Theresa Kopajtic

Opioid Activation of Toll-Like Receptor 4 Contributes to Drug Reinforcement

Opioid action was thought to exert reinforcing effects solely via the initial agonism of opioid receptors. Here, we present evidence for an additional novel contributor to opioid reward: the innate immune pattern-recognition receptor, toll-like receptor 4 (TLR4), and its MyD88-dependent signaling. Blockade of TLR4/MD2 by administration of the nonopioid, unnatural isomer of naloxone, (+)-naloxone (rats), or two independent genetic knock-outs of MyD88-TLR4-dependent signaling (mice), suppressed opioid-induced conditioned place preference. (+)-Naloxone also reduced opioid (remifentanil) self-administration (rats), another commonly used behavioral measure of drug reward. Moreover, pharmacological blockade of morphine-TLR4/MD2 activity potently reduced morphine-induced elevations of extracellular dopamine in rat nucleus accumbens, a region critical for opioid reinforcement. Importantly, opioid-TLR4 actions are not a unidirectional influence on opioid pharmacodynamics, since TLR4−/− mice had reduced oxycodone-induced p38 and JNK phosphorylation, while displaying potentiated analgesia. Similar to our recent reports of morphine-TLR4/MD2 binding, here we provide a combination of in silico and biophysical data to support (+)-naloxone and remifentanil binding to TLR4/MD2. Collectively, these data indicate that the actions of opioids at classical opioid receptors, together with their newly identified TLR4/MD2 actions, affect the mesolimbic dopamine system that amplifies opioid-induced elevations in extracellular dopamine levels, therefore possibly explaining altered opioid reward behaviors. Thus, the discovery of TLR4/MD2 recognition of opioids as foreign xenobiotic substances adds to the existing hypothesized neuronal reinforcement mechanisms, identifies a new drug target in TLR4/MD2 for the treatment of addictions, and provides further evidence supporting a role for central proinflammatory immune signaling in drug reward.

Identification of a dopamine transporter ligand that blocks the stimulant effects of cocaine.

There is a large unmet medical need for cocaine addiction treatments. Studies have indicated that the dopamine transporter (DAT) is the primary biological target of cocaine, and most drugs that have DAT affinity have behavioral effects like those of cocaine. However, analogs of benztropine have high DAT affinity and behavioral effects that show varying degrees of similarity to cocaine. We now report the discovery that a benztropine analog, JHW007, with high affinity for the DAT does not have cocaine-like behavioral effects and antagonizes the effects of cocaine. JHW007 occupied the DAT in vivo more slowly than did cocaine and had not reached an apparent plateau up to 270 min after injection. The in vivo binding of cocaine to the DAT suggested rate of DAT occupancy as an important contributor to its behavioral effects, and the slow association with the DAT may provide an explanation for JHW007 being relatively devoid of cocaine-like behavioral effects. The antagonism of cocaine suggests that DAT ligands with reduced cocaine-like activity can function as cocaine antagonists and suggests JHW007 as a lead for discovery of cocaine-abuse pharmacotherapeutics.

R-MODAFINIL (ARMODAFINIL): A UNIQUE DOPAMINE UPTAKE INHIBITOR AND POTENTIAL MEDICATION FOR PSYCHOSTIMULANT ABUSE

BACKGROUND (±)-Modafinil has piqued interest as a treatment for attention-deficit/hyperactivity disorder and stimulant dependence. The R-enantiomer of modafinil might have unique pharmacological properties that should be further investigated. METHODS (±)-Modafinil and its R-(-)- and S-(+)-enantiomers were synthesized and tested for inhibition of [(3)H] dopamine (DA) uptake and [(3)H]WIN 35428 binding in human dopamine transporter (DAT) wild-type and mutants with altered conformational equilibria. Data were compared with cocaine and the atypical DA uptake inhibitor, JHW 007. R- and S-modafinil were also evaluated in microdialysis studies in the mouse nucleus accumbens shell and in a cocaine discrimination procedure. RESULTS (±)-, R-, and S-modafinil bind to the DAT and inhibit DA uptake less potently than cocaine, with R-modafinil having approximately threefold higher affinity than its S-enantiomer. Molecular docking studies revealed subtle differences in binding modes for the enantiomers. R-modafinil was significantly less potent in the DAT Y156F mutant compared with wild-type DAT, whereas S-modafinil was affected less. Studies with the Y335A DAT mutant showed that the R- and S-enantiomers tolerated the inward-facing conformation better than cocaine, which was further supported by [2-(trimethylammonium)ethyl]-methanethiosulfonate reactivity on the DAT E2C I159C. Microdialysis studies demonstrated that both R- and S-modafinil produced increases in extracellular DA concentrations in the nucleus accumbens shell less efficaciously than cocaine and with a longer duration of action. Both enantiomers fully substituted in mice trained to discriminate cocaine from saline. CONCLUSIONS R-modafinil displays an in vitro profile different from cocaine. Future trials with R-modafinil as a substitute therapy with the potential benefit of cognitive enhancement for psychostimulant addiction are warranted.

DAT isn’t all that: cocaine reward and reinforcement require Toll-like receptor 4 signaling

The initial reinforcing properties of drugs of abuse, such as cocaine, are largely attributed to their ability to activate the mesolimbic dopamine system. Resulting increases in extracellular dopamine in the nucleus accumbens (NAc) are traditionally thought to result from cocaine’s ability to block dopamine transporters (DATs). Here we demonstrate that cocaine also interacts with the immunosurveillance receptor complex, Toll-like receptor 4 (TLR4), on microglial cells to initiate central innate immune signaling. Disruption of cocaine signaling at TLR4 suppresses cocaine-induced extracellular dopamine in the NAc, as well as cocaine conditioned place preference and cocaine self-administration. These results provide a novel understanding of the neurobiological mechanisms underlying cocaine reward/reinforcement that includes a critical role for central immune signaling, and offer a new target for medication development for cocaine abuse treatment.

Sigma receptor agonists: Receptor binding and effects on mesolimbic dopamine neurotransmission assessed by microdialysis

BACKGROUND Subtypes of sigma (σ) receptors, σ₁ and σ₂, can be pharmacologically distinguished, and each may be involved in substance-abuse disorders. σ-Receptor antagonists block cocaine place conditioning and σ-receptor agonists are self-administered in rats that previously self-administered cocaine. Self-administration of abused drugs has been related to increased dopamine (DA) neurotransmission, however, σ-receptor agonist effects on mesolimbic DA are not fully characterized. METHODS Receptor-binding studies assessed affinities of σ-receptor ligands for σ-receptor subtypes and the DA transporter; effects on DA transmission in the rat nucleus accumbens shell were assessed using in vivo microdialysis. RESULTS Cocaine (.1-1.0 mg/kg intravenous [IV]), the nonselective σ(½)-receptor agonist DTG (1.0-5.6 mg/kg IV), and the selective σ₁-receptor agonist PRE-084 (.32-10 mg/kg IV) dose-dependently increased DA to ∼275%, ∼150%, and ∼160% maxima, respectively. DTG-induced stimulation of DA was antagonized by the nonselective σ(½)-receptor antagonist BD 1008 (10 mg/kg intraperitoneal [IP]) and the preferential σ₂-receptor antagonist SN 79 (1-3 mg/kg IP), but not by the preferential σ₁-receptor antagonist, BD 1063 (10-30 mg/kg IP). Neither PRE-084 nor cocaine was antagonized by BD 1063 or BD 1008. CONCLUSIONS σ-Receptor agonists stimulated DA in a brain area critical for reinforcing effects of cocaine. DTG effects on DA appear to be mediated by σ₂-receptors rather than σ₁-receptors. However, DA stimulation by cocaine or PRE-084 does not likely involve σ-receptors. The relatively low potency on DA transmission of the selective σ₁-receptor agonist, PRE-084, and its previously reported potent reinforcing effects, suggest a dopamine-independent reinforcing pathway that may contribute to substance-abuse disorders.

A Role for Sigma Receptors in Stimulant Self Administration and Addiction

Sigma1 receptors (σ1Rs) represent a structurally unique class of intracellular proteins that function as chaperones. σ1Rs translocate from the mitochondria-associated membrane to the cell nucleus or cell membrane, and through protein-protein interactions influence several targets, including ion channels, G-protein-coupled receptors, lipids, and other signaling proteins. Several studies have demonstrated that σR antagonists block stimulant-induced behavioral effects, including ambulatory activity, sensitization, and acute toxicities. Curiously, the effects of stimulants have been blocked by σR antagonists tested under place-conditioning but not self-administration procedures, indicating fundamental differences in the mechanisms underlying these two effects. The self administration of σR agonists has been found in subjects previously trained to self administer cocaine. The reinforcing effects of the σR agonists were blocked by σR antagonists. Additionally, σR agonists were found to increase dopamine concentrations in the nucleus accumbens shell, a brain region considered important for the reinforcing effects of abused drugs. Although the effects of the σR agonist, DTG, on dopamine were obtained at doses that approximated those that maintained self administration behavior those of another agonist, PRE-084 required higher doses. The effects of DTG were antagonized by non-selective or a preferential σ2R antagonist but not by a preferential σ1R antagonist. The effects of PRE-084 on dopamine were insensitive to σR antagonists. The data suggest that the self administration of σR agonists is independent of dopamine and the findings are discussed in light of a hypothesis that cocaine has both intracellular actions mediated by σRs, as well as extracellular actions mediated through conventionally studied mechanisms. The co-activation and potential interactions among these mechanisms, in particular those involving the intracellular chaperone σRs, may lead to the pernicious addictive effects of stimulant drugs.

Decreases in cocaine self-administration with dual inhibition of the dopamine transporter and σ receptors.

Sigma receptor (σR) antagonists attenuate many behavioral effects of cocaine but typically not its reinforcing effects in self-administration procedures. However, the σR antagonist rimcazole and its N-propylphenyl analogs, [3-(cis-3,5-dimethyl-4-[3-phenylpropyl]-1-piperazinyl)-propyl]diphenylamine hydrochloride (SH 3-24) and 9-[3-(cis-3,5-dimethyl-4-[3-phenylpropyl]-1-piperazinyl)-propyl]carbazole hydrobromide (SH 3-28), dose-dependently decreased the maximal rates of cocaine self-administration without affecting comparable responding maintained by food reinforcement. In contrast, a variety of σR antagonists [N-phenethylpiperidine oxalate (AC927), N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(1-pyrrolidinyl)ethylamine dihydrobromide (BD 1008), N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(dimethylamino) ethylamine dihydrobromide (BD 1047), N-[2-(3,4-dichlorophenyl) ethyl]-4-methylpiperazine dihydrochloride (BD 1063), and N,N-dipropyl-2-[4-methoxy-3-(2-phenylethoxy)phenyl]-ethylamine monohydrochloride (NE-100)] had no effect on cocaine self-administration across the range of doses that decreased rates of food-maintained responding. Rimcazole analogs differed from selective σR antagonists in their dual affinities for σRs and the dopamine transporter (DAT) assessed with radioligand binding. Selective DAT inhibitors and σR antagonists were studied alone and in combination on cocaine self-administration to determine whether actions at both σRs and the DAT were sufficient to reproduce the effects of rimcazole analogs. Typical DAT inhibitors [2β-carbomethoxy-3β-(4-fluorophenyl)tropane (WIN 35,428), methylphenidate, and nomifensine] dose-dependently shifted the cocaine dose-effect curve leftward. Combinations of DAT inhibitor and σR antagonist doses that were behaviorally inactive alone decreased cocaine self-administration without effects on food-maintained responding. In addition, whereas the DAT inhibitors were self-administered at rates similar to those of cocaine, neither rimcazole analogs nor typical σR antagonists (NE-100 and AC927) maintained responding above control levels across a wide range of doses. These findings suggest that the unique effects of rimcazole analogs are due to dual actions at the DAT and σRs and that a combined target approach may have utility in development of medical treatments for cocaine abuse.

Effects of Muscarinic M1 Receptor Blockade on Cocaine-Induced Elevations of Brain Dopamine Levels and Locomotor Behavior in Rats

Cholinergic muscarinic systems have been shown to influence dopaminergic function in the central nervous system. In addition, previous studies of benztropine analogs that inhibit dopamine uptake and show antagonism at muscarinic receptors show these drugs to be less effective than cocaine in producing its various prototypic effects such as locomotor stimulation. Because previous pharmacological studies on these topics have used nonselective M1 antagonists, we examined the interactions of preferential M1 muscarinic antagonists and cocaine. Dose-dependent increases in extracellular levels of dopamine in selected brain areas, the nucleus accumbens (NAc) shell and core, and the prefrontal cortex, were produced by cocaine but not by the preferential M1 antagonists telenzepine and trihexyphenidyl. When administered with cocaine, however, both M1 antagonists dose-dependently increased the effects of cocaine on dopamine in the NAc shell, and these effects were selective in that they were not obtained in the NAc core or in the prefrontal cortex. Telenzepine also increased locomotor activity, although the effect was small compared with that of cocaine. The locomotor stimulant effects of trihexyphenidyl, in contrast, approached those of cocaine. Telenzepine attenuated, whereas trihexyphenidyl enhanced the locomotor stimulant effects of cocaine, with neither drug facilitating cocaine-induced stereotypy. The present results indicate that preferential antagonist effects at muscarinic M1 receptors do not uniformly alter all of the effects of cocaine, nor do they explain the differences in effects of cocaine and benztropine analogs, and that the alterations in dopamine levels in the NAc shell do not predict the behavioral effects of the interactions with cocaine.

Cocaine‐like neurochemical effects of antihistaminic medications

The pattern of activation of dopamine (DA) neurotransmission in the nucleus accumbens (NAc) of rats produced by H1 histamine antagonists which have behavioral effects like those of psychostimulant drugs was examined. Diphenhydramine and (+)‐chlorpheniramine were compared with triprolidine, a potent and selective H1 antagonist and (−)‐chlorpheniramine which is less active than its enantiomer at H1 receptors. Affinities of the drugs to DA, serotonin, and norepinephrine transporters at H1 receptors and potencies for DA uptake inhibition in striatal synaptosomes were determined to assess mechanisms by which the compounds increased DA levels. Intravenous diphenhydramine (1.0–3.0 mg/kg) (+)‐ and (−)‐chlorpheniramine (1.0–5.6 mg/kg) but not triprolidine (1.0–3.0 mg/kg) elicited a cocaine‐like pattern of stimulation of DA transmission with larger effects in the NAc shell than core. The absence of stereospecific effects with chlorpheniramine enantiomers along with the lack of an effect with triprolidine suggest that the effects on DA transmission were not related to H1 receptor antagonism. Although in vivo potencies were not directly related to DA transporter affinities, it is hypothesized that actions at that site modulated by other actions, possibly those at the serotonin transporter, are primarily responsible for the neurochemical actions of the drugs on DA neurotransmission and might underlie the occasional misuse of these medications.

N-substituted phenyltropanes as in vivo binding ligands for rapid imaging studies of the dopamine transporter:

Variously substituted phenyltropanes are proven as superb binding ligands for the dopamine transporter (DAT). In this study, we examine four N‐substituted phenyltropanes which are derivatives of RTI‐55 as in vivo binding ligands in mice. In this series, the methyl group on the nitrogen was replaced by a propyl (RTI‐310), an allyl (RTI‐311), a butyl (RTI‐312), or a fluoropropyl (RTI‐313) group. The in vitro binding potencies of these compounds at rat striatal DAT varied somewhat but were about 1 nM. While these compounds did not display marked selectivity for the dopamine transporter, they were more selective than RTI‐55. Injection of the radiolabeled compound into mice resulted in striatal‐to‐cerebellar ratios that varied from about 4.5–6.5. The ratios peaked most rapidly for RTI‐311 and RTI‐313, at about 20 min. Pharmacological inhibition studies indicated that these compounds were binding to DATs in the striatum, as expected. These findings suggest that some compounds of this type may be excellent in vivo binding ligands for rapid imaging studies of the DAT. Synapse 25:345–349, 1997.