High Affinity For Dopamine Transporter Research Articles

Substantia nigral dopamine transporter uptake in dementia with Lewy bodies

Nigrostriatal dopaminergic degeneration is a pathological hallmark of dementia with Lewy bodies (DLB). To identify the subregional dopamine transporter (DAT) uptake patterns that improve the diagnostic accuracy of DLB, we analyzed N-(3-[18F] fluoropropyl)-2β-carbomethoxy-3β-(4-iodophenyl)-nortropane (FP-CIT) PET in 51 patients with DLB, in 36 patients with mild cognitive impairment with Lewy body (MCI-LB), and in 40 healthy controls (HCs). In addition to a high affinity for DAT, FP-CIT show a modest affinity to serotonin or norepinephrine transporters. Specific binding ratios (SBRs) of the nigrostriatal subregions were transformed to age-adjusted z-scores (zSBR) based on HCs. The diagnostic accuracy of subregional zSBRs were tested using receiver operating characteristic (ROC) curve analyses separately for MCI-LB and DLB versus HCs. Then, the effect of subregional zSBRs on the presence of clinical features and gray matter (GM) density were evaluated in all patients with MCI-LB or DLB as a group. ROC curve analyses showed that the diagnostic accuracy of DLB based on the zSBR of substantia nigra (area under the curve [AUC], 0.90) or those for MCI-LB (AUC, 0.87) were significantly higher than that based on the zSBR of posterior putamen for DLB (AUC, 0.72) or MCI-LB (AUC, 0.65). Lower zSBRs in nigrostriatal regions were associated with visual hallucination, severe parkinsonism, and cognitive dysfunction, while lower zSBR of substantia nigra was associated with widespread GM atrophy in DLB and MCI-LB patients. Taken together, our results suggest that evaluation of nigral DAT uptake may increase the diagnostic accuracy of DLB and MCI-LB than other striatal regions.

Acute and chronic bupropion treatment does not prevent morphine-induced conditioned place preference in mice

A substantial barrier to the treatment of Opioid Use Disorder (OUD) is the elevated relapse rates in affected patients, and a significant contributor to these events of relapse is exposure to cues and contexts that are intensely associated with prior drug abuse. The neurotransmitter dopamine plays a key role in reward-related behaviors, and previous studies have illustrated that dopamine hypofunction in periods of abstinence serves to prompt drug craving and seeking. We hypothesized that restoration of dopaminergic signaling could attenuate drug-seeking behaviors. Therefore, we investigated whether use of an FDA-approved drug, bupropion, an inhibitor of the dopamine transporter (DAT), or a dopamine uptake inhibitor with high affinity for DAT, JHW 007, was able to decrease preference for a drug-paired context. In these experiments, mice underwent 5 days of non-contingent morphine (10 mg/kg) exposure in a conditioned place preference (CPP) paradigm. We found that systemic injection of bupropion (20 mg/kg, i. p.) or intracranial injection of JHW 007 into the nucleus accumbens shell did not prevent the expression of morphine CPP. We then investigated whether chronic bupropion treatment (via implanted osmotic pumps) would influence morphine CPP. We observed that chronic bupropion treatment for 21 days following morphine conditioning did not attenuate the prolonged preference for morphine-paired contexts. Overall, with our dose and paradigm, neither acute nor chronic bupropion diminishes morphine CPP. Continued studies should address FDA-approved medications and their potential for recovery in OUD patients.

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.

TRODAT SPECT in patients with idiopathic REM sleep behaviour disorder.

Objective to determine the value of dopamine transporter (DAT) neuroimaging radiotracer in a group of idiopathic rapid eye movement sleep behavior disorder (iRBD) patients regarding the development of a synucleinopathy. Methods: 6 retrospectively selected patients with clinical and polysomnographic diagnosis of iRBD, on treatment or not, were submitted to a single-photon emission computerized tomography (SPECT) with TRODAT. Results: Five from six patients have an abnormal test showing reduction in DAT density measured by the linkage potential on SPECT. Conclusions: TRODAT crosses the blood brain barrier, has a high affinity for DAT and is capture by SPECT. The decreased uptake of DAT tracers means a reduction in dopaminergic activity which suggest the possibility of Parkinson Disease. We have tried to reinforce iRBD as a marker of neurodegenerative disease and suggest SPECT with TRODAT as an easy method in our country to follow longitudinally these patients.

The in vivo pharmacodynamic and biopharmaceutical characterization of a novel dual mechanism, rapid‐acting antidepressant

Rational design of lead compounds targeting monoamine transporters is critical to developing novel therapeutics for treating psychiatric and substance abuse disorders. A 3‐D dopamine transporter (DAT) computer model was used to virtually screen a small molecule library for high DAT affinity drug‐like compounds. One hit, coded “MI‐4,” inhibited human dopamine, norepinephrine, and serotonin transporters in vitro. In vivo administration in mice (i.p.) induced robust, dose‐dependent antidepressant‐like effects in behavioral models of mood responsive to acute administration (tail suspension and forced swim tests) and chronic administration (novelty‐induced hypophagia test, chronic social‐defeat stress). Importantly, MI‐4 exhibited minimal abuse liability in behavioral and neurological models (conditioned place preference and dopamine in vivo microdialysis). To facilitate pharmacokinetic analysis, a rapid and highly selective reversed‐phase HPLC method for of MI‐4 was developed and validated according to ICH guidelines. Analysis in human, rat and mouse plasma resulted in a distinct single peak of MI‐4 with USP tailing factor < 2.0 using the MI‐4 analog ifenprodil as an internal standard. The stability of MI‐4 incubated in simulated fasted and fed‐state gastric fluid was 98–100% after 3 hrs and 91–99% after 6 hrs. The Papp value of MI‐4 (0.5 mM) across caco‐2 monolayers after one hour was 33.2×10−6 cm/s, indicating high membrane permeability. Moreover, oral administration (p.o.) of MI‐4 in mice resulted in dose‐ and time‐dependent reductions in tail suspension test immobility scores comparable to the established antidepressants fluoxetine and desipramine, indicating oral efficacy for antidepressant‐like effects. MI‐4 was found to be Ro‐25‐6981, an ifenprodil analog and reputed NMDA antagonist. These data suggest that Ro‐25‐6981, previously known for rapid‐acting antidepressant activity, may also functionally inhibit monoamine reuptake and produce sustained antidepressant effects in vivo. This demonstrates, as proof of principle, the viability of combining glutamatergic and monoaminergic mechanisms to produce rapid and sustained antidepressant‐like effects.

Novel and High Affinity 2-[(Diphenylmethyl)sulfinyl]acetamide (Modafinil) Analogues as Atypical Dopamine Transporter Inhibitors.

The development of pharmacotherapeutic treatments of psychostimulant abuse has remained a challenge, despite significant efforts made toward relevant mechanistic targets, such as the dopamine transporter (DAT). The atypical DAT inhibitors have received attention due to their promising pharmacological profiles in animal models of cocaine and methamphetamine abuse. Herein, we report a series of modafinil analogues that have an atypical DAT inhibitor profile. We extended SAR by chemically manipulating the oxidation states of the sulfoxide and the amide functional groups, halogenating the phenyl rings, and/or functionalizing the terminal nitrogen with substituted piperazines, resulting in several novel leads such as 11b, which demonstrated high DAT affinity (Ki = 2.5 nM) and selectivity without producing concomitant locomotor stimulation in mice, as compared to cocaine. These results are consistent with an atypical DAT inhibitor profile and suggest that 11b may be a potential lead for development as a psychostimulant abuse medication.

Rapid and sustained antidepressant properties of an NMDA antagonist/monoamine reuptake inhibitor identified via transporter-based virtual screening

Rational design of lead compounds targeting monoamine transporters (MATs) is critical to developing novel therapeutics to treat psychiatric disorders including depression and substance abuse. A 3-D dopamine transporter (DAT) computer model was used to virtually screen a commercially available small molecule library for high DAT affinity drug-like compounds. One hit, coded “MI-4”, inhibited human dopamine, norepinephrine, and serotonin transporters in vitro. In vivo administration in mice induced robust, dose-dependent antidepressant-like behaviors in learned helplessness models (tail suspension and forced swim tests). Moreover, chronic administration (21day, 10mg/kg, bid) reduced drinking latencies comparable to fluoxetine (10mg/kg, bid) in the novelty-induced hypophagia test, which requires chronic treatment to produce antidepressant-like effects. MI-4 (10mg/kg, bid) produced rapid (three-day) antidepressant-like effects in the social avoidance test following 10days of social defeat stress. Unlike ketamine, chronic administration of MI-4 increased social interaction scores while improving resiliency to the mood-altering effects of stress to over 70%. Importantly, MI-4 exhibited minimal abuse liability in behavioral and neurological models (conditioned place preference and dopamine in vivo microdialysis). MI-4 was found to be Ro-25-6981, an ifenprodil analog and reputed NMDA antagonist. The data suggest that Ro-25-6981, previously known for rapid-acting glutamatergic antidepressant actions, may also functionally inhibit monoamine reuptake and produces sustained antidepressant effects in vivo. This demonstrates, as proof of principle, the viability of combining these mechanisms to produce rapid and sustained antidepressant-like effects. Overall, these findings suggest MAT computational model-based virtual screening is a viable method for identifying antidepressant lead compounds of unique scaffold.

Relationship between rate of drug uptake in brain and behavioral pharmacology of monoamine transporter inhibitors in rhesus monkeys

Although inhibition of dopamine transporters (DAT) and the subsequent increase in dopamine clearly play a role in the effects of psychomotor stimulants, the reinforcing effectiveness of DAT inhibitors varies. Previous studies suggest that pharmacokinetic and pharmacodynamic properties of these drugs account for this variability. The present studies compared the time course and behavioral effects of five phenyltropane analogs of cocaine with high affinity for DAT and varying time courses of action in rhesus monkeys. The rate of drug uptake in putamen was measured using positron emission tomography neuroimaging. The rank order of the time to peak drug uptake was cocaine < RTI-336 < RTI-150 < RTI-113 < RTI-177. Cocaine and all five analogs fully substituted for the cocaine cue in animals trained to discriminate cocaine from saline. All of the drugs were self-administered under a progressive-ratio schedule of drug self-administration and reinstated previously extinguished self-administration maintained under a second-order schedule. The time to peak drug uptake corresponded closely with the time to peak discriminative stimulus effects, and there was a trend for the time of peak drug uptake to correspond negatively with the peak number of drug infusions. Collectively, these results indicate that the rate of drug entry in brain can play an important role in the behavioral pharmacology of psychomotor stimulants.

Dopamine Transporter (DAT) Inhibitors Alleviate Specific Parkinsonian Deficits in Monkeys: Association with DAT Occupancy in Vivo

Viable dopamine neurons in Parkinson's disease express the dopamine transporter (DAT) and release dopamine (DA). We postulated that potent DAT inhibitors, with low affinity for the serotonin transporter (SERT), may elevate endogenously released extracellular dopamine levels to provide therapeutic benefit. The therapeutic potential of eight DAT inhibitors was investigated in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated cynomolgus monkeys (Macaca fascicularis), with efficacy correlated with DAT occupancy as determined by positron emission tomography imaging in striatum. Four potent DAT inhibitors, with relatively high norepinephrine transporter, but low SERT affinities, that occupied the DAT improved activity in parkinsonian monkeys, whereas three high-affinity DAT inhibitors with low DAT occupancy did not. 2beta-Carbomethoxy-3alpha-(3,4-dichlorophenyl)-7beta-hydroxy-8-methyl-8-azabicyclo[3.2.1.]octane (O-1163) occupied the DAT but had short-lived pharmacological effects. The benztropine analog difluoropine increased general activity, improved posture, reduced body freeze, and produced sleep disturbances at high doses. (1R)-2beta-(1-Propanoyl)-3alpha-(4-fluorophenyl)tropane (O-1369) alleviated parkinsonian signs in advanced parkinsonian monkeys, by increasing general activity, improving posture, reducing body freeze, and sedation, but not significantly reducing bradykinesia or increasing locomotor activity. In comparison with the D(2)-D(3) DA receptor agonist quinelorane, O-1369 elicited oral/facial dyskinesias, whereas quinelorane did not improve posture or reduce balance and promoted stereotypy. In conclusion, DAT inhibitors with therapeutic potential combine high DAT affinity in vitro and high DAT occupancy of brain striatum in vivo with enduring day-time effects that do not extend into the nighttime. Advanced parkinsonian monkeys (80% DAT loss) respond more effectively to DAT inhibitors than mild parkinsonian monkeys (46% DAT loss). The therapeutic potential of dopamine transport inhibitors for Parkinson's disease warrants preclinical investigation.

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.

Structure-activity relationships at monoamine transporters for a series of N-substituted 3alpha-(bis[4-fluorophenyl]methoxy)tropanes: comparative molecular field analysis, synthesis, and pharmacological evaluation.

The development of structure-activity relationships (SAR) with divergent classes of monoamine transporter ligands and comparison of their effects in animal models of cocaine abuse have provided insight into the complex relationship among structure, binding profiles, and behavioral activity. Many 3alpha-(diphenylmethoxy)tropane (benztropine) analogues are potent dopamine uptake inhibitors but exhibit behavioral profiles that differ from those of cocaine and other compounds in this class. One of the most potent and dopamine transporter (DAT) selective N-substituted benztropine analogues (N-(4-phenyl-n-butyl)-3alpha-(bis[4-fluorophenyl]methoxy)tropane, 1c) is devoid of cocaine-like behaviors in rodent models but is also highly lipophilic (cLogD = 5.01), which compromises its water solubility and may adversely affect its pharmacokinetic properties. To further explore the SAR in this series and ultimately to design dopamine uptake inhibitors with favorable lipophilicities for drug development, a comparative molecular field analysis (CoMFA) was performed on a set of benztropine analogues previously synthesized in our laboratory. The CoMFA field analysis on the statistically significant (r2(cv) = 0.632; r2(ncv) = 0.917) models provided valuable insight into the structural features required for optimal binding to the DAT, which was used to design a series of novel benztropine analogues with heteroatom substitutions at the tropane N-8. These compounds were evaluated for binding at DAT, serotonin (SERT) and norepinephrine (NET) transporters, and muscarinic M1 receptors in rat brain. Inhibition of [3H]DA uptake in synaptosomes was also evaluated. Most of the analogues showed high DAT affinity (12-50 nM), selectivity (10- to 120-fold), potent inhibition of dopamine uptake, and lower lipophilicities as predicted by cLogD values.

Review of the pharmacological and clinical profile of rimcazole.

Rimcazole is a carbazole derivative that acts in part as a sigma receptor antagonist. Wellcome Research Laboratories introduced this compound during the 1980s when it was hypothesized to be a novel antipsychotic with an improved side effect profile. However, subsequent clinical trials demonstrated that rimcazole lacked efficacy in schizophrenic patients and it is now primarily used as an experimental tool. In addition to its actions as a sigma receptor antagonist, rimcazole also has high affinity for dopamine transporters, and in recent years it has served as a lead compound for the development of novel dopamine transporter ligands. Although rimcazole cannot be considered a selective ligand for sigma receptors, the recent development of other selective agonists and antagonists for sigma receptors have aided in clarifying the involvement of these receptors in the actions of rimcazole. Many of the physiological and behavioral effects of rimcazole can in fact be ascribed to its action as a sigma receptor antagonist, although there are exceptions. Rimcazole is likely to have a continued role in elucidating sigma receptor function in either in vitro or in vivo systems where sigma receptor-mediated effects can be studied independently of the influence of dopamine and serotonin transporters.

Cocaine and other indirect-acting monoamine agonists differentially attenuate a naltrexone discriminative stimulus in morphine-treated rhesus monkeys.

Monoaminergic drugs can modify opioid withdrawal in nonhumans, and cocaine is reported to attenuate opioid withdrawal in humans. Drug discrimination was used to examine whether s.c. cocaine or other indirect-acting monoamine agonists attenuate morphine (3.2 mg/kg/day) withdrawal induced by naltrexone and by 27 h of morphine deprivation. Naltrexone-precipitated withdrawal was attenuated not only by morphine but also by cocaine, amphetamine, and imipramine. However, reversal of naltrexone-precipitated withdrawal was greater for morphine than for any of the indirect-acting monoamine agonists. Attenuation of the naltrexone discriminative stimulus by indirect-acting monoamine agonists was pharmacologically selective insofar as drugs lacking affinity for monoamine transporters (ketamine and triazolam) were without effect. Twenty-seven hours of morphine deprivation occasioned naltrexone-lever responding and decreased response rate, and both effects were reversed by morphine, cocaine, and amphetamine and not by imipramine, desipramine, ketamine, and triazolam. Thus, indirect-acting monoamine agonists attenuate some (e.g., discriminative) aspects of naltrexone-precipitated withdrawal, whereas only indirect-acting agonists with high affinity for dopamine transporters attenuate deprivation-induced withdrawal. These results suggest that dopamine is differentially involved in naltrexone- and deprivation-induced withdrawal and support the notion that opioid-dependent individuals use stimulants, in part, to attenuate withdrawal.

1-[1-(2-Benzo[ b]thiopheneyl)cyclohexyl]piperidine hydrochloride (BTCP) yields two active primary metabolites in vivo : Identification and quantification of BTCP primary metabolites in mice plasma, urine, and brain and their affinity for the neuronal dopamine transporter

1-[1-(2-Benzo[ b]thiopheneyl)cyclohexyl]piperidine hydrochloride (BTCP) and cocaine bind to the neuronal dopamine transporter (DAT) to strongly inhibit dopamine (DA) reuptake. Although similar to acute administration, cocaine and BTCP produce sensitization and tolerance, respectively, on chronic administration. We previously found that liver microsomes produced two primary metabolites from BTCP with a high affinity for DAT. Because such metabolites, if produced in vivo, could account for the pharmacological difference with cocaine, it was important to compare BTCP biotransformations in vitro and in vivo. Therefore, we identified and quantified BTCP and primary metabolites in mice urine, plasma, and brain after acute i.p. administration. The low recovery yield suggest that BTCP might behave like its close analogue, phencyclidine, with long-term storage of metabolites. Two active metabolites found in vitro were found in mice brain with estimated half-life values similar to that of BTCP (∼0.3 h). Although respective brain concentrations were 20 and 40 times lower than that of BTCP, their potency to displace in vivo [ 3H]BTCP bound to the DAT was 50 and 10 times higher, respectively, than that of BTCP. They could, therefore, contribute to the inhibition of DA transport and play an important role in BTCP pharmacology. They could also explain the differences between BTCP and cocaine on repeated administration.

3H]β-CIT: a radioligand for dopamine transporters in rat brain tissue

[ 3H]2-β-carbomethoxy-3-β-[4′-iodophenyl]tropane (β-CIT) was prepared and evaluated. With rat forebrain tissue, [ 3H]β-CIT showed high affinity for dopamine transporters (DAT), with selectivity for DAT over norepinephrine transporters, but not serotonin transporters, as well as DAT-stereoselectivity with β-CIT, amphetamine and methylphenidate. Affinity and selectivity for 53 compounds assayed with [ 3H]β-CIT and standard DAT radioligand [ 3H]GBR-12935 were highly correlated ( r>0.95). [ 3H]β-CIT is proposed as a useful, high-affinity DAT radioprobe.