useful approach for examining different receptor types mediating the effects of these two drugs.

Pharmacological Comparison of Mitragynine and 7-Hydroxymitragynine: In Vitro Affinity and Efficacy for µ-Opioid Receptor and Opioid-Like Behavioral Effects in Rats.

Relationships between µ-opioid receptor (MOR) efficacy and effects of mitragynine and 7-hydroxymitragynine are not fully established. We assessed in vitro binding affinity and efficacy and discriminative stimulus effects together with antinociception in rats. The binding affinities of mitragynine and 7-hydroxymitragynine at MOR (Ki values 77.9 and 709 nM, respectively) were higher than their binding affinities at κ-opioid receptor (KOR) or δ-opioid receptor (DOR). [35S]guanosine 5'-O-[γ-thio]triphosphate stimulation at MOR demonstrated that mitragynine was an antagonist, whereas 7-hydroxymitragynine was a partial agonist (Emax = 41.3%). In separate groups of rats discriminating either morphine (3.2 mg/kg) or mitragynine (32 mg/kg), mitragynine produced a maximum of 72.3% morphine-lever responding, and morphine produced a maximum of 65.4% mitragynine-lever responding. Other MOR agonists produced high percentages of drug-lever responding in the morphine and mitragynine discrimination assays: 7-hydroxymitragynine (99.7% and 98.1%, respectively), fentanyl (99.7% and 80.1%, respectively), buprenorphine (99.8% and 79.4%, respectively), and nalbuphine (99.4% and 98.3%, respectively). In the morphine and mitragynine discrimination assays, the KOR agonist U69,593 produced maximums of 72.3% and 22.3%, respectively, and the DOR agonist SNC 80 produced maximums of 34.3% and 23.0%, respectively. 7-Hydroxymitragynine produced antinociception; mitragynine did not. Naltrexone antagonized all of the effects of morphine and 7-hydroxymitragynine; naltrexone antagonized the discriminative stimulus effects of mitragynine but not its rate-decreasing effects. Mitragynine increased the potency of the morphine discrimination yet decreased morphine antinociception. Here we illustrate striking differences in MOR efficacy, with mitragynine having less than 7-hydroxymitragynine. SIGNIFICANCE STATEMENT: At human µ-opioid receptor (MOR) in vitro, mitragynine has low affinity and is an antagonist, whereas 7-hydroxymitragynine has 9-fold higher affinity than mitragynine and is an MOR partial agonist. In rats, intraperitoneal mitragynine exhibits a complex pharmacology including MOR agonism; 7-hydroxymitragynine has higher MOR potency and efficacy than mitragynine. These results are consistent with 7-hydroxymitragynine being a highly selective MOR agonist and with mitragynine having a complex pharmacology that combines low efficacy MOR agonism with activity at nonopioid receptors.

Exploring the Chemistry of Alkaloids from Malaysian Mitragyna speciosa (Kratom) and the Role of Oxindoles on Human Opioid Receptors.

Ten indole and oxindole alkaloids (1-10) were isolated from the freshly collected leaves of Malaysian Mitragyna speciosa (Kratom). The chemical structures of these compounds were established on the basis of extensive 1D and 2D NMR and HRMS data analysis. The spectroscopic data of mitragynine oxindole B (4) are reported herein for the first time. The spatial configuration of mitragynine oxindole B (4) was confirmed by single-crystal X-ray diffraction. Simultaneous quantification of the isolated alkaloids in the M. speciosa leaf specimens collected from different locations in the northern region of Peninsular Malaysia was also performed using UPLC-MS/MS. The oxindole alkaloids (1-4) and the indole alkaloid (10) were assessed for binding affinity at opioid receptors. Corynoxine (1) showed high binding affinity to µ-opioid receptors with a Ki value of 16.4 nM. Further, corynoxine (1) was 1.8-fold more potent than morphine in rats subjected to a nociceptive hot plate assay. These findings have important implications for evaluating the combined effects of the minor oxindole alkaloids in the overall therapeutic activity of M. speciosa.

The discriminative stimulus effects of epibatidine in C57BL/6J mice.

The α4ß2* nicotinic acetylcholine receptor (nAChR) subtypes are targeted for the development of smoking cessation aids, and the use of drug discrimination in mice provides a robust screening tool for the identification of drugs acting through nAChRs. Here, we established that the α4ß2* nAChR agonist epibatidine can function as a discriminative stimulus in mice. Male C57BL/6J mice discriminated epibatidine (0.0032 mg/kg, subcutaneously) and were tested with agonists varying in selectivity and efficacy for α4ß2* nAChRs. The discriminative stimulus effects of epibatidine were characterized with the nonselective, noncompetitive nicotinic antagonist mecamylamine, with the selective ß2-substype-containing nAChR antagonist dihydro-ß-erythroidine hydrobromide (DHßE), and the α7 antagonist methyllycaconitine (MLA). Nicotine (0.32-1.0 mg/kg, subcutaneously), the partial nAChR agonist cytisine (1.0-5.6 mg/kg, subcutaneously), and the α7 nAChR agonist N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-chlorobenzamide (10-56 mg/kg, intraperitoneally) produced no more than 33% epibatidine-appropriate responding. The partial α4ß2* nAChR agonists varenicline and 2'-fluoro-3'-(4-nitro-phenyl)deschloroepibatidine produced 61 and 69% epibatidine-appropriate responding, respectively. DHßE and mecamylamine, but not MLA, significantly antagonized the discriminative stimulus effects of epibatidine. These results show that epibatidine may be trained as a discriminative stimulus in mice and has utility in elucidating the in-vivo pharmacology of α4ß2* nAChR ligands.

The sigma-1 receptor modulates dopamine transporter conformation and cocaine binding and may thereby potentiate cocaine self-administration in rats.

The dopamine transporter (DAT) regulates dopamine (DA) neurotransmission by recapturing DA into the presynaptic terminals and is a principal target of the psychostimulant cocaine. The sigma-1 receptor (σ1R) is a molecular chaperone, and its ligands have been shown to modulate DA neuronal signaling, although their effects on DAT activity are unclear. Here, we report that the prototypical σ1R agonist (+)-pentazocine potentiated the dose response of cocaine self-administration in rats, consistent with the effects of the σR agonists PRE-084 and DTG (1,3-di-o-tolylguanidine) reported previously. These behavioral effects appeared to be correlated with functional changes of DAT. Preincubation with (+)-pentazocine or PRE-084 increased the Bmax values of [3H]WIN35428 binding to DAT in rat striatal synaptosomes and transfected cells. A specific interaction between σ1R and DAT was detected by co-immunoprecipitation and bioluminescence resonance energy transfer assays. Mutational analyses indicated that the transmembrane domain of σ1R likely mediated this interaction. Furthermore, cysteine accessibility assays showed that σ1R agonist preincubation potentiated cocaine-induced changes in DAT conformation, which were blocked by the specific σ1R antagonist CM304. Moreover, σ1R ligands had distinct effects on σ1R multimerization. CM304 increased the proportion of multimeric σ1Rs, whereas (+)-pentazocine increased monomeric σ1Rs. Together these results support the hypothesis that σ1R agonists promote dissociation of σ1R multimers into monomers, which then interact with DAT to stabilize an outward-facing DAT conformation and enhance cocaine binding. We propose that this novel molecular mechanism underlies the behavioral potentiation of cocaine self-administration by σ1R agonists in animal models.

Dopamine Transporter Dynamics of N-Substituted Benztropine Analogs with Atypical Behavioral Effects.

Atypical dopamine transporter (DAT) inhibitors, despite high DAT affinity, do not produce the psychomotor stimulant and abuse profile of standard DAT inhibitors such as cocaine. Proposed contributing features for those differences include off-target actions, slow onsets of action, and ligand bias regarding DAT conformation. Several 3α-(4',4''-difluoro-diphenylmethoxy)tropanes were examined, including those with the following substitutions: N-(indole-3''-ethyl)- (GA1-69), N-(R)-2''-amino-3''-methyl-n-butyl- (GA2-50), N-2''aminoethyl- (GA2-99), and N-(cyclopropylmethyl)- (JHW013). These compounds were previously reported to have rapid onset of behavioral effects and were presently evaluated pharmacologically alone or in combination with cocaine. DAT conformational mode was assessed by substituted-cysteine accessibility and molecular dynamics (MD) simulations. As determined by substituted-cysteine alkylation, all BZT analogs except GA2-99 showed bias for a cytoplasmic-facing DAT conformation, whereas cocaine stabilized the extracellular-facing conformation. MD simulations suggested that several analog-DAT complexes formed stable R85-D476 "outer gate" bonds that close the DAT to extracellular space. GA2-99 diverged from this pattern, yet had effects similar to those of other atypical DAT inhibitors. Apparent DAT association rates of the BZT analogs in vivo were slower than that for cocaine. None of the compounds was self-administered or stimulated locomotion, and each blocked those effects of cocaine. The present findings provide more detail on ligand-induced DAT conformations and indicate that aspects of DAT conformation other than "open" versus "closed" may facilitate predictions of the actions of DAT inhibitors and may promote rational design of potential treatments for psychomotor-stimulant abuse.

σ Receptor Effects of N-Substituted Benztropine Analogs: Implications for Antagonism of Cocaine Self-Administration.

Several N-substituted benztropine (BZT) analogs are atypical dopamine transport inhibitors as they have affinity for the dopamine transporter (DAT) but have minimal cocaine-like pharmacologic effects and can block numerous effects of cocaine, including its self-administration. Among these compounds, N-methyl (AHN1-055), N-allyl (AHN2-005), and N-butyl (JHW007) analogs of 3α-[bis(4'-fluorophenyl)methoxy]-tropane were more potent in antagonizing self-administration of cocaine and d-methamphetamine than in decreasing food-maintained responding. The antagonism of cocaine self-administration (0.03-1.0 mg/kg per injection) with the above BZT analogs was reproduced in the present study. Further, the stimulant-antagonist effects resembled previously reported effects of pretreatments with combinations of standard DAT inhibitors and σ1-receptor (σ1R) antagonists. Therefore, the present study examined binding of the BZT analogs to σRs, as well as their in vivo σR antagonist effects. Each of the BZT analogs displaced radiolabeled σR ligands with nanomolar affinity. Further, self-administration of the σR agonist DTG (0.1-3.2 mg/kg/injection) was dose dependently blocked by AHN2-005 and JHW007 but potentiated by AHN1-055. In contrast, none of the BZT analogs that were active against DTG self-administration was active against the self-administration of agonists at dopamine D1-like [R(+)-SKF 81297, (±)-SKF 82958 (0.00032-0.01 mg/kg per injection each)], D2-like [R(-)-NPA (0.0001-0.0032 mg/kg per injection), (-)-quinpirole (0.0032-0.1 mg/kg per injection)], or µ-opioid (remifentanil, 0.0001-0.0032 mg/kg per injection) receptors. The present results indicate that behavioral antagonist effects of the N-substituted BZT analogs are specific for abused drugs acting at the DAT and further suggest that σR antagonism contributes to those actions.

A Role for Sigma Receptors in Stimulant Self-Administration and Addiction.

Sigma receptors (σRs) are structurally unique proteins that function intracellularly as chaperones. Historically, σRs have been implicated as modulators of psychomotor stimulant effects and have at times been proposed as potential avenues for modifying stimulant abuse. However, the influence of ligands for σRs on the effects of stimulants, such as cocaine or methamphetamine, in various preclinical procedures related to drug abuse has been varied. The present paper reviews the effects of σR agonists and antagonists in three particularly relevant procedures: stimulant discrimination, place conditioning, and self-administration. The literature to date suggests limited σR involvement in the discriminative-stimulus effects of psychomotor stimulants, either with σR agonists substituting for the stimulant or with σR antagonists blocking stimulant effects. In contrast, studies of place conditioning suggest that administration of σR antagonists or down-regulation of σR protein can block the place conditioning induced by stimulants. Despite place conditioning results, selective σR antagonists are inactive in blocking the self-administration of stimulants. However, compounds binding to the dopamine transporter and blocking σRs can selectively decrease stimulant self-administration. Further, after self-administration of stimulants, σR agonists are self-administered, an effect not seen in subjects without that specific history. These findings suggest that stimulants induce unique changes in σR activity, and once established, the changes induced create redundant, and dopamine independent reinforcement pathways. Concomitant targeting of both dopaminergic pathways and σR proteins produces a selective antagonism of those pathways, suggesting new avenues for combination chemotherapies to specifically combat stimulant abuse.

Blockade of Cocaine or σ Receptor Agonist Self Administration by Subtype-Selective σ Receptor Antagonists.

The identification of sigma receptor (σR) subtypes has been based on radioligand binding and, despite progress with σ1R cellular function, less is known about σR subtype functions in vivo. Recent findings that cocaine self administration experience will trigger σR agonist self administration was used in this study to assess the in vivo receptor subtype specificity of the agonists (+)-pentazocine, PRE-084 [2-(4-morpholinethyl) 1-phenylcyclohexanecarboxylate hydrochloride], and 1,3-di-o-tolylguanidine (DTG) and several novel putative σR antagonists. Radioligand binding studies determined in vitro σR selectivity of the novel compounds, which were subsequently studied for self administration and antagonism of cocaine, (+)-pentazocine, PRE-084, or DTG self administration. Across the dose ranges studied, none of the novel compounds were self administered, nor did they alter cocaine self administration. All compounds blocked DTG self administration, with a subset also blocking (+)-pentazocine and PRE-084 self administration. The most selective of the compounds in binding σ1Rs blocked cocaine self administration when combined with a dopamine transport inhibitor, either methylphenidate or nomifensine. These drug combinations did not decrease rates of responding maintained by food reinforcement. In contrast, the most selective of the compounds in binding σ2Rs had no effect on cocaine self administration in combination with either dopamine transport inhibitor. Thus, these results identify subtype-specific in vivo antagonists, and the utility of σR agonist substitution for cocaine self administration as an assay capable of distinguishing σR subtype selectivity in vivo. These results further suggest that effectiveness of dual σR antagonism and dopamine transport inhibition in blocking cocaine self administration is specific for σ1Rs and further support this dual targeting approach to development of cocaine antagonists.

A role for sigma receptors in stimulant self-administration and addiction.

Sigma-1 receptors (σ1Rs) are structurally unique intracellular proteins that function as chaperones. σ1Rs translocate from the mitochondria-associated membrane to other subcellular compartments, and can influence a host of targets, including ion channels, G-protein-coupled receptors, lipids, and other signaling proteins. Drugs binding to σRs can induce or block the actions of σRs. Studies indicate that stimulant self-administration induces the reinforcing effects of σR agonists, because of dopamine transporter actions. Once established, the reinforcing effects of σR agonists are independent of dopaminergic mechanisms traditionally thought to be critical to the reinforcing effects of stimulants. Self-administered doses of σR agonists do not increase dopamine concentrations in the nucleus accumbens shell, a transmitter and brain region considered important for the reinforcing effects of abused drugs. However, self-administration of σR agonists is blocked by σR antagonists. Several effects of stimulants have been blocked by σR antagonists, including the reinforcing effects, assessed by a place-conditioning procedure. However, the self-administration of stimulants is largely unaffected by σR antagonists, indicating fundamental differences in the mechanisms underlying these two procedures used to assess the reinforcing effects. When σR antagonists are administered in combination with dopamine uptake inhibitors, an effective and specific blockade of stimulant self-administration is obtained. Actions of stimulant drugs related to their abuse induce unique changes in σR activity and the changes induced potentially create redundant and, once established, independent reinforcement pathways. Concomitant targeting of both dopaminergic pathways and σR proteins produces a selective antagonism of stimulant self-administration, suggesting new avenues for combination chemotherapies to specifically combat stimulant abuse.

The Reinforcing Effects of Nicotine in Humans and Nonhuman Primates: A Review of Intravenous Self-Administration Evidence and Future Directions for Research.

INTRODUCTION: Cigarette smoking is largely driven by the reinforcing properties of nicotine. Intravenous (IV) self-administration procedures are the gold standard for investigating the reinforcing effects of psychoactive drugs. The goal of this review was to examine the results of published investigations of the reinforcing effects of nicotine measured using IV self-administration procedures in humans and nonhuman primates. RESULTS: The body of literature using nonhuman primate subjects indicates nicotine functions as a positive reinforcer when available for self-administration via IV catheters. However, it can also be difficult to establish IV nicotine self-administration in nonhuman primates and sometimes supplemental strategies have been required (e.g., priming injections or food deprivation) before subjects acquire the behavior. Although the body of literature using human subjects is limited, the evidence indicates nicotine functions as a reinforcer via the IV route of administration in adult cigarette smokers. Rates of nicotine self-injection can be variable across subjects and responding is sometimes inconsistent across sessions in both humans and nonhuman primates. CONCLUSIONS: The Family Smoking Prevention and Tobacco Control Act, enacted in 2009, gave the Food and Drug Administration regulatory authority over the manufacture, marketing, and distribution of tobacco products. Research examining the threshold reinforcing doses for initiation and maintenance of nicotine self-administration, comparisons of the reinforcing effects of nicotine in adolescent versus adult subjects, investigations of gender differences in the reinforcing effects of nicotine, and studies of the abuse liability of non-nicotine tobacco product constituents and their ability to alter the reinforcing effects of nicotine will inform potential tobacco regulatory actions.

Preclinical efficacy of N-substituted benztropine analogs as antagonists of methamphetamine self-administration in rats.

Atypical dopamine-uptake inhibitors have low abuse potential and may serve as leads for development of cocaine-abuse treatments. Among them, the benztropine (BZT) derivatives, N-butyl (JHW007), N-allyl (AHN2-005), and N-methyl (AHN1-055) analogs of 3α-[bis(4'-fluorophenyl)methoxy]-tropane dose-dependently decreased cocaine self-administration without effects on food-maintained responding. Our study examined selectivity by assessing their effects on self-administration of other drugs. As with cocaine, each BZT analog (1.0-10.0 mg/kg i.p.) dose-dependently decreased maximal self-administration of d-methamphetamine (0.01-0.32 mg/kg/infusion) but was inactive against heroin (1.0-32.0 µg/kg/infusion) and ketamine (0.032-1.0 mg/kg/infusion) self-administration. Further, standard dopamine indirect-agonists [WIN35,428 ((-)-3ß-(4-fluorophenyl)-tropan-2-ß-carboxylic acid methyl ester tartrate), d-amphetamine (0.1-1.0 mg/kg i.p., each)] dose-dependently left-shifted self-administration dose-effect curves for d-methamphetamine, heroin, and ketamine. Noncompetitive NMDA-glutamate receptor/channel antagonists [(+)-MK-801 (0.01-0.1 mg/kg i.p.), memantine (1.0-10.0 mg/kg i.p.)] also left-shifted dose-effect curves for d-methamphetamine and ketamine (but not heroin) self-administration. The µ-agonists [dl-methadone and morphine (1.0-10.0 mg/kg i.p., each)] dose-dependently decreased maximal self-administration of µ-agonists (heroin, remifentanil) but not d-methamphetamine or ketamine self-administration. The µ-agonist-induced decreases were similar to the effects of BZT analogs on stimulant self-administration and effects of food prefeeding on responding maintained by food reinforcement. Radioligand-binding and behavioral studies suggested that inhibition of dopamine transporters and σ receptors were critical for blocking stimulant self-administration by BZT-analogs. Thus, the present results suggest that the effects of BZT analogs on stimulant self-administration are similar to effects of µ-agonists on µ-agonist self-administration and food prefeeding on food-reinforced responding, which implicates behavioral mechanisms for these effects and further supports development of atypical dopamine uptake inhibitors as medications for stimulant abuse.

2-isoxazol-3-phenyltropane derivatives of cocaine: molecular and atypical system effects at the dopamine transporter.

The present study examined RTI-371 [3ß-(4-methylphenyl)-2ß-[3-(4-chlorophenyl)-isoxazol-5-yl]tropane], a phenyltropane cocaine analog with effects distinct from cocaine, and assessed potential mechanisms for those effects by comparison with its constitutional isomer, RTI-336 [3ß-(4-chlorophenyl)-2ß-[3-(4-methylphenyl)-isoxazol-5-yl]tropane]. In mice, RTI-371 was less effective than cocaine and RTI-336 in stimulating locomotion, and incompletely substituted (∼60% maximum at 5 minutes or 1 hour after injection) in a cocaine (10 mg/kg i.p.)/saline discrimination procedure; RTI-336 completely substituted. In contrast to RTI-336, RTI-371 was not self-administered, and its pretreatment (1.0-10 mg/kg i.p.) dose-dependently decreased maximal cocaine self-administration more potently than food-maintained responding. RTI-336 pretreatment dose-dependently left-shifted the cocaine self-administration dose-effect curve. Both RTI-336 and RTI-371 displaced [(3)H]WIN35,428 [[(3)H](-)-3ß-(4-fluorophenyl)-tropan-2ß-carboxylic acid methyl ester tartrate] binding to striatal dopamine transporters (DATs) with Ki values of 10.8 and 7.81 nM, respectively, and had lower affinities at serotonin or norepinephrine transporters, or muscarinic and σ receptors. The relative low affinity at these sites suggests the DAT as the primary target of RTI-371 with minimal contributions from these other targets. In biochemical assays probing the outward-facing DAT conformation, both RTI-371 and RTI-336 had effects similar to cocaine, suggesting little contribution of DAT conformation to the unique pharmacology of RTI-371. The locomotor-stimulant effects of RTI-371 (3.0-30 mg/kg i.p.) were comparable in wild-type and knockout cannabinoid CB1 receptor (CB1R) mice, indicating that previously reported CB1 allosteric effects do not decrease cocaine-like effects of RTI-371. DAT occupancy in vivo was most rapid with cocaine and least with RTI-371. The slow apparent association rate may allow compensatory actions that in turn dampen cocaine-like stimulation, and give RTI-371 its unique pharmacologic profile.