The GABA transporter and its inhibitors.

GABA is the major inhibitory neurotransmitter in the brain and GABA re- uptake from the synaptic cleft is one important mechanism in the regulation of GABA activity. Inhibition of the re-uptake of GABA by potent and selective inhibitors of the GABA transporter enhances GABA activity. This property can be used therapeutically in for instance epilepsy or psychiatric disorders. In this paper putative structures of the GABA transporter, its mechanism of action, the progress made in the search for its amino acids involved in the binding of substrates and the SAR of inhibitors acting at the transporter will be discussed. To date only highly selective GAT-1 inhibitors are available. These compounds are lipophilic derivatives of (R)-nipecotic acid and guvacine. The most potent inhibitors of the cloned human GAT-1 are NNC-711 (IC50 = 0.04 mM) and tiagabine (IC50 = 0.07 mM). A diheteroarylvinyloxy analogue of tiagabine, 5 times more potent than tiagabine, has been reported recently. For the GAT-2, GAT-3 and BGT-1 subtypes only compounds with a small preference for one of the subtypes have been published.

Serotonin, dopamine and norepinephrine transporters in the central nervous system and their inhibitors.

An overview is presented on progress made in the research on neuronal transporters of serotonin, dopamine and norepinephrine in the central nervous system. Tools developed by molecular biology, such as expression of cloned transporters, their mutants and chimera in non-neuronal cells offered the opportunity to study the putative domains for binding of substrates and uptake inhibitors and discover factors in the regulation of the transporter function. The study of the distribution of monoamine transporters in human brain became possible by the development of selective radiolabelled transport inhibitors. The relationships between the chemical structure of the uptake inhibitors and the affinity for the monoamine transporters is reported, and the (potential) therapeutic applications of the compounds are discussed.

5-HT(3) receptor antagonists and anxiety; a preclinical and clinical review.

The present paper reviews the evidence for anxiolytic activity of 5-HT(3) receptor antagonists in animal models of anxiety and in clinical trials in humans. Compared to the established anxiolytics (benzodiazepine receptor agonists and, to a lesser extent, 5-HT(1A) receptor agonists) 5-HT(3) receptor antagonists display a different anxiolytic profile. They are anxiolytic in a limited number of animal anxiety models. If active, they often are very potent and display bell-shaped dose response curves, whereas the ratio between therapeutic activity and side effects appears remarkably large. 5-HT(3) receptor antagonists remain active after chronic dosing and no indications for tolerance, dependence or rebound effects were found, which seems to make these drugs an attractive alternative to the benzodiazepines. However, the large body of animal data indicating a complete lack of psychotropic activity of 5-HT(3) receptor antagonists weakens the prediction of anxiolytic activity in these drugs. Human data are also controversial; some investigators have reported positive effects in anxiety disorders (panic disorder, GAD), others did not. It can be concluded that 5-HT(3) receptor antagonists do not represent a breakthrough in the treatment of various anxiety disorders, as initially suggested.

The 5-HT1A receptor and its ligands: structure and function.

An overview is presented on progress made in research on 5-HT1A receptors and their ligands since their discovery in 1983. Molecular biology has offered new tools, for example cloned 5-HT1A receptors, their mutants and chimeras to study structure and function. Many compounds, belonging to different chemical classes, display high affinity and selectivity for 5-HT1A receptors. The majority of these compounds are agonists or partial agonists, full antagonists are still scarce. Agonists and partial agonists are active in various animal models of anxiety and depression. Partial receptor agonists have been proven to be effective in general anxiety disorder and depression in man. Potential therapeutic applications for 5-HT1A receptor antagonists are evaluated, for example, in cognition disorders.

Functional characteristics of a series of N4-substituted 1-(2,3-dihydro-1,4-benzodioxin-5-yl)piperazines as 5-HT1A receptor ligands. Structure-activity relationships.

The agonistic/antagonistic profile of a series of 10 N4-substituted 1-(2,3-dihydro-1,4-benzodioxin-5-yl)piperazines is evaluated in the in vitro adenylyl cyclase assay. The profile is severely affected by the characteristics of the N4-substituents ranging from full agonism (benzamidoethyl derivative 1), mixed agonism/antagonism (phthalimidobutyl derivative 7) to predominantly antagonism (saccharinbutyl derivate 9). A novel full antagonist 10, as potent as WAY 100635, is obtained by substitution of Cl at C-7 of the benzodioxinyl moiety in 9.

Partial agonism of the nonselective adenosine receptor agonist 8-butylaminoadenosine at the A1 receptor in vivo.

The cardiovascular effects of the nonselective adenosine receptor agonist 8-butylaminoadenosine (BAA) were quantified in conscious normotensive rats. The potency and intrinsic activity for the A1 and A2a receptor were determined separately. The rats received a short intravenous infusion of 100 mg/kg BAA in combination with a continuous infusion of either the A1-selective antagonist 8-cyclopentyltheophylline (CPT) (8 micrograms/min/kg), the A2a-selective antagonist 8-(3-chlorostyryl)caffeine (CSC) (32 micrograms/min/kg) or the vehicle. Heart rate (HR) and mean arterial pressure (MAP) were recorded continuously as pharmacodynamic indices for adenosine receptor activation. The MAP/HR ratio was derived as an additional cardiovascular parameter. This ratio reflects changes in total peripheral resistance on the assumption that stroke volume remains constant. During the infusion of CSC, the potency and intrinsic activity for the A1 receptor were estimated by relating the negative chronotropic effect to BAA blood concentrations. The sigmoidal curve yielded a potency value based on unbound concentrations (EC50,u) of 1.9 +/- 0.4 micrograms/ml (mean +/- S.E.; n = 5). The maximal reduction (Emax) in HR (-85 +/- 9 beats/min) was significantly smaller than the values reported for full agonists. During A1 blockade, EC50,u values of 5.5 +/- 0.8 and 6.0 +/- 0.8 micrograms/ml were observed for the A2a receptor-mediated reductions in blood pressure and MAP/HR, respectively (mean +/- SE; n = 5). The Emax values for the hypotensive effect and the reduction in MAP/HR (-55 +/- 3 beats/min and -9.7 +/- 0.6 x 10(-2) mm Hg/ beats/min, respectively) were similar to those of a full A2a receptor agonist. This study shows that BAA is a partial agonist for the adenosine A1 receptor and a full agonist for the A2a receptor.

Pharmacokinetic-haemodynamic relationships of 2-chloroadenosine at adenosine A1 and A2a receptors in vivo.

1. The purpose of the present study was to develop an experimental strategy for the quantification of the cardiovascular effects of non-selective adenosine receptor ligands at the adenosine A1 and A2a receptor in vivo. 2-Chloroadenosine (CADO) was used as a model compound. 2. Three groups of normotensive conscious rats received an short intravenous infusion of 1.4 mg kg-1 CADO during constant infusions of the A1-selective antagonist, 8-cyclopentyltheophylline (CPT; 20 micrograms min-1 kg-1), the A2a-selective antagonist, 8-(3-chlorostyryl) caffeine (CSC; 32 micrograms min-1 kg-1) or the vehicle. The heart rate (HR) and mean arterial blood pressure (MAP) were recorded continuously during the experiment and serial arterial blood samples were taken for analysis of drug concentrations. The ratio MAP/HR was also calculated, which may reflect changes in total peripheral resistance on the assumption that no changes in stroke volume occur. 3. During the infusion of CPT, CADO produced a reduction in both blood pressure and MAP/HR by activation of the A2a receptor. The concentration-effect relationships were described according to the sigmoidal Emax model, yielding potencies based on free drug concentrations (EC50,u) of 61 and 68 ng ml-1 (202 and 225 nM) for the reduction of blood pressure and MAP/HR, respectively. During the infusion of CSC, an EC50,u value of 41 ng ml-1 (136 nM) was observed for the A1 receptor-mediated reduction in heart rate. The in vivo potencies correlated with reported receptor affinities (Ki(A1) = 300 nM and Ki(A2a) = 80 nM). The maximal reductions in MAP/HR and heart rate were comparable to those of full agonists, with the Emax values of -12 +/- 1 x 10(-2) mmHg b.p.m.-1 and -205 b.p.m. respectively. 4. It is concluded that this integrated pharmacokinetic-pharmacodynamic approach can be used to obtain quantitative information on the potency and intrinsic activity of new non-selective adenosine receptor agonists at different receptor subtypes in vivo.

Quantification of the in vivo potency of the adenosine A2 receptor antagonist 8-(3-chlorostyryl)caffeine.

The purpose of the present study was to quantify the in vivo potency of the selective adenosine A2a antagonist CSC [8-(3-chlorostyryl)caffeine]. Four groups of conscious, normotensive rats received a continuous i.v. infusion of 0, 6, 12 and 24 micrograms/min/kg of CSC. During a steady-state infusion of CSC, the animals received 1000 micrograms/kg of the adenosine A2a receptor agonist CGS 21680C [the sodium salt of 2-p-(2-carboxyethyl) phenylethylamino-5'-N-ethylcarboxamidoadenosine] i.v. over 15 min. During the experiment, the mean arterial pressure and the heart rate were recorded continuously and arterial blood samples were taken for the analysis of drug concentrations. For each individual rat, the CGS 21680C-provoked reduction in blood pressure was related to the blood concentration of the agonist according to the sigmoidal Emax model. The presence of CSC produced a parallel shift of the concentration-hypotensive effect curve to the right, indicating competitive interaction of the compounds. Infusion of 0, 6, 12 and 24 micrograms/min/kg of CSC resulted in steady-state concentrations of 0, 85 +/- 7, 210 +/- 20 and 400 +/- 40 ng/ml, and apparent EC50 values of CGS 21680C based on free concentrations (EC50,u) of 4.8 +/- 1.1, 7.2 +/- 0.5, 32 +/- 6 and 57 +/- 10 ng/ml, respectively (mean +/- S.E., n = 6, 6, 5 and 6). The relationship between the CSC concentration and the apparent EC50 was quantified according to a competitive pharmacodynamic interaction model.(ABSTRACT TRUNCATED AT 250 WORDS)

A series of N4-imidoethyl derivatives of 1-(2,3-dihydro-1,4-benzodioxin-5-yl)piperazine as 5-HT1A receptor ligands: synthesis and structure-affinity relationships.

A series of unsubstituted and substituted succinimido, maleimido, and glutarimidoethyl derivatives of eltoprazine (3) was synthesized and tested for affinity for the 5-HT1A receptor in rat brain homogenates. The unsubstituted compounds have a moderate affinity for the receptor, while the affinity considerably increases by substitution at or enlargement of these cyclic ring systems. A good correlation was found between the inhibition constant Ki (expressed as pKi) and the lipophilicity (clogP). No correlation was observed between the pKi or pKi+ (local inhibition constant) and the basicity of the N4-nitrogen atom.

Deoxyribose analogues of N6-cyclopentyladenosine (CPA): partial agonists at the adenosine A1 receptor in vivo.

1. The purpose of the present study was to quantify the cardiovascular effects of the 2'-, 3'-, 5'-deoxyribose analogues of the selective adenosine A1 receptor agonist, N6-cyclopentyladenosine (CPA) in vivo. The blood concentration-effect relationships of the compounds were assessed in individual rats and correlated to their receptor binding characteristics. 2. The pharmacokinetics and pharmacodynamics of the compounds were determined after a single intravenous infusion of 0.80 mg kg (-1) (63 micromol kg(-1)of 2' dCPA. The heart rate (HR) and mean arterial blood pressure (MAP) were monitored continuously during the experiment and serial arterial blood samples were taken for analysis of drug concentration. 3. The relationship between blood concentrations and the reductions in both heart rate and blood pressure were described according to the sigmoidal Emax model. For the bradycardiac effect, the potencies based on free drug concentrations (EC50,u) of 5'dCPA, 3'dCPA, 2'dCPAin blood were 5.9 +/- 1.7, 18 +/- 4 and 260 +/- 70 ng ml (-1) (19 +/- 6, 56 +/- 11 and 830 +/- 210 nM), respectively, and correlated well with the adenosine A1 receptor affinity in vitro. The Emax value of 2'dCPA was significantly less than those of the other compounds, suggesting that this compound may be regarded as a partial agonist when compared to the other analogues. The rank order of the maximal reduction in heart rate of the compounds corresponded well with the order of the GTP-shifts, as determined in vitro. 4. It is concluded that deoxyribose derivatives of CPA may be partial agonists for the adenosine A1 receptor and may serve as tools for further investigation of adenosine receptor partial agonism in vivo.

Ribose-modified adenosine analogues as potential partial agonists for the adenosine receptor.

We have adopted a practical three-step route for the synthesis of 2'- and 3'-deoxy analogues of N6-substituted adenosines: protection of the hydroxyl groups, replacement of the N6-amino by a better leaving group, and combined deprotection and N6-amination in the last step. This route was used to synthesize deoxy analogues of CPA, CHA, and R- and S-PIA. The compounds were tested on the adenosine A1 and A2a receptors in our search for partial agonists for these receptors. The GTP shift was used as an in vitro measure for the intrinsic activity of these compounds; the in vivo intrinsic activities of the deoxy analogues of CPA and R-PIA were determined in the rat cardiovascular system. Thus, it was shown that the hydroxyl groups are determinants for the affinity and intrinsic activity of these analogues. Removal of the 2'- and 3'-hydroxyl groups affects affinity and intrinsic activity, whereas removal of the 5'-hydroxyl group decreases only affinity.

Suramin analogs, divalent cations and ATP gamma S as inhibitors of ecto-ATPase.

Ecto-nucleotidases are plasma membrane-bound enzymes that sequentially dephosphorylate extracellular nucleotides such as ATP. This breakdown of ATP and other nucleotides obscures the characterization and classification of P2 (nucleotide) receptors. We therefore studied suramin and several of its analogs, divalent cations and ATP gamma S for their ability to inhibit ecto-ATPase in human blood cells. Suramin itself and Ni2+ were the more potent, non-competitive inhibitors with micromolar affinity. ATP gamma S also displayed micromolar affinity and inhibited ecto-ATPase competitively. The data obtained with the divalent cations demonstrate that coordination of the phosphate chain but not the N7 of the adenine ring is required for the breakdown of ATP by ecto-ATPase. Divalent cations that coordinate both the phosphate chain and N7 inhibit ecto-ATPase in a non-competitive manner.

Pharmacokinetic-pharmacodynamic modelling of the cardiovascular effects of R- and S-N6-phenylisopropyladenosine in conscious normotensive rats.

Recently, the commercially available adenosine receptor agonist S-N6-phenylisopropyladenosine (S-PIA) has been demonstrated to be contaminated with the more potent R-diastereomer. The potency of S-PIA may therefore have been overestimated in previously published in vivo studies. Our objective was to determine the potency of both diastereomers in conscious normotensive rats by using an integrated pharmacokinetic-pharmacodynamic model. In a cross-over designed study, the animals received i.v. infusions of 177 micrograms/kg (0.51 mumol/kg) R-N6-phenylisopropyladenosine (R-PIA) and 4000 micrograms/kg (11.6 mumol/kg) S-PIA. The infusion of S-PIA corresponded to a simultaneous administration of 3823 micrograms/kg (11.1 mumol/kg) and 177 micrograms/kg (0.51 mumol/kg) of the S- and R-diastereomer, respectively. During the experiment, time courses of heart rate and blood pressure were recorded continuously. Serial arterial blood samples were collected and concentrations were determined by using a stereoselective high-performance liquid chromatography assay. After administration of R-PIA, the individual concentration-effect relationships could be quantified by the sigmoidal Emax model, yielding an EC50 value of 24 +/- 3 ng/ml for the reduction in heart rate (mean +/- S.E., n = 12). After administration of S-PIA, a similar EC50 value was obtained when heart rate was correlated to concentrations of R-PIA. Modelling of the concentration-effect data according to a competitive interaction model did not yield pharmacodynamic parameter estimates for S-PIA. In conclusion, the cardiovascular effects observed after infusion of S-PIA may be attributed entirely to the presence of R-PIA.

Relative binding orientations of adenosine A1 receptor ligands--a test case for Distributed Multipole Analysis in medicinal chemistry.

The electrostatic properties of adenosine-based agonists and xanthine-based antagonists for the adenosine A1 receptor were used to assess various proposals for their relative orientation in the unknown binding site. The electrostatic properties were calculated from distributed multipole representations of SCF wavefunctions. A range of methods of assessing the electrostatic similarity of the ligands were used in the comparison. One of the methods, comparing the sign of the potential around the two molecules, gave inconclusive results. The other approaches, however, provided a mutually complementary and consistent picture of the electrostatic similarity and dissimilarity of the molecules in the three proposed relative orientations. This was significantly different from the results obtained previously with MOPAC AM1 point charges. In the standard model overlay, where the aromatic nitrogen atoms of both agonists and antagonists are in the same position relative to the binding site, the electrostatic potentials are so dissimilar that binding to the same receptor site is highly unlikely. Overlaying the N6-region of adenosine with that near C8 of theophylline (the N6-C8 model) produces the greatest similarity in electrostatic properties for these ligands. However, N6-cyclopentyladenosine (CPA) and 1,3-dipropyl-8-cyclopentyl-xanthine (DPCPX) show greater electrostatic similarity when the aromatic rings are superimposed according to the flipped model, in which the xanthine ring is rotated around its horizontal axis. This difference is mainly attributed to the change in conformation of N6-substituted adenosines and could result in a different orientation for theophylline and DPCPX within the receptor binding site. However, it is more likely that DPCPX also binds according to the N6-C8 model, as this model gives the best steric overlay and would be favoured by the lipophilic forces, provided that the binding site residues could accommodate the different electrostatic properties in the N6/N7-region. Finally, we have shown that Distributed Multipole Analysis (DMA) offers a new, feasible tool for the medicinal chemist, because it provides the use of reliable electrostatic models to determine plausible relative binding orientations.

Pharmacokinetic modelling of the haemodynamic effects of the A2a adenosine receptor agonist CGS 21680C in conscious normotensive rats.

1. The aim of the present investigation was to determine the relationship between the blood concentration and haemodynamic effects of the adenosine A2a receptor agonist, CGS 21680C (the sodium salt of 2-p-(2-carboxyethyl)phenylethylamino-5'-N-ethylcarboxamidoadeno sin e) in conscious normotensive rats. 2. Chronically cannulated rats were randomly assigned to three groups which received 300, 1000 or 3000 micrograms kg-1 (0.56, 1.9 or 5.6 mumol kg-1) of CGS 21680C intravenously over 15 min. The mean arterial blood pressure (MAP) and heart rate (HR) were monitored continuously during the experiment and serial arterial blood samples were taken for analysis of drug concentration. The ratio MAP/HR was also calculated, which may reflect changes in total peripheral resistance on the assumption that no changes in stroke volume occur. 3. For each individual rat the reduction in mean arterial pressure was related to the blood concentration according to the sigmoidal Emax model. The concentration-effect relationships were consistent for the different treatment groups. The potency based on free drug concentrations (EC50,u) was 5.8 ng ml-1 (11 nM) (mean +/- s.e.; n = 19) and correlated well with the reported adenosine A2a receptor affinity (Ki 19 nM). In comparison with the reduction in blood pressure, CGS 21680C exhibited a greater potency for the reduction of the ratio MAP/HR. 4. It is concluded that estimates can be obtained for the potency and intrinsic activity of adenosine A2a receptor agonists in vivo by pharmacokinetic-pharmacodynamic analysis of mean arterial pressure data in a rat model. In future studies, total peripheral resistance may also be useful as a pharmacodynamic parameter for A24 activation, provided that possible changes of the stroke volume are also assessed.

Molecular modelling of CCK-A receptors.

Recently, the primary structure of the cholecystokinin A-type (CCK-A) receptor has been determined. From the Kyte-Doolittle-predicted hydrophobic stretches of this sequence and the transmembrane domains of bacteriorhodopsin, a membrane-bound protein of known tertiary structure, a three-dimensional model of the membrane-embedded part of this receptor was built. Subsequently, the modelled receptor pore was searched for a binding site that matches the structural and conformational characteristics of the parent classes of the antagonists devazepide and lorglumide. In addition, the binding mode of hybrid analogues of these reference compounds was examined. The proposed antagonist, binding site includes regions in which hydrophobic, hydrogen-bonding and aromatic interactions stabilize the receptor-ligand complex.

Structure-affinity relationship studies on 5-HT1A receptor ligands. 2. Heterobicyclic phenylpiperazines with N4-aralkyl substituents.

Structure-affinity relationship (SAR) studies for the 5-HT1A receptor site are presented for two series of heterobicyclic phenylpiperazines with N4-aralkyl substituents: 4-aralkyl derivatives of 1-(2,3-dihydro-1,4-benzodioxin-5-yl)piperazine (3) and 1-(benzo[b]furan-7-yl)piperazine (4). Their affinities for 5-HT1A receptors range from 0.15 to 28 nM and thus emphasize the importance of N4-substitution. By combining the SAR of these N4-aralkyl series with the recently published SAR of the N4-alkyl-substituted phenylpiperazines, the nature of the interaction of the N4-substituted phenylpiperazines and the 5-HT1A receptor was further examined using comparative molecular field analysis (CoMFA). To discriminate between two postulated hypotheses, CoMFA models were built and validated utilizing cross-validation, bootstrapping, and randomizing techniques. The model based on a N4-substituent alignment in which all N4-substituents are equally oriented in space was selected for further evaluation. According to the CoMFA/PLS analysis, the steric and electrostatic field properties contribute in a 98:2 ratio to the affinity found for the 5-HT1A receptor. Increasing steric bulk was found to be positively as well as negatively related to affinity depending on the distance of the bulk's center from the N4-nitrogen. The location of these steric CoMFA contour levels are well defined in space when the defined alignment rules are followed. Because CoMFA does not take hydrogen bonding into account, this could indicate that the contribution of the amide function (its ability to interact through hydrogen bonding), as present in the N4-substituents, to affinity is of minor importance.

Novel competitive antagonists for P2 purinoceptors.

Binding of the radioligand [35S]adenosine 5'-O-(2-thiodiphosphate) (ADP beta 35S) to P2 gamma purinoceptors on turkey erythrocyte membranes was used to determine the affinity of suramin and various suramin congeners belonging to different structure classes (large urea, small urea, dibenzamides and benzamides) for these receptors. Suramin was shown to be a competitive antagonist with a Ki value of 7.3 +/- 2.2 microM. The simple benzamide compound XAMR0721 (8-(3,5-dinitrophenylene carbonylimino)-1,3,5-naphthalene trisulfonate, trisodium salt) displays a high affinity for the P2 gamma purinoceptor (Ki value of 19 +/- 6 microM). Similar to suramin, compound XAMR0721 is a competitive antagonist at P2 gamma purinoceptors. In contrast to suramin, which is a potent inhibitor of the ecto-nucleotidase activity in human blood cells (44 +/- 2% residual activity at 100 microM), compound XAMR0721 is hardly active in this assay (93 +/- 1% residual activity at 100 microM). So XAMR0721, the first competitive antagonist for P2 purinoceptors that is able to discriminate between P2 purinoceptor affinity and ecto-nucleotidase activity, is an interesting pharmacological tool for the characterization of P2 purinoceptor mediated effects.

Pharmacokinetic-pharmacodynamic relationship of the cardiovascular effects of adenosine A1 receptor agonist N6-cyclopentyladenosine in the rat.

The purpose of the investigation was to develop a pharmacokinetic-pharmacodynamic model for the characterization of the cardiovascular effects of the adenosine A1 receptor agonist N6-cyclopentyladenosine (CPA) in individual normotensive rats. After the i.v. administration of 200 micrograms/kg (0.60 mumol/kg) of CPA, the time course of heart rate and arterial blood pressure was monitored in conjunction with serial blood sampling. Potential interference of the concentration-cardiovascular effect relationship by the development of acute tolerance or the formation of (inter)active metabolites was investigated by infusion of CPA with different rates and by determination of blood concentrations both by high-performance liquid chromatography and radioreceptor assay. In the individual rats the concentration-hemodynamic effect relationships were satisfactorily modeled according to the sigmoidal Emax pharmacodynamic model. For the negative chronotropic effect, the pharmacodynamic parameters proved to be independent of the infusion rate, indicating the absence of development of acute tolerance during the experiment. Potency (EC50) and intrinsic efficacy (Emax) were 2.7 +/- 0.5 ng/ml and -209 +/- 10 bpm, respectively (mean +/- S.E., n = 17). The concentrations of CPA as determined by the radioreceptor assay were identical to those determined by high-performance liquid chromatography, thereby excluding the formation of (inter)active metabolites. It is concluded that on the basis of this integrated pharmacokinetic-pharmacodynamic model, with the negative chronotropic effect as a pharmacodynamic endpoint, estimates of the potency and the intrinsic efficacy of adenosine A1 receptor agonists in vivo can be obtained after the administration of a single dose.

Nucleoside transport inhibition and platelet aggregation in human blood: R75231 and its enantiomers, draflazine and R88016.

In this study, we determined whether R75231, (+/-)-2-(aminocarbonyl)-N-(4-amino-2,6-dichlorophenyl)-4-[5,5-bis( 4-fluoro- phenyl)pentyl]-1-piperazineacetamide, and its two enantiomers, all nucleoside transport inhibitors, could play a role as anti-aggregatory agents. First, we determined the binding characteristics of [3H]nitrobenzylthioinosine, also a nucleoside transport inhibitor, on intact human erythrocytes. The Kd value was 0.27 +/- 0.04 nM and the Bmax was 23.5 +/- 5.1 pmol/10(9) erythrocytes. Second, we studied the ability of R75231 and its enantiomers R88021 ((-)-R75231, or draflazine) and R88016 ((+)-R75231), to displace [3H]nitrobenzylthioinosine. R75231 had an IC50 value of 2.2 +/- 0.3 nM. R88021 was twice as potent as R75231 and R88016 was approximately 20-fold less potent than R75231. Finally, the ability of these nucleoside transport inhibitors to enhance anti-aggregatory effects of adenosine was examined in whole human blood. Adenosine alone, 10 microM, had no effect on ADP-induced platelet aggregation. However, in the presence of 1 microM R75231, 10 microM of adenosine inhibited the aggregatory response completely. Dose-response curves indicated that the IC50 values of draflazine and R88016 were approximately 0.5 microM and 10 microM, respectively. R75231 and its enantiomers are valuable research tools to assess the role of the nucleoside transporter. Moreover, R75231 and draflazine (R88021) may prove to be useful as anti-aggregatory agents.