Synthesis and Pharmacological Evaluation of Enantiopure N-Substituted Ortho-c Oxide-Bridged 5-Phenylmorphans.

The design of enantiopure stereoisomers of N-2-phenylcyclopropylmethyl-substituted ortho-c oxide-bridged phenylmorphans, the E and Z isomers of an N-cinnamyl moiety, and N-propyl enantiomers were based on combining the most potent oxide-bridged phenylmorphan (the ortho-c isomer) with the most potent N-substituent that we previously found with a 5-(3-hydroxy)phenylmorphan (i.e., N-2-phenylcyclopropyl methyl moieties, N-cinnamyl, and N-propyl substituents). The synthesis of the eight enantiopure N-2-phenylcyclopropylmethyl ortho-c oxide-bridged phenylmorphans and six additional enantiomers of the N-substituted ortho-c oxide-bridged phenylmorphans (N-E and Z-cinnamyl compounds, and N-propyl compounds) was accomplished. The synthesis started from common intermediates (3R,6aS,11aS)-10-methoxy-1,3,4,5,6,11a-hexahydro-2H-3,6a-methano-benzofuro[2,3-c]azocine (+)-6 and its enantiomer, (3S, 6aR, 11aR)-(-)-6, respectively. The enantiomers of ±-6 were obtained through salt formation with (S)-(+)- and (R)-(-)-p-methylmandelic acid, and the absolute configuration of the (R)-(-)-p-methylmandelate salt of (3S, 6aR, 11aR)-(-)-6 was determined by single-crystal X-ray analysis. The enantiomeric secondary amines were reacted with N-(2-phenylcyclopropyl)methyl derivatives, 2-(E)-cinnamyl bromide, and (Z)-3-phenylacrylic acid. These products led to all of the desired N-derivatives of the ortho-c oxide-bridged phenylmorphans. Their opioid receptor binding affinity was measured. The compounds with MOR affinity < 50 nM were examined for their functional activity in the forskolin-induced cAMP accumulation assay. Only the enantiomer of the N-phenethyl ortho-c oxide-bridged phenylmorphan ((-)-1), and only the (3S,6aR,11aR)-2-(((1S,2S)-2-phenylcyclopropyl)methyl)-1,3,4,5,6,11a-hexahydro-2H-3,6a-methanobenzofuro[2,3-c]azocin-10-ol isomer ((+)-17), and the N-phenylpropyl derivative ((-)-25) had opioid binding affinity < 50 nM. Both (-)-1 and (-)-25 were partial agonists in the cAMP assay, with the former showing high potency and low efficacy, and the latter with lower potency and less efficacy. Most interesting was the N-2-phenylcyclopropylmethyl (3S,6aR,11aR)-2-(1S,2S)-enantiomer ((+)-17). That compound had good MOR binding affinity (Ki = 11.9 nM) and was found to have naltrexone-like potency as a MOR antagonist (IC50 = 6.92 nM).

The Intriguing Effects of Substituents in the N-Phenethyl Moiety of Norhydromorphone: A Bifunctional Opioid from a Set of "Tail Wags Dog" Experiments.

(-)-N-Phenethyl analogs of optically pure N-norhydromorphone were synthesized and pharmacologically evaluated in several in vitro assays (opioid receptor binding, stimulation of [35S]GTPγS binding, forskolin-induced cAMP accumulation assay, and MOR-mediated ß-arrestin recruitment assays). "Body" and "tail" interactions with opioid receptors (a subset of Portoghese's message-address theory) were used for molecular modeling and simulations, where the "address" can be considered the "body" of the hydromorphone molecule and the "message" delivered by the substituent (tail) on the aromatic ring of the N-phenethyl moiety. One compound, N-p-chloro-phenethynorhydromorphone ((7aR,12bS)-3-(4-chlorophenethyl)-9-hydroxy-2,3,4,4a,5,6-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-7(7aH)-one, 2i), was found to have nanomolar binding affinity at MOR and DOR. It was a potent partial agonist at MOR and a full potent agonist at DOR with a δ/µ potency ratio of 1.2 in the ([35S]GTPγS) assay. Bifunctional opioids that interact with MOR and DOR, the latter as agonists or antagonists, have been reported to have fewer side-effects than MOR agonists. The p-chlorophenethyl compound 2i was evaluated for its effect on respiration in both mice and squirrel monkeys. Compound 2i did not depress respiration (using normal air) in mice or squirrel monkeys. However, under conditions of hypercapnia (using air mixed with 5% CO2), respiration was depressed in squirrel monkeys.

Exploring 1-adamantanamine as an alternative amine moiety for metabolically labile azepane ring in newly synthesized benzo[d]thiazol-2(3H)one σ receptor ligands.

In this work we report the structure-activity relationships, binding properties, and metabolic stability studies of a series of benzo[d]thiazol-2(3H)one as sigma receptors (σRs) ligands. Specifically, to improve the metabolic stability of the cyclic amine fragment of our lead compound (SN56), the metabolically unstable azepane ring was replaced with a 1-adatamantamine moiety. Within the synthesized analogs, compound 12 had low nanomolar affinity for the σ1R (K i = 7.2 nM) and moderate preference (61-fold) over the σ2R. In vitro metabolic stability studies showed a slight improvement of the metabolic stability for 7-12, even though an extensive metabolism in rat liver microsomes is being observed. Furthermore, metabolic soft spot identification of 12 suggested that the N-methyl group of the adamantyl moiety is a major site of metabolism.

Modulation of opioid receptor affinity and efficacy via N-substitution of 9ß-hydroxy-5-(3-hydroxyphenyl)morphan: Synthesis and computer simulation study.

The enantiomers of a variety of N-alkyl-, N-aralkyl-, and N-cyclopropylalkyl-9ß-hydroxy-5-(3-hydroxyphenyl)morphans were synthesized employing cyanogen bromide and K2CO3 to improve the original N-demethylation procedure. Their binding affinity to the µ-, δ-, and κ-opioid receptors (ORs) was determined and functional (GTPγ35S) assays were carried out on those with reasonable affinity. The 1R,5R,9S-enantiomers (1R,5R,9S)-(-)-5-(3-hydroxyphenyl)-2-(4-nitrophenethyl)-2-azabicyclo[3.3.1]nonan-9-ol (1R,5R,9S-16), (1R,5R,9S)-(-) 2-cinnamyl-5-(3-hydroxyphenyl)-2-azabicyclo[3.3.1]nonan-9-ol (1R,5R,9S-20), and (1R,5R,9S)-(-)-5-(3-hydroxyphenyl)-2-(4-(trifluoromethyl)phenethyl)-2-azabicyclo[3.3.1]nonan-9-ol (1R,5R,9S-15), had high affinity for the µ-opioid receptor (e.g., 1R,5R,9S-16: Ki=0.073, 0.74, and 1.99nM, respectively). The 1R,5R,9S-16 and 1R,5R,9S-15 were full, high efficacy µ-agonists (EC50=0.74 and 18.5nM, respectively) and the former was found to be a partial agonist at δ-OR and an antagonist at κ-OR, while the latter was a partial agonist at δ-OR and κ-OR in the GTPγ35S assay. The enantiomer of 1R,5R,9S-16, (+)-1S,5S,9R-16 was unusual, it had good affinity for the µ-OR (Ki=26.5nM) and was an efficacious µ-antagonist (Ke=29.1nM). Molecular dynamics simulations of the µ-OR were carried out with the 1R,5R,9S-16 µ-agonist and the previously synthesized (1R,5R,9S)-(-)-5-(9-hydroxy-5-(3-hydroxyphenyl-2-phenylethyl)-2-azabicyclo[3.3.1]nonane (1R,5R,9S-(-)-NIH 11289) to provide a structural basis for the observed high affinities and efficacies. The critical roles of both the 9ß-OH and the p-nitro group are elucidated, with the latter forming direct, persistent hydrogen bonds with residues deep in the binding cavity, and the former interacting with specific residues via highly structured water bridges.

2-Substituted 3ß-Aryltropane Cocaine Analogs Produce Atypical Effects without Inducing Inward-Facing Dopamine Transporter Conformations.

Previous structure-activity relationship studies indicate that a series of cocaine analogs, 3ß-aryltropanes with 2ß-diarylmethoxy substituents, selectively bind to the dopamine transporter (DAT) with nanomolar affinities that are 10-fold greater than the affinities of their corresponding 2α-enantiomers. The present study compared these compounds to cocaine with respect to locomotor effects in mice, and assessed their ability to substitute for cocaine (10 mg/kg, i.p.) in rats trained to discriminate cocaine from saline. Despite nanomolar DAT affinity, only the 2ß-Ph2COCH2-3ß-4-Cl-Ph analog fully substituted for cocaine-like discriminative effects. Whereas all of the 2ß compounds increased locomotion, only the 2ß-(4-ClPh)PhCOCH2-3ß-4-Cl-Ph analog had cocaine-like efficacy. None of the 2α-substituted compounds produced either of these cocaine-like effects. To explore the molecular mechanisms of these drugs, their effects on DAT conformation were probed using a cysteine-accessibility assay. Previous reports indicate that cocaine binds with substantially higher affinity to the DAT in its outward (extracellular)- compared with inward-facing conformation, whereas atypical DAT inhibitors, such as benztropine, have greater similarity in affinity to these conformations, and this is postulated to explain their divergent behavioral effects. All of the 2ß- and 2α-substituted compounds tested altered cysteine accessibility of DAT in a manner similar to cocaine. Furthermore, molecular dynamics of in silico inhibitor-DAT complexes suggested that the 2-substituted compounds reach equilibrium in the binding pocket in a cocaine-like fashion. These behavioral, biochemical, and computational results show that aryltropane analogs can bind to the DAT and stabilize outward-facing DAT conformations like cocaine, yet produce effects that differ from those of cocaine.

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.

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.

Stimulants as specific inducers of dopamine-independent σ agonist self-administration in rats.

A previous study showed that cocaine self-administration induced dopamine-independent reinforcing effects of σ agonists mediated by their selective actions at σ1 receptors (σ1Rs), which are intracellularly mobile chaperone proteins implicated in abuse-related effects of stimulants. The present study assessed whether the induction was specific to self-administration of cocaine. Rats were trained to self-administer the dopamine releaser, d-methamphetamine (0.01-0.32 mg/kg per injection), the µ-opioid receptor agonist, heroin (0.001-0.032 mg/kg per injection), and the noncompetitive N-methyl-d-aspartate receptor/channel antagonist ketamine (0.032-1.0 mg/kg per injection). As with cocaine, self-administration of d-methamphetamine induced reinforcing effects of the selective σ1R agonists PRE-084 [2-(4-morpholinethyl)1-phenylcyclohexanecarboxylate hydrochloride] and (+)-pentazocine (0.032-1.0 mg/kg per injection, each). In contrast, neither self-administration of heroin nor ketamine induced PRE-084 or (+)-pentazocine (0.032-10 mg/kg per injection, each) self-administration. Although the σ1R agonists did not maintain responding in subjects with histories of heroin or ketamine self-administration, substitution for those drugs was obtained with appropriate agonists (e.g., remifentanil, 0.1-3.2 µg/kg per injection, for heroin and (5S,10R)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine ((+)-MK 801; dizocilpine), 0.32-10.0 µg/kg per injection, for ketamine). The σR antagonist N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(1-pyrrolidinyl)ethylamine dihydrobromide (BD 1008; 1.0-10 mg/kg) dose-dependently blocked PRE-084 self-administration but was inactive against d-methamphetamine, heroin, and ketamine. In contrast, PRE-084 self-administration was affected neither by the dopamine receptor antagonist (+)-butaclamol (10-100 µg/kg) nor by the opioid antagonist (-)-naltrexone (1.0-10 mg/kg), whereas these antagonists were active against d-methamphetamine and heroin self-administration, respectively. The results indicate that experience specifically with indirect-acting dopamine agonists induces reinforcing effects of previously inactive σ1R agonists. It is further suggested that induced σ1R reinforcing mechanisms may play an essential role in treatment-resistant stimulant abuse, suggesting new approaches for the development of effective medications for its treatment.

N-substituted benztropine analogs: selective dopamine transporter ligands with a fast onset of action and minimal cocaine-like behavioral effects.

Previous studies suggested that differences between the behavioral effects of cocaine and analogs of benztropine were related to the relatively slow onset of action of the latter compounds. Several N-substituted benztropine analogs with a relatively fast onset of effects were studied to assess whether a fast onset of effects would render the effects more similar to those of cocaine. Only one of the compounds increased locomotor activity, and the increases were modest compared with those of 10 to 20 mg/kg cocaine. In rats trained to discriminate 10 mg/kg cocaine from saline none of the compounds produced more than 40% cocaine-like responds up to 2 h after injection. None of the compounds produced place-conditioning when examined up to 90 min after injection, indicating minimal abuse liability. The compounds had 5.6 to 30 nM affinities at the dopamine transporter (DAT), with uniformly lower affinities at norepinephrine and serotonin transporters (from 490-4600 and 1420-7350 nM, respectively). Affinities at muscarinic M(1) receptors were from 100- to 300-fold lower than DAT affinities, suggesting minimal contribution of those sites to the behavioral effects of the compounds. Affinities at histaminic H(1) sites were from 11- to 43-fold lower than those for the DAT. The compounds also had affinity for sigma, 5-hydroxytryptamine(1) (5-HT(1)), and 5-HT(2) receptors that may have contributed to their behavioral effects. Together, the results indicate that a slow onset of action is not a necessary condition for reduced cocaine-like effects of atypical DAT ligands and suggest several mechanisms that may contribute to the reduced cocaine-like efficacy of these compounds.

Dopamine transporter-dependent and -independent striatal binding of the benztropine analog JHW 007, a cocaine antagonist with low abuse liability.

The benztropine analog N-(n-butyl)-3α-[bis(4'-fluorophenyl)methoxy]-tropane (JHW 007) displays high affinity for the dopamine transporter (DAT), but unlike typical DAT ligands, has relatively low abuse liability and blocks the effects of cocaine, including its self-administration. To determine sites responsible for the cocaine antagonist effects of JHW 007, its in vitro binding was compared with that of methyl (1R,2S,3S,5S)-3-(4-fluorophenyl)-8-methyl-8-azabicyclo[3.2.1]octane-2-carboxylate (WIN 35428) in rats, mice, and human DAT (hDAT)-transfected cells. A one-site model, with K(d) values of 4.21 (rat) and 8.99 nM (mouse) best fit the [(3)H]WIN 35428 data. [(3)H]JHW 007 binding best fit a two-site model (rat, 7.40/4400 nM; mouse, 8.18/2750 nM), although a one-site fit was observed with hDAT membranes (43.7 nM). Drugs selective for the norepinephrine and serotonin transporters had relatively low affinity in competition with [(3)H]JHW 007 binding, as did drugs selective for other sites identified previously as potential JHW 007 binding sites. The association of [(3)H]WIN 35428 best fit a one-phase model, whereas the association of [(3)H]JHW 007 best fit a two-phase model in all tissues. Because cocaine antagonist effects of JHW 007 have been observed previously soon after injection, its rapid association observed here may contribute to those effects. Multiple [(3)H]JHW 007 binding sites were obtained in tissue from mice lacking the DAT, suggesting these as yet unidentified sites as potential contributors to the cocaine antagonist effects of JHW 007. Unlike WIN 35428, the binding of JHW 007 was Na(+)-independent. This feature of JHW 007 has been linked to the conformational status of the DAT, which in turn may contribute to the antagonism of cocaine.

Pharmacodynamics and pharmacokinetics of the novel synthetic opioid, U-47700, in male rats.

Novel synthetic opioids are appearing in recreational drug markets worldwide as adulterants in heroin or ingredients in counterfeit analgesic medications. Trans-3,4-dichloro-N-[2-(dimethylamino)cyclohexyl]-N-methyl-benzamide (U-47700) is an example of a non-fentanyl synthetic opioid linked to overdose deaths. Here, we examined the pharmacodynamics and pharmacokinetics of U-47700 in rats. Male Sprague-Dawley rats were fitted with intravenous (i.v.) catheters and subcutaneous (s.c.) temperature transponders under ketamine/xylazine anesthesia. One week later, rats received s.c. injections of U-47700 HCl (0.3, 1.0 or 3.0 mg/kg) or saline, and blood samples (0.3 mL) were withdrawn via i.v. catheters at 15, 30, 60, 120, 240, 480 min post-injection. Pharmacodynamic effects were assessed at each blood withdrawal, and plasma was assayed for U-47700 and its metabolites by liquid chromatography tandem mass spectrometry. U-47700 induced dose-related increases in hot plate latency (ED50 = 0.5 mg/kg) and catalepsy (ED50 = 1.7 mg/kg), while the 3.0 mg/kg dose also caused hypothermia. Plasma levels of U-47700 rose linearly as dose increased, with maximal concentration (Cmax) achieved by 15-38 min. Cmax values for N-desmethyl-U-47700 and N,N-didesmethyl-U-47700 were delayed but reached levels in the same range as the parent compound. Pharmacodynamic effects were correlated with plasma U-47700 and its N-desmethyl metabolite. Using radioligand binding assays, U-47700 displayed high affinity for µ-opioid receptors (Ki = 11.1 nM) whereas metabolites were more than 18-fold weaker. Our data reveal that U-47700 induces typical µ-opioid effects which are related to plasma concentrations of the parent compound. Given its high potency, U-47700 poses substantial risk to humans who are inadvertently exposed to the drug.

Cocaine-like neurochemical effects of antihistaminic medications.

The pattern of activation of dopamine (DA) neurotransmission in the nucleus accumbens (NAc) of rats produced by H(1) histamine antagonists which have behavioral effects like those of psychostimulant drugs was examined. Diphenhydramine and (+)-chlorpheniramine were compared with triprolidine, a potent and selective H(1) antagonist and (-)-chlorpheniramine which is less active than its enantiomer at H(1) receptors. Affinities of the drugs to DA, serotonin, and norepinephrine transporters at H(1) 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 H(1) 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.

Pharmacological Research as a Key Component in Mitigating the Opioid Overdose Crisis.

The United States is experiencing an epidemic of opioid overdose deaths. Many of the recent fatalities are associated with illicitly manufactured fentanyl, which is being added to heroin and counterfeit pain pills. The crisis is further exacerbated by the emergence of an increasing number of novel synthetic opioids (NSOs), including various fentanyl analogs and non-fentanyl compounds that display potent agonist actions at the µ-opioid receptor. Importantly, most users are unaware of their exposure to fentanyl and NSOs. Stemming the tide of opioid-related fatalities will require a coordinated multidisciplinary response from policy makers, law enforcement personnel, first responders, treatment providers, family members, and scientists. To this end, basic research in pharmacology can contribute significantly to mitigating the crisis through efforts to characterize the biological effects of NSOs, discover more effective antidotes for overdose rescue, and develop safer medications for treating addiction and alleviating pain.

Abuse liability of mitragynine assessed with a self-administration procedure in rats.

RATIONALE: Substantial use of the plant kratom for psychoactive effects has driven interest in its abuse liability. Several place conditioning studies suggest abuse liability of the active ingredient mitragynine, though studies of its self-administration have not been published. METHODS: Binding of mitragynine to rat brain mu, kappa, and delta opioid receptors was compared to that for heroin and morphine. Self-administration of mitragynine, heroin, methamphetamine, or saline was assessed during single-session substitutions in rats trained to self-administer methamphetamine (0.022 mg/kg/injection, i.v.) during 1-h daily sessions. RESULTS: Mitragynine had > 2- or ~ 16-fold greater affinity for the mu opioid receptor than, respectively, for kappa or delta opioid receptors. Its affinity for the mu receptor was ~ 200-fold less than that for morphine. In rats trained to self-administer methamphetamine, saline substitutions significantly decreased the number of responses, whereas different doses of methamphetamine (0.002-0.068 mg/kg/injection) or heroin (0.001-0.03 mg/kg/injection) maintained self-administration with maximal responding at 0.022 or 0.01 mg/kg/injection, respectively. In contrast, no dose of mitragynine maintained response rates greater than those obtained with saline. Presession mitragynine treatment (0.1 to 3.0 mg/kg) decreased response rates maintained by heroin but had little effect on responding maintained by methamphetamine across the same range of doses. CONCLUSIONS: These results suggest a limited abuse liability of mitragynine and potential for mitragynine treatment to specifically reduce opioid abuse. With the current prevalence of opioid abuse and misuse, it appears currently that mitragynine is deserving of more extensive exploration for its development or that of an analog as a medical treatment for opioid abuse.

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.

Mechanisms of amphetamine action illuminated through optical monitoring of dopamine synaptic vesicles in Drosophila brain.

Amphetamines elevate extracellular dopamine, but the underlying mechanisms remain uncertain. Here we show in rodents that acute pharmacological inhibition of the vesicular monoamine transporter (VMAT) blocks amphetamine-induced locomotion and self-administration without impacting cocaine-induced behaviours. To study VMAT's role in mediating amphetamine action in dopamine neurons, we have used novel genetic, pharmacological and optical approaches in Drosophila melanogaster. In an ex vivo whole-brain preparation, fluorescent reporters of vesicular cargo and of vesicular pH reveal that amphetamine redistributes vesicle contents and diminishes the vesicle pH-gradient responsible for dopamine uptake and retention. This amphetamine-induced deacidification requires VMAT function and results from net H(+) antiport by VMAT out of the vesicle lumen coupled to inward amphetamine transport. Amphetamine-induced vesicle deacidification also requires functional dopamine transporter (DAT) at the plasma membrane. Thus, we find that at pharmacologically relevant concentrations, amphetamines must be actively transported by DAT and VMAT in tandem to produce psychostimulant effects.

Mazindol analogues as potential inhibitors of the cocaine binding site at the dopamine transporter.

A series of mazindol (2) and homomazindol (3) analogues with a variety of electron-donating and electron-withdrawing groups in the pendant aryl group and the benzo ring C, as well as H, methoxy, and alkyl groups replacing the hydroxyl group were synthesized, and their binding affinities at the dopamine transporter (DAT) on rat or guinea pig striatal membranes were determined. Several active analogues were also evaluated for their ability to block uptake of DA, 5-HT, and NE and inhibit binding of [(125)I] RTI-55 at HEK-hDAT, HEK-hSERT, and HEK-hNET cells. Mazindane (26) was found to be a pro-drug, oxidizing (5-H --> 5-OH) to mazindol on rat striatal membranes and HEK-hDAT cells. The 4',7,8-trichloro analogue (38) of mazindol was the most potent and selective ligand for HEK-hDAT cells (DAT K(i) = 1.1 nM; SERT/DAT = 1283 and NET/DAT = 38). Experimental results strongly favor the cyclic or ol tautomers of 2 and 3 to bind more tightly at the DAT than the corresponding keto tautomers.

Benzo- and cyclohexanomazindol analogues as potential inhibitors of the cocaine binding site at the dopamine transporter.

A series of mazindol (1), homomazindol (2), and bishomomazindol (3) derivatives with a benzo or cyclohexano ring fused at various sites were prepared as part of an SAR study to determine the effect of increased aliphatic and aromatic lipophilicity on selected in vitro assays used to identify potential cocaine-like and cocaine antagonism activity. Very good (IC(50) = 2-3 nM) inhibition of [(3)H] WIN 35,428 and [(125)I] RTI-55 binding on rat or guinea pig striatal membranes and HEK cells expressing cDNA for the human dopamine transporter (HEK-hDAT) was shown by the 8,9-benzomazindol 25 and 9,10-benzohomomazindol 28. All new compounds were weaker inhibitors of [(3)H] DA uptake in HEK-hDAT cells than 1 and 2. No improvement in the binding selectivity ratio (SERT/DAT and NET/DAT) was found when compared to 2. Compounds 25and 28 showed a considerable increase versus 1 in uptake/binding discrimination ratios at the DAT (311.0 and 182.1 vs 0.9), SERT (33.6 and 127.3 vs 1.9), and NET (7.3 and 10.0 vs 0.3).

Relations between stimulation of mesolimbic dopamine and place conditioning in rats produced by cocaine or drugs that are tolerant to dopamine transporter conformational change.

RATIONALE: Dopamine transporter (DAT) conformation plays a role in the effectiveness of cocaine-like and other DAT inhibitors. Cocaine-like stimulants are intolerant to DAT conformation changes having decreased potency in cells transfected with DAT constructs that face the cytosol compared to wild-type DAT. In contrast, analogs of benztropine (BZT) are among compounds that are less affected by DAT conformational change. METHODS: We compared the displacement of radioligand binding to various mammalian CNS sites, acute stimulation of accumbens shell dopamine levels, and place conditioning in rats among cocaine and four BZT analogs with Cl substitutions on the diphenyl-ether system including two with carboalkoxy substitutions at the 2-position of the tropane ring. RESULTS: Binding assays confirmed high-affinity and selectivity for the DAT with the BZT analogs which also produced significant stimulation of mesolimbic dopamine efflux. Because BZT analogs produced temporal patterns of extracellular dopamine levels different from those by cocaine (3-10 mg/kg, i.p.), the place conditioning produced by BZT analogs and cocaine was compared at doses and times at which both the increase in dopamine levels and rates of increase were similar to those produced by an effective dose of cocaine. Despite this equilibration, none of the BZT analogs tested produced significant place conditioning. CONCLUSIONS: The present results extend previous findings suggesting that cocaine-like actions are dependent on a binding equilibrium that favors the outward conformational state of the DAT. In contrast, BZT analogs with reduced dependence on DAT conformation have reduced cocaine-like behavioral effects and may prove useful in development of medications for stimulant abuse.