Structural basis for action by diverse antidepressants on biogenic amine transporters.

The biogenic amine transporters (BATs) regulate endogenous neurotransmitter concentrations and are targets for a broad range of therapeutic agents including selective serotonin reuptake inhibitors (SSRIs), serotonin-noradrenaline reuptake inhibitors (SNRIs) and tricyclic antidepressants (TCAs). Because eukaryotic BATs are recalcitrant to crystallographic analysis, our understanding of the mechanism of these inhibitors and antidepressants is limited. LeuT is a bacterial homologue of BATs and has proven to be a valuable paradigm for understanding relationships between their structure and function. However, because only approximately 25% of the amino acid sequence of LeuT is in common with that of BATs, and as LeuT is a promiscuous amino acid transporter, it does not recapitulate the pharmacological properties of BATs. Indeed, SSRIs and TCAs bind in the extracellular vestibule of LeuT and act as non-competitive inhibitors of transport. By contrast, multiple studies demonstrate that both TCAs and SSRIs are competitive inhibitors for eukaryotic BATs and bind to the primary binding pocket. Here we engineered LeuT to harbour human BAT-like pharmacology by mutating key residues around the primary binding pocket. The final LeuBAT mutant binds the SSRI sertraline with a binding constant of 18 nM and displays high-affinity binding to a range of SSRIs, SNRIs and a TCA. We determined 12 crystal structures of LeuBAT in complex with four classes of antidepressants. The chemically diverse inhibitors have a remarkably similar mode of binding in which they straddle transmembrane helix (TM) 3, wedge between TM3/TM8 and TM1/TM6, and lock the transporter in a sodium- and chloride-bound outward-facing open conformation. Together, these studies define common and simple principles for the action of SSRIs, SNRIs and TCAs on BATs.

Effects of acute administration of mazindol on brain energy metabolism in adult mice.

OBJECTIVES: Mazindol is a sympathomimetic amine, widely used as an anorectic agent in the treatment of obesity. This drug causes psychostimulant effects because of its pharmacological profile similar to amphetamine, acting like a monoamine reuptake inhibitor. However, the mechanisms underlying the action of mazindol are still not clearly understood. METHODS: Swiss mice received a single acute administration of mazindol (0.25, 1.25 and 2.5 mg/kg, ip) or saline. After 2 h, the animals were killed by decapitation; the brain was removed and used for the evaluation of activities of mitochondrial respiratory chain complexes, Krebs cycle enzymes and creatine kinase. RESULTS: Acute administration of mazindol decreased complex I activity only in the hippocampus. Complex IV activity was increased in the cerebellum (2.5 mg/kg) and cerebral cortex (0.25 mg/kg). Citrate synthase activity was increased in the cerebellum (1.25 mg/kg) and cerebral cortex (1.25 mg/kg), and creatine kinase activity was increased in the cerebellum (1.25 mg/kg). CONCLUSION: We suggest that the inhibition of complex I in the hippocampus only and activation of complex IV, citrate synthase and creatine kinase occurs because of a stimulus effect of mazindol in the central nervous system, which causes a direct impairment on energy metabolism.

Efficacy and safety of the metformin-mazindol anorectic combination in rat.

The current study investigates the anorectic interaction and safety of the mazindol-metformin combination in rats. Isobologram and interaction index were used to determine anorectic interaction between mazindol and metformin in the sweetened milk model. The safety profile of the mazindol-metformin combination was determined by measuring anxiety, blood pressure, hematic biometry and blood chemistry. An acute dose of mazindol and metformin administered per os, individually or as a mixture, has reduced the milk consumption in rats in a dose-dependent manner. Theoretical effective dose 40 (ED40t) did not differ from the experimental effective dose 40 (ED40e) obtained with the mazindol-metformin mixture in the anorexia experiments, by Student's t-test. In addition, the interaction index confirmed the additive anorectic effect between both drugs. A single oral dose of ED40e mazindol-metformin mixture induced anxiolysis in the elevated plus-maze test. Moreover, oral administration of mazindol-metformin combination for 3 months significantly decreased glycemia, but not blood pressure nor other parameters of hematic biometry and blood chemistry. Results suggest that mazindol-metformin combination exerts an additive anorectic effect, as well as anxiolytic and hypoglycemic properties. Mazindol-metformin combination might be useful in obese patients with anxiety disorders or diabetes risk factors.

Apomorphine offers new insight into dopaminergic neuron vulnerability in mesencephalic cultures.

The mechanism by which the dopamine neurons of the substantia nigra pars compacta degenerate in Parkinson's disease, is partly unknown. Dopamine could be implicated in this phenomenon, and in order to explain its toxicity several hypotheses have been suggested. The similarity between apomorphine and dopamine as regards their chemical, pharmacological and toxicological properties provided a basis for investigating the nature of the toxicity of the former agent. In this study we describe some effects of apomorphine on mouse mesencephalic cell cultures at relatively low concentrations (from 0.5 to 2.5microM), apomorphine produced a neurotrophic effect, consisting of a 60% increase in dopaminergic neuron survival as measured by [(3)H] dopamine uptake. At high concentrations (over 20microM), however, apomorphine induced an increasing cytotoxic effect, as measured by the marked decrease in [(3)H] dopamine uptake, and by the direct observation of the dopaminergic neurons after TH immunostaining. This study may offer a new strategy for investigating the mechanisms underlying DA neuron vulnerability.

Systemic mazindol reduces food intake in rats via suppression of meal size and meal number.

The aim of the present study was to determine the impact of the appetite suppressant mazindol on meal pattern in rats. Meal patterns were monitored in adult male rats after mazindol dosing during the first three hours of the dark cycle using automated feeding chambers (BioDAQ). Mazindol (0, 0.25, 1.25 and 2.5 mg/kg, IP) produced a dose-dependent hypophagia and hypodipsia. Meal size and meal number were significantly suppressed by mazindol. The meal pattern findings indicate that mazindol inhibits eating in the rat via a suppression of both meal size and meal number.

Increased thirst and plasma arginine vasopressin levels during 2-deoxy-D-glucose-induced glucoprivation in humans.

Insulin-induced hypoglycemia by unknown mechanism(s) increases plasma arginine vasopressin (AVP) levels in humans. Mechanisms for increased AVP levels during central nervous system glucoprivation were investigated by administering 20-min i.v. infusions of 2-deoxy-d-glucose (50 mg/kg), a competitive inhibitor of glucose utilization, or normal saline (sham), to 24 normal volunteers. Some of the infusions were administered in combination with neuropharmacological blocking agents (placebo). The behavioral, physiological, metabolic, and hormonal correlates of 2-deoxy-d-glucose (2DG)-induced gluco-privation and AVP secretion were studied in a group (n = 5) pretreated for 1 wk with either mazindol (1 mg per os three times per day), a potent norepinephrine and dopamine-reuptake blocker, or placebo. A second group (n = 5) received either propranolol (3 mg/3 min followed by 80 mug/min) or normal saline infusion before and during 2DG administration. With 2DG alone, plasma AVP levels increased from 1.3+/-0.3 pg/ml at base line to a peak of 4.5+/-1.4 pg/ml at 60 min and remained elevated for 150 min. From 30 to 180 min after 2DG administration, the 2DG-infused volunteers increased their water intake in comparison with sham-infused volunteers. Marked increases in epinephrine and slight increases in norepinephrine were associated with increases in plasma glucose and renin activity and decreases in plasma potassium. Plasma sodium and osmolality increased transiently and mean arterial pressure (MAP) fell. These changes, however, were small and inconstant and could not account for the observed increases in thirst and AVP levels. Pretreatment with mazindol prevented the decrease in MAP and the increase in plasma renin activity (PRA) following 2DG infusions without modifying increased thirst, water intake, or AVP responses to glucoprivation. Pretreatment with propranolol effectively blocked beta-adrenoreceptors as evidenced by increased MAP and plasma epinephrine, and abolition of the RPA increases during 2DG-induced glycoprivation, but did not suppress AVP and thirst responses. A cervical cord-sectioned patient lacking descending sympathetic out-flow had a potentiated thirst response to 2DG-induced glucoprivation in the absence of increases in sodium, catecholamines, and PRA. Thus 2DG administration activates mechanisms for increased thirst and AVP which are unrelated to changes in peripheral catecholamines, MAP, PRA, and osmolality.

Fluctuation of the dopamine uptake inhibition potency of cocaine, but not amphetamine, at mammalian cells expressing the dopamine transporter.

Cocaine, amphetamines and other psychostimulants inhibit synaptic dopamine uptake by interfering with dopamine transporter (DAT) function. The resultant potentiation of dopaminergic neurotransmission is associated with psychostimulant addiction. Fluctuations in dopamine uptake inhibition potency (DUIP) were observed for classical DAT blockers including cocaine, mazindol, methylphenidate (Ritalintrade mark) and benztropine in CHO cells expressing wild type DAT; cocaine potency also decreased in DAT-expressing non-neuronal COS-7 cells and neuronal N2A neuroblastoma cells. In contrast, the DAT substrate (+)-amphetamine did not display this DUIP fluctuation. In parallel experiments, no fluctuation was observed for the apparent binding affinities of these 5 drugs. The DUIP decrease appeared to correlate with an increase in cell surface DAT expression level, as measured by B(max) values and confocal microscopy. The fact that the DUIP profile of amphetamine diverged from that of the classical DAT blockers is consistent with the idea of fundamental differences between the mechanisms of abused psychostimulant DAT substrates and inhibitors. Identification of the cellular factors that underlie the DAT inhibitor DUIP fluctuation phenomenon may be relevant to anti-psychostimulant drug discovery efforts.

Measurement of psychological state changes at low dopamine transporter occupancy following a clinical dose of mazindol.

RATIONALE: The beneficial effects of psychostimulant drugs in the treatment of psychiatric disorders occur because they increase the extracellular dopamine concentration by inhibiting re-uptake of extracellular dopamine at dopamine transporters. However, the psychological effects at low dopamine transporter occupancy have not been well demonstrated. OBJECTIVES: The purpose of the study was to evaluate the psychological effects, dopamine transporter occupancy, and dopamine release induced by a single oral administration of a clinical dose of mazindol. METHODS: Ten healthy male volunteers were orally administered a placebo and a clinical dose of mazindol (1.5 mg) on separate days. The psychological effects of mazindol were assessed using a visual analogue scale to detect alterations in the state of consciousness. The amount of blockade of dopamine transporters was assessed using positron emission tomography with [18F]FE-PE2I and extracellular dopamine release was measured as the amount of change in [11C]raclopride binding. RESULTS: Following administration of a clinical dose of mazindol, the dopamine transporters were blocked by 24-25 %, and the binding potential of [11C]raclopride was reduced by 2.8-4.6 %. The differences of a score measuring derealisation and depersonalization associated with a positive basic mood were significantly correlated with the change in the [11C]raclopride binding in the limbic striatum. CONCLUSIONS: A subtle alteration in the state of consciousness was detected with a correlation to the changes in the [11C]raclopride binding, which implies that a subtle alteration in extracellular dopamine concentration in the limbic striatum by a small amount of dopamine transporter occupancy can affect the state of consciousness. TRIAL REGISTRATION HTTPS://UPLOAD.UMIN.AC.JP/CGI-OPEN-BIN/CTR_E/CTR_VIEW.CGI?RECPTNO=R000009703 : UMIN000008232.

Reevaluation of anti-obesity action of mazindol and elucidation of its effect on the reward system.

In this study, we evaluated the preventive effect of mazindol on the development of obesity and sought to elucidate the drug's effects on the reward system. In mice, body weight gain and hyperphagia induced by high-fat diet (HFD) were decreased by 38.6% and 13.9%, respectively, by subcutaneous infusion of mazindol (1.5mg/kg/day) for 28days. A single intraperitoneal administration of mazindol (1.5mg/kg) significantly reduced lipid preference, as assessed using the two-bottle preference paradigm (vehicle, 89.98±1.66%; mazindol, 75.65±5.47%; p<0.05). In addition, the conditioned place preference (CPP) test demonstrated that mazindol (1.5mg/kg) significantly decreased CPP score for HFD as compared with vehicle (vehicle, 330.44±58.61s; mazindol, 144.72±43.02s; p<0.05). Moreover, at the dose required for these effects, mazindol did not elicit abuse potential or induce psychostimulant-like behavior. These results confirm that mazindol prevents diet-induced obesity without addictive behavior and demonstrate that its action is mediated at least in part via the reward system, advancing our understanding of mazindol in clinical practice.

Clinical and biochemical effects of catecholamine depletion on antidepressant-induced remission of depression.

BACKGROUND: Most hypotheses of the therapeutic mechanism of action of antidepressant drugs have focused on the role of the monoamines. We examined the effect of catecholamine depletion on antidepressant-induced remission. METHOD: The tyrosine hydroxylase inhibitor alpha-methylparatyrosine and the antihistamine diphenhydramine hydrochloride were administered, during separate test sessions, to depressed patients in remission maintained with either norepinephrine reuptake inhibitors (desipramine [n = 7] or mazindol [n = 2]) or serotonin reuptake inhibitors (fluoxetine hydrochloride [n = 9] or sertraline hydrochloride [n = 1]). Because of considerable sedation associated with alpha-methylparatyrosine testing, diphenhydramine was used as an active control rather than an inactive placebo. The effects of alpha-methylparatyrosine and diphenhydramine on depression, anxiety, and plasma catecholamine metabolites were assessed. RESULTS: alpha-Methylparatyrosine produced similar significant decreases in plasma 3-methoxy-4-hydroxyphenylethyleneglycol and homovanillic acid levels in the treatment groups. alpha-Methylparatyrosine produced a robust increase in depressive symptoms on the Hamilton Depression Rating Scale, including depressed mood, decreased concentration, anhedonia, loss of interest, and feelings of worthlessness, helplessness, and hopelessness, in the desipramine-mazindol but not in the fluoxetine-sertraline group. Diphenhydramine had no effects on mood in either treatment group. CONCLUSIONS: The therapeutic effects of norepinephrine reuptake inhibitors, but not serotonin reuptake inhibitors, are reversed by catecholamine depletion. Considered with previous reports that serotonin depletion produces depressive relapses in patients in remission maintained with serotonin reuptake inhibitors, but not norepinephrine reuptake inhibitors, these findings suggest that antidepressants may not work via a single monoamine-related mechanism.

Dopamine transporters participate in the physiological regulation of prolactin.

Three populations of hypothalamic neuroendocrine dopaminergic (NEDA) neurons, arising from the arcuate and periventricular nuclei of the hypothalamus release dopamine (DA) that acts at the pituitary gland to regulate the secretion of PRL. It is generally accepted that NEDA neurons lack functional DA transporters (DATs), which are responsible for uptake of DA from the synaptic cleft into the presynaptic axon terminal. This study localized DATs to the hypothalamo-pituitary axis and evaluated the effect of DAT blockade on the hypothalamo-pituitary regulation of PRL. After 7 days of treatment with cocaine (a nonspecific amine transporter blocker) or mazindol (a specific DAT blocker), the relative abundance of PRL messenger RNA (mRNA) in the anterior lobe (AL) of OVX rats was significantly decreased, whereas the relative abundance of tyrosine hydroxylase mRNA in the hypothalamus was significantly increased. The effect of cocaine or mazindol administration on DA turnover and serum PRL concentration was examined in estradiol (E2)-treated OVX rats. E2 administration (i.v.) resulted in a significant increase in serum PRL within 4 h; however, cocaine or mazindol administration abolished the E2-induced increase of PRL. Cocaine or mazindol significantly increased the concentration of DA at the site of the axon terminals within the median eminence (ME), intermediate lobe (IL) and neural lobe (NL), indicating blockade of uptake. Because formation of DOPAC requires uptake of DA, concentrations of DOPAC in the ME, IL and NL decreased following treatment with either cocaine or mazindol. These data, together with the presence of immunopositive DAT in the ME, pituitary stalk, IL, and NL, suggest that a functional DAT system is present within all three populations of NEDA neurons. Moreover, similarity between the effects of cocaine and mazindol treatment indicate that blockade of the DAT, but not other amine transporters, is responsible for suppression of PRL gene expression and secretion. Blockade of DATs prevent uptake of DA into NEDA neurons and consequently increases the amount of DA that diffuses into the portal vasculature and reaches the AL. These data provide evidence that DATs play a physiological role in the regulation of DA release from and TH expression in NEDA neurons and consequently PRL secretion and PRL gene expression and further support our previous observation that the regulation of PRL secretion involves all three populations of NEDA neurons.

The contribution of increased thermogenesis to the effect of anorectic drugs on body composition in mice.

The study investigated whether changes in body composition of normal and genetically obese C57BL/6J (ob/ob) mice caused by the anorectic drugs phentermine, diethylpropion, fenfluramine, and mazindol are entirely due to reduced food intake. Mice were dosed daily (25 mg/kg po) for 28 days after which time carcass composition was determined. Compared to controls fed at libitum, reductions in food intake were for phentermine, 7%; fenfluramine, 17%; diethylpropion, 17%, whereas reduction in body lipid content were for phentermine, 16%; mazindol, 18%; fenfluramine, 8%; diethylpropion, 10%. Since diet restriction by 22% (in the absence of treatment with any drug) resulted in a body lipid content 12% below that of controls fed ad libitum, these results suggest that some of the lipid loss caused by phentermine and possibly mazindol is due to increased energy expenditure. In support of this conclusion, phentermine and mazindol increased energy expenditure in normal mice by 35% compared to untreated controls in the 6 h after dosing but diethylpropion and fenfluramine had little or no effect. Determination of the carcass composition of the normal mice confirmed that phentermine has a metabolic antiobesity effect. Fenfluramine had an unexpected effect on carcass composition in normal mice.

Clinical and basic aspects of an anorexiant, mazindol, as an antiobesity agent in Japan.

The Japanese Mazindol study group investigated the action of an anorexiant, mazindol, and found that it reduced food intake by directly suppressing neurons in the lateral hypothalamus, inhibited gastric acid secretion, increased motor activity, decreased glucose absorption, and inhibited insulin secretion. It thus appears that the main effect of mazindol is to decrease food intake through suppressing feeding centers in the hypothalamus. A multicenter open study of mazindol in Japan revealed that loss of body weight and relative body weight in 14 wk were 4.6 kg and 9.2%, respectively, with suppression of appetite in the majority of obese patients. A multicenter double-blind study demonstrated that mazindol was superior to the placebo in the treatment of simple obesity. We also suggest that mazindol is effective in the maintenance of reduced body weight after obesity therapy and in the treatment of obesity-related diseases such as diabetes, hypertension, or hyperlipidemia.

Cerebrospinal fluid of Parkinson's disease patients inhibits the growth and function of dopaminergic neurons in culture.

We report the possible existence of an inhibitory factor in the CSF of Parkinson's disease patients that inhibits the function and growth of dopaminergic neurons in rat mesencephalic culture. After 40 hours' exposure to the < 10 kd fraction of CSF from PD patients, the high-affinity dopamine uptake was 66% of that of cultures exposed to CSF from controls. However, the number of dopaminergic neurons remained unchanged at this time. After 90 hours' exposure to the < 10 kd fraction of CSF from PD patients, the number of dopaminergic neurons decreased to 10% of that in cultures exposed to CSF from controls, and the size of the remaining dopaminergic neurons in the culture became smaller. This inhibitory factor did not affect the growth of other types of neurons. The chemical nature of this inhibitory factor is under investigation.

Serotonergic and catecholaminergic reuptake inhibitors have opposite effects on the ultrasonic isolation calls of rat pups.

Rat pups emit a highly stereotyped and well-characterized distress call in the ultrasonic range when socially isolated. We compared the modulatory influence of catecholamines and indoleamines on rat pup ultrasonic calls using pharmacologic probes. Administration of low doses of monoamine reuptake inhibitors produced significant, selective changes in the calls emitted by isolated 10-day-old pups. Acute administration of clomipramine (CMI; relatively 5-HT specific) reduced the rate of calling at low doses (1.0 and 5.0 mg/kg [3.2 and 18.0 mmol/kg] SC) but had reduced efficacy at higher doses (10 and 20 mg/kg [32.0 and 63.7 mmol/kg] SC). Motor activity and rectal body temperature were unaffected at these doses. Similarly, low doses of other 5-HT-selective uptake inhibitors, such as paroxetine (1.0 mg/kg [3.1 mmol/kg] SC) and citalopram (1.0 mg/kg [3.09 mmol/kg] SC), virtually eliminated isolation calling. The effects of CMI were not antagonized by either naltrexone (0.1 mg/kg [0.28 mmol/kg] SC) or Ro 15-1788 (5.0 mg/kg [16.5 mmol/kg] SC). Desipramine (DMI; norepinephrine [NE] specific) significantly increased calling rates at all doses tested (1.0 to 10.0 mg/kg [3.8 to 75 mmol/kg] SC). These effects were associated with significant reductions in body temperature, but not motor activity. Similar increases in the rate of isolation calling, reduction in rectal body temperature, and an increase in motor activity were produced by low doses of mazindol (0.5 mg/kg [1.75 mmol/kg] SC) and nortriptyline (1.0 mg/kg [19 mmol/kg] SC). In an additional study, the chronic effects of CMI and desipramine were evaluated with treatment beginning within 24 hours after birth.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of 5-HT(2a) and 5-HT(2B/2C) receptors in the behavioral interactions between serotonin and catecholamine reuptake inhibitors.

Dysfunction of monoamine neurotransmission seems to contribute to such pathopsychological states as depression, schizophrenia, and drug abuse. The present study examined the effects of the selective serotonin (5-hydroxytryptamine; 5-HT) reuptake inhibitor (SSRI) and antidepressant fluvoxamine on locomotor activity in rats following administration of the catecholamine reuptake inhibitor mazindol. Mazindol (1 mg/kg) did not alter locomotor activity; whereas, fluvoxamine (20 mg/kg) given alone induced a brief period of hypomotility. Hyperactivity was elicited in a dose-related manner when fluvoxamine (5-20 mg/kg) was combined with mazindol (1 mg/kg). The hyperactivity elicited by fluvoxamine (20 mg/kg) plus mazindol (1 mg/kg) was significantly attenuated by the 5-HT(2A) receptor antagonist M100907 (2 mg/kg) and potentiated by the 5-HT(2B/2C) receptor antagonist SB 206553 (2 mg/kg). Neither antagonist significantly altered basal activity. The hyperactivity evoked by the combination of fluvoxamine and mazindol seems to be mediated in part by 5-HT(2A) receptors; whereas, 5-HT(2B/2C) receptors may serve to limit this effect. Thus, the balance of activation between 5-HT(2A) and 5-HT(2B/2C) receptors seems to contribute to the expression of locomotor hyperactivity evoked via combination of a 5-HT and a catecholamine reuptake inhibitor. A disruption in this balance may contribute to the expression of affective disorders, schizophrenia, and drug abuse.

The effects of mazindol on local cerebral glucose utilization in rats.

The effects of mazindol (1 mg or 10 mg/animal, p.o.) on local cerebral utilization of glucose were studied by the quantitative autoradiographic [14C]2-deoxyglucose method in conscious adult male rats. Significant increases in local cerebral utilization of glucose were observed 2 hr after administration of 10 mg of mazindol in 10 out of 37 anatomically discrete regions examined. These 10 areas included regions rich in dopaminergic receptors (substantia nigra, globus pallidus), and also regions with few dopaminergic receptors (cerebral cortex, thalamus, cerebellum). Only the habenular nucleus showed a significant decrease in utilization of glucose induced by the administration of 10 mg of mazindol. No significant changes in local cerebral utilization of glucose were observed following the administration of 1 mg of mazindol. The fact that the pattern of utilization of glucose observed in this study resembled that produced by apomorphine, a putative dopaminergic agonist, indicates that the pharmacological effects of mazindol are related to the dopaminergic system.

Differences in the temperature dependence of drug interaction with the noradrenaline and serotonin transporters.

High affinity, sodium-dependent binding of [3H]mazindol is associated with the noradrenergic transporter while the binding of [3H]imipramine and [3H]paroxetine are associated with the serotonin transporter, e.g. in human platelets. In general radioligand binding studies to the monoamine transporters are performed at a temperature different from the physiological temperature (37 degrees C) at which uptake occurs. Previously reported data show a temperature dependence for tricyclic but not for nontricyclic inhibitors of the uptake of serotonin in their interaction with radioligand binding to the serotonin transporter. In the present study, both tricyclic and nontricyclic inhibitors of the uptake of noradrenaline were shown to have equal affinity for the binding of [3H]mazindol to the noradrenergic transporter at 0 degrees and 25 degrees C. Moreover, whereas serotonin transporter substrates inhibit the binding of [3H]imipramine/paroxetine in human platelets in an essentially temperature-independent manner (0-37 degrees C), noradrenaline transporter substrates are of higher affinity in inhibiting the binding of [3H]mazindol at 0 degrees C than at 25 degrees C. At 0 degrees C, substrate affinity for inhibition of the binding of [3H]mazindol to the noradrenaline transporter in the salivary gland of the rat approximates to the substrate affinity for transport by the noradrenaline carrier. The present data point to significant differences in the interaction of both substrates and inhibitors with the noradrenergic and serotonergic transporters, as studied using the radioligand binding. Thus, the pharmacological profile of the serotonin transporter, in terms of substrate and inhibitor affinity, is best studied using radioligand binding at 37 degrees C, whereas radioligand binding to the noradrenaline transporter is most representative at 0 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

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).