Abstract
The benzofurane (+)-S 14297, the benzamide nafadotride, the aminoindane U 99194 and the arylpiperazine GR 103,691 have been proposed as “selective” antagonists at dopamine D3vs.D2 receptors. Herein, we compared their in vitroaffinities and in vivo actions to those of the aminotetralin D3 antagonists (+)-AJ 76 and (+)-UH 232. Affinities at recombinant, human (h)D3 and/or hD2 sites were determined by employing the mixed D2/D3antagonist [125I]-iodosulpride and the preferential D3 ligands [3H]-(+)-PD 128,907 and [3H]-(+)-S 14297. [3H]-(+)-PD 128,907, [3H]-(+)-S 14297 and [125I]-iodosulpride yielded an essentially identical pattern of displacement at D3 sites, which suggests that they recognize the same population of receptors. The rank order of potency (Ki values in nM vs.[3H]-(+)-PD 128,907) was GR 103,691 (0.4) ≈ nafadotride (0.5) > haloperidol (2) ≈ (+)-UH 232 (3) ≈ (+)-S 14297 (5) > (+)-AJ 76 (26) > U 99194 (160). The rank order of preference (Ki ratio, D2:D3) for D3 receptors (labeled by [3H]-PD 128,907)vs. D2 sites (labeled by [125I]-iodosulpride) was (+)-S 14297 (61) ≈ GR 103,691 (60) > U 99194 (14) > nafadotride (9) ≈ (+)-UH 232 (8) ≈ (+)-AJ 76 (6) > haloperidol (0.2). (+)-S 14297 and GR 103,691 also showed greater than 100-fold selectivity at dopamine hD3vs. hD4 and hD1 sites. However, GR 103,691 showed marked affinity for serotonin1A receptors (5.8 nM) and alpha-1 adrenoceptors (12.6 nM). In vivo, all antagonists except GR 103,691 prevented the induction of hypothermia by (+)-PD 128,907 (0.63 mg/kg s.c.) and a further preferential D3 agonist, (+)-7-OH-DPAT (0.16 mg/kg s.c.). On the other hand, haloperidol, (+)-AJ 76, (+)-UH 232 and nafadotride all induced catalepsy in rats, whereas (+)-S 14297, U 99194 and GR 103,691 were inactive. Haloperidol, (+)-AJ 76, (+)-UH 232, nafadotride and (weakly) U 99194 also enhanced prolactin secretion and striatal dopamine synthesis, whereas (+)-S 14297 and GR 103,691 were inactive. However, despite its high affinity at 5-HT1A receptors andalpha-1 adrenoceptors, both of which are present on raphe-localized serotonergic neurons, GR 103,691 (0.5 mg/kg i.v.) failed to influence their basal firing rate or the inhibition of their electrical activity by the 5-HT1A agonist (±)-8-OH-DPAT (0.005 mg/kg i.v.), a result that casts doubt on its activity in vivo. In conclusion, both (+)-S 14297 and GR 103,691 are markedly selective ligands that permit the characterization of actions at hD3vs. hD2 receptors in vitro, but (+)-S 14297 appears to be of greater utility for the evaluation of their functional significance in vivo. Nevertheless, to develop a better understanding of the respective roles of dopamine D3 and D2 receptors, we need additional, chemically diverse antagonists of improved potency and selectivity.
DA is implicated in the modulation of several physiological functions, including endocrine secretion, motor behavior, thermoregulation and mood (Creese and Fraser, 1987). Its actions are expressedvia at least five different receptor types. On the basis of their molecular structure and pharmacological properties, these receptors have been divided into two families: D1-like, including D1 and D5 receptors, and D2-like, including D2, D3 and D4 receptors (Sokoloff and Schwartz, 1995; Strange, 1993). As concerns the D2 receptor family, the low level of expression of mRNA encoding D4 receptors in rodent and human tissue, and the hitherto lack of selective radioligands for D4 sites, have complicated the elucidation of their functional significance, although selective D4 antagonists are now becoming available (Boyfield et al., 1996; Bristowet al., 1997; Kulagowski et al., 1996; Merchantet al., 1996; Millan et al., 1996). In contrast, both in situ hybridization and polymerase chain reaction amplification studies have allowed for the localization of mRNA encoding D3 receptors in rodent and human brain, and the use of antibodies directed against D3 receptors has permitted localization of the corresponding protein (Bouthenet et al., 1991; Landwehrmeyer et al., 1993; Lévesqueet al., 1992). These approaches, together with studies of the preferential D3 agonists [3H]-(+)-PD 128,907 and [3H]-(+)-7-OH-DPAT (Hall et al., 1996; Herroelen et al., 1994), suggest that D3sites are predominantly localized in limbic regions such as the olfactory tubercles, the nucleus accumbens and the islands of Calleja. This restricted D3 receptor distribution contrasts with the broad distribution of D2 receptors (Gehlert et al., 1992; Larson and Ariano, 1995; Levant et al., 1993; Murray et al., 1994; Richtand et al., 1995). In addition, a minor population of D3 autoreceptors may exist with D2 autoreceptors on dopaminergic cell bodies of the ventral tegmental area and substantia nigra, pars compacta (Diazet al., 1995; Gobert et al., 1995; Koeltzowet al., 1998; Meador-Woodruff et al., 1996).
Despite the utility of gene knockout and antisense strategies (Acciliet al., 1996; Baik et al., 1995; Carta et al., 1996, Tepper et al., 1997), ligands that interact selectively with D3vs. D2 receptors are essential for an improved knowledge of their localization, modulation and functional significance. In the absence of appropriate tissue preparations, the characterization of novel ligands has generally been performed by using mammalian cell lines transfected with human or rodent D2 or D3 receptor subtypes and radioligands such as [125I]-iodosulpride or [3H]-spiperone that do not differentiate these sites (Chio et al., 1993; Millan et al., 1995; Sokoloffet al., 1992; Tang et al., 1994). On the basis of such studies, the aminotetralins (+)-AJ 76 and (+)-UH 232 were found to display a modest (about 2–5-fold) preference for D3 over D2 sites (Lévesque, 1996; Sokoloff et al., 1992; Van Vliet et al., 1996). Subsequently, several antagonists with improved selectivity at D3vs.D2 sites have been identified: the aminoindane U 99194 (Waters et al., 1993), the benzofurane (±)-S 11566 and its active (+)-isomer (+)-S 14297 (Gobert et al., 1995, 1996;Millan et al., 1994; 1995; Morris et al., 1997), the arylpiperazine GR 103,691 (Murray et al., 1995) and the benzamide derivative nafadotride (Sautel et al., 1996). Of these, (+)-S 14297 has been extensively employed in the functional evaluation of the potential significance of D3 receptorsin vivo. It has been shown that (+)-S 14297 inhibits the hypothermia induced by dopaminergic agonists such as (+)-7-OH-DPAT and quinpirole in rodents, which suggests an involvement of D3receptors in the control of CT (Millan et al., 1994; 1995;Rivet et al., 1996). At comparable doses, (+)-S 14297 neither modifies PRL secretion nor induces catalepsy in rats, two responses reflecting blockade of D2 receptors (Broccoet al., 1995; Millan et al., 1995; 1997). Interestingly, (+)-S 14297 does not modify the synthesis or release of DA in cerebral tissues, and it fails to modify the activity of ventral tegmental area-localized dopaminergic neurons (Gobert et al., 1995; Lejeune and Millan, 1995; Millan et al., 1995; Rivet et al., 1994; 1996). These observations suggest that D2 rather than D3 autoreceptors tonically inhibit the activity of central dopaminergic neurons. Nevertheless, (+)-S 14297 attenuates the inhibitory influence of (+)-7-OH-DPAT and (+)-PD 128,907 upon DA release and synthesis, which suggests that D3 (auto)receptors may contribute to the “phasic” inhibition of dopaminergic pathways (Gobert et al., 1995;Lejeune and Millan, 1995; Rivet et al., 1994).
Evidently, there now exist several antagonists and agonists of potential utility for the in vitro and in vivofunctional characterization of actions mediated by D3receptors. However, there has not yet been any comparative evaluation of the properties of these ligands. Thus, using the radioligands [3H]-(+)-S 14297 and [3H]-(+)-PD 128,907, both of which have recently been radiolabeled (Akunne et al., 1995; Newman-Tancredi et al., 1995), as well as the mixed D2/D3 antagonist [125I]-iodosulpride, the present study undertook a comparative evaluation of the binding profiles of (+)-S 14297, nafadotride, U 99194, GR 103,691, (+)-AJ 76, (+)-UH 232 and haloperidol at recombinant hD2 and hD3 receptors. In addition, we examined their in vivo actions in models of putative activity at D3 receptors (hypothermia) and D2 receptors (catalepsy, PRL secretion and DA synthesis). These analyses allowed for the classification of drugs in terms of 1) their rank order of potency at hD3 receptors and 2) their rank order of selectivity for hD3vs.hD2 receptors. In addition, a direct comparison of drug doses in putative models of D3 as compared with D2 receptor-mediated activity was possible.
Material and Methods
Binding at cloned, human D2 and D3receptors.
Binding studies were carried out at recombinant hD2 receptors (Receptor Biology, Beltsville, MA) and hD3 receptors (J.-C. Schwartz, INSERM, Paris, France) expressed in CHO cell lines as described previously (Millan et al., 1995). Briefly, affinities at hD2 and hD3 receptors were determined using [125I]-iodosulpride (0.1 and 0.2 nM for D2and D3, respectively), [3H]-(+)-S 14297 (7 nM for D3) and [3H]-(+)-PD 128,907 (2 nM for D3). Nonspecific binding was defined using 10 μM raclopride. Binding conditions for [125I]-iodosulpride and [3H]-(+)-S 14297 were as previously described (Newman-Tancredi et al., 1995; Sokoloff et al., 1992), and they were the same for [3H]-(+)-PD 128,907 except that incubations were carried out for 90 min.
Binding at other sites.
Binding conditions at other receptor sites were as described in Millan et al. (1995). For each receptor, the tissue, radioligand and ligands used to define nonspecific binding were as follows: hD4 receptors (4-repeat variant) expressed in CHO cells; [3H]-spiperone (0.2 nM); haloperidol 10 μM; rat striatal D2 receptors; [3H]-spiperone (0.1 nM), raclopride 10 μM; rat hippocampal 5-HT1A receptors and cloned, human serotonin (h5-HT1A) receptors expressed in CHO cells; [3H]-(±)-8-OH-DPAT (0.4 nM); 5-HT 10 μM; cloned, human muscarinic (hM1) receptors expressed in CHO cells; [3H]-N-methylscopolamine (0.5 nM); atropine 10 μM; rat frontal cortex alpha-1 adrenoceptors; [3H]-prazosin (0.2 nM); phentolamine 10 μM.
Influence on CT.
For evaluation of the influence of drugs alone on CT, basal CT was determined, drug or vehicle was administered, and CT was determined again 30 min later. This time corresponds to that at which (+)-PD 128,9078 and (+)-7-OH-DPAT exert their maximal influence on CT (Salmi et al., 1993; Dekeyne, A., unpublished observation). The difference between basal and postdrug values was calculated. For antagonist studies, the procedure was as follows. Basal CT was measured, drug or vehicle was administered and (+)-7-OH-DPAT, (+)-PD 128,907 or vehicle was administered 30 min later. A further 30 min later, CT was reevaluated and the difference from basal values calculated. The percent of drug inhibition of the actions of (+)-7-OH-DPAT and (+)-PD 128,907 was computed as 1-[(antagonist + agonist) − vehicle alone)/(vehicle + agonist) − vehicle alone)] × 100. Drug potency was expressed as ID50 95% CL.
Evaluation of catalepsy.
Catalepsy was determined in rats using a previously described procedure (Waldmeier and Delini-Stula, 1979). The animals were placed in a position whereby the left and right hind paws were crossed over the ipsilateral front paws; the time during which this position was maintained was determined up to a maximum of 30 sec. The mean value of three consecutive tests, separated by intervals of 1 min, was determined. Cataleptogenic potency was expressed in terms of AD50 (95% CL)—that is, the dose required for the induction of a half-maximal response (equivalent to 15 sec).
Evaluation of PRL secretion.
As described previously (Gobertet al., 1995), we measured PRL 30 min after drug administration in plasma by radioimmunoassay, using a highly specific (<0.5% cross-reactivity to all other hormones examined) antibody against rat PRL (Amersham, Buckingham, England). The detection limit was 70 pg/tube, and the intravariation and interassay variation were 5% and 15%, respectively. Because there is no theoretical limit to PRL levels in plasma, the MED was defined as the lowest dose significantly different (P < .05) from vehicle control values in Dunnett’s test after ANOVA.
Evaluation of DA turnover.
The method we used was described previously (Gobert et al., 1995). The effect of drugs alone was determined 30 min after their s.c. injection. The striatum, nucleus accumbens, olfactory tubercle and frontal cortex were examined (the inclusion of a portion of cingulate cortex in “frontal” cortex should be noted).
Tissues were homogenized in 500 μl of 0.1 M HClO4containing 0.5% Na2S2O5 and 0.5% EDTA Na2 and centrifuged at 15,000 × g for 15 min at 4°C. Supernatants were diluted in the mobile phase and injected into a HPLC column (Hypersil ODS 5 μm, C18, 150 × 4.6 mm (Thermo Quest, Les Ulis, France) maintained at 25°C). The mobile phase of the HPLC was KH2PO4, 100 mM; EDTA Na2, 0.1 mM; sodium octylsulphonate, 0.5 mM; methanol 5% adjusted to pH 3.15 with PO4H3. The flow rate was 1 ml/min. Electrochemical detection was performed using a Waters M460 detector with a working potential of 850 mV against an Ag/AgCl reference. Quantitative determinations were made by comparison with appropriate external standards. Levels of DA and DOPAC were expressed as a function of the tissue content of protein, with bovine serum albumin (Sigma Chemical Co., St. Louis, MO) as the standard. As an index of turnover, the ratio of DOPAC:DA was calculated. For each experiment, the mean levels of DA and DOPAC and the DOPAC:DA ratios were determined. They were expressed relative to values in animals treated with vehicle (defined as 100%). The influence of drugs was expressed as a percentage thereof. Drug potency was expressed as AD50 (95% CL)—that is, an increase in DOPAC:DA ratios to 150% relative to vehicle values.
Electrical activity of serotonergic neurons in the DRN.
As described in detail previously (Lejeune et al., 1994), rats were anesthetized with chloral hydrate (500 mg/kg s.c.). The femoral vein was cannulated and the rat placed in a stereotaxic apparatus. A tungsten electrode was lowered into the DRN, and the firing activity was measured by means of a Spike 2 analysis system obtained from Cambridge Electronic Design (Cambridge, U.K.). Drug effects were quantified over 60-sec bins at their time of maximal effect (1–2 min) after their injection i.v. Administered alone, GR 103,691 was injected in cumulative doses every 2 min. It was also injected at a single dose 2 min after administration of the 5-HT1A receptor agonist (±)-8-OH-DPAT (0.005 mg/kg i.v.). Firing rates were expressed as a percentage of basal, preinjection values (defined as 100%). The mean, basal firing rate was 1.1 Hz.
Data analysis and statistics.
All binding data were analysed with nonlinear regression (“Prism,” GraphPad, San Diego, CA).Ki values were calculated according to the Cheng-Prussof equation, Ki = IC50/(1 + L/Kd ), where IC50 is the concentration of compound that gives 50% inhibition of radioligand binding, L is the concentration of radioligand and Kd is the dissociation constant of the radioligand. In vivo dose-response curves were initially analyzed by ANOVA followed by Dunnett’s test, for which the level of significance was set at P < .05. As indicated above, MEDs, ID50 values, AD50 values or AD150 values were determined to estimate drug potencies. MOEs of drugs were also calculated.
Drugs and chemicals.
[125I]-Iodosulpride (2000 Ci/mmol) and [3H]-(+)-PD 128,907 (90–130 Ci/mmol) were purchased from Amersham, and [3H]-(+)-S 14297 (145 Ci/mmol) was radiolabeled by C.E.A (Gif-sur-Yvette, France). Forin vitro binding studies, drugs were dissolved in water or in dimethylsulfoxide. For in vivo studies, drugs were dissolved in sterile water, plus a few drops of lactic acid if necessary, and pH was adjusted to as close to neutrality (>5.0) as possible. Drugs were injected s.c., and doses are expressed in terms of the base. Drug sources, salts and structures were as follows: (+)-AJ 76 and (+)-UH 232 (Tocris Cookson, Southampton, England); (+)-7-OH-DPAT HCl (J. Besselièvre, CNRS, Paris, France); (+)-PD 128,907 HCl and (±)-8-OH-DPAT HBr (Research Biochemicals, Inc., Natick, MA) and haloperidol base (Sigma, Chesnes, France). (±)-S 11566 HCl, (+)-S 14297 dibenzoyltartrate, (−)-S 17777 dibenzoyltartrate, U 99194 HCl, GR 103,691 and nafadotride were synthesized by Servier chemists (J.-L. Peglion and G. Lavielle).
Results
In vitro ligand binding at hD3 and hD2 receptors.
In an initial series of experiments, we investigated the binding characteristics of [3H]-(+)-PD 128,907 to hD3 receptors. [3H]-(+)-PD 128,907 associated rapidly and monophasically with a t1/2 of 21 ± 3 min. Dissociation of [3H]-(+)-PD 128,907 from hD3 receptors was biphasic with a t1/2value for the first component of the isotherm of 58 ± 8 min (k−1 = 0.013 ± 0.002 min−1). The second component of the isotherm dissociated slowly, with 27% ± 1% of binding still remaining after 6 hr of incubation. An estimate of the Kd derived from the association and dissociation (rapid component) constants yielded a value of 0.6 nM. In saturation binding experiments, [3H]-(+)-PD 128,907 yielded a Kd value at hD3 receptors of 1.61 ± 0.31 nM. The Bmax value (6.06 ± 0.59 pmol/mg) was similar to that determined with [125I]-iodosulpride in the same membrane preparation (not shown). Antagonist competition binding isotherms at hD3receptors were derived for the preferential D3 receptor agonist [3H]-(+)-PD 128,907, the D3antagonist [3H]-(+)-S 14297 and the D2/D3 antagonist [125I]-iodosulpride (fig.1). Ki values at hD3 receptors were similar regardless of the radioligand used (table 1). The rank order of antagonist potency against [3H]-(+)-PD 128,907 binding at hD3 sites was GR 103,691 > nafadotride > haloperidol > (+)-UH 232 > (+)-S 14297 > (±)-S 11566 > (+)-AJ 76 > U 99194 > (−)-S 17777. The agonists (+)-7-OH-DPAT and (+)-PD 128,907 yielded biphasic isotherms at hD2 receptors (table 1, legend). The selectivity ratios (Ki , hD2/Ki , hD3) for the three radioligands are shown in table 1. (±)-S 11566, its isomer (+)-S 14297 and GR 103,691 exhibited the highest selectivity for hD3 receptors. Affinities at hD3 receptors correlated positively between radioligands with coefficients (r values) of +0.98 to +0.99. The affinity of the antagonists at other key receptor subtypes was determined.Ki values are shown for hD4, h5-HT1A and hM1 receptors and for ratalpha-1 adrenoceptors (table2). (+)-AJ 76 was only 8-fold more selective for hD3 than for hD4 receptors, and GR 103,691 was only 11-fold more selective for hD3 than for h5-HT1A receptors. (+)-S 14297 exhibited modest affinity at hM1 receptors, for which its affinity was 30-fold less than at hD3 receptors labeled by [3H]-PD 128,907 (Millan et al., 1995).
Competition binding of dopaminergic antagonists at cloned, hD3 and hD2 receptors expressed in CHO cells. [3H]-(+)-PD 128,907, hD3 (panel A), [3H]-(+)-S 14297, hD3 (panel B) and [125I]-iodosulpride, hD3 (panel C). Competition binding experiments at hD2 receptors were carried out against [125I]-iodosulpride (panel D). Points shown are means of triplicate determinations from a single, representative experiment performed at least three times.
Affinities of dopaminergic antagonists at hD3, hD2 and rat D2 receptors determined by competition with different radioligands
Binding affinities (Ki values) of dopaminergic ligands at key receptors
Electrical activity of DRN-localized serotonergic neurons.
Administered at a dose of 5 μg/kg i.v., (±)-8-OH-DPAT completely abolished the firing rate of DRN serotonergic neurons (−100.0 ± 0.0% relative to vehicle values, defined as 0%). Further, thealpha-1 adrenoceptor antagonist prazosin (150 μg/kg i.v.) reduced firing levels to −77.9 ± 6.4% of vehicle values. Both effects were significant in Student’s two-tailed t test (P < .05). By contrast, administered in incremental doses up to 500 μg/kg i.v., GR 103,691 failed (±0.0 ± 5.5%) to modify the firing rate of these neurons. Similarly, at a dose of 250 μg/kg i.v., GR 103,691 did not modify the influence of 8-OH-DPAT on DRN firing (−100.0 ± 0.0%).
Blockade of (+)-PD 128,907- and (+)-7-OH-DPAT-induced hypothermia.
Both (+)-PD 128,907 and (+)-7-OH-DPAT dose-dependently elicited hypothermia (fig.2). Their actions were prevented by (±)-S 11566, as well as by its active eutomer (+)-S 14297, whereas the inactive distomer (−)-S 17777 was ineffective (table3; fig. 3). (+)-AJ 76, (+)-UH 232, nafadotride and U 99194 also antagonized (+)-PD 128,907- and (+)-7-OH-DPAT-induced hypothermia, whereas GR 103,691 was inactive (table 3; fig. 3). Haloperidol was active at low doses. Antagonist potencies in blocking PD 128,907- and (+)-7-OH-DPAT-induced hypothermia were highly correlated (r = 0.97, P < .01).
Dose-dependent induction of hypothermia by the preferential agonists at hD3 receptors, (+)-PD 128,907 and (+)-7-OH-DPAT. The data represent the difference between basal and post-treatment values and are means ± S.E.M. (n≥ 5 per value). ANOVA results: (+)-PD 128,907, F(6,31) = 55.8, P < .01 and (+)-7-OH-DPAT, F(4,38) = 51.1, P < .01. * Significantly different from vehicle values in Dunnett’s test after ANOVA; P < .05.
Inhibition of (+)-PD 128,907- and (+)-7-OH-DPAT-induced hypothermia by dopaminergic antagonists
Dose-dependent blockade of (+)-PD 128,907-induced hypothermia by dopaminergic antagonists. Antagonists were given 30 min before (+)-PD 128,907 (0.63 mg/kg s.c.) (closed symbols) or vehicle (open symbols). Data are means ± S.E.M. (n ≥ 4 per value). ANOVA results follow. Panel A: (±)-S 11566/vehicle,F(3,25) = 4.4, P < .05; (±)-S 11506/(+)-PD 128,907,F(3,15) = 24.5, P < .001; (+)-S 14297/vehicle,F(3,33) = 8.5, P < .001; (+)-S 14297/(+)-PD 128,907,F(3,15) = 12.4, P < .001; (−)-S 17777/vehicle,F(2,12) = 3.1, P > .05; (−)-S 17777/(+)-PD 128,907,F(3,16) = 3.7, P < .05. Panel B: (+)-UH 232/vehicle,F(3,10) = 0.3, P > .05; (+)-UH 232/(+)-PD 128,907,F(3,17) = 15.7, P < .01; (+)-AJ 76/vehicle,F(3,30) = 4.0, P < .05; (+)-AJ 76/(+)-PD 128,907,F(3,14) = 12.5, P < .01; nafadotride/vehicle,F(3,13) = 1.6, P > .05. Panel C: nafadotride/(+)-PD 128,907, F(4,18) = 19.4, P < .01; U 99,194/vehicle,F(3,11) = 0.2, P > .05; U 99,194/(+)-PD 128,907,F(4,26) = 11.1, P < .01 and GR 103,691/(+)-PD 128,907,F(3,13) = 0.8, P > .05. * Significantly different from vehicle/(+)-PD 128,907 values in Dunnett’s test after ANOVA: P < .05.
Induction of catalepsy, PRL secretion and DA synthesis by dopaminergic antagonists.
Haloperidol potently elicited catalepsy, increased plasma PRL levels and elevated DA synthesis (tables4 and 5; fig. 4). (+)-AJ 76, (+)-UH 232 and nafadotride likewise induced both PRL secretion and DA synthesis (tables 4 and 5; fig. 4). U 99194 failed to elicit catalepsy and increased PRL release and DA turnover only at very high doses (40.0 mg/kg). (+)-S 14297 and GR 103,691 were devoid of activity in each of these models (tables 4 and 5; fig. 4).
Induction of catalepsy and PRL secretion by dopaminergic antagonists
Influence of dopaminergic antagonists on DA turnover
Dose-dependent induction by dopaminergic antagonists of catalepsy (panel A), PRL secretion (panel B) and striatal DA synthesis (panel C). Data are means ± S.E.M. (n≥ 4 per value). ANOVA results follow. Catalepsy: (+)-AJ 76,F(4,26) = 4.8, P < .05; GR 103,691, F(3,18) = 0.6, P > .05; haloperidol, F(4,43) = 58.3, P < .05; nafadotride, F(3,26) = 11.8, P < .05; (+)-UH 232,F(3,19) = 11.7, P < .05; U 99194, F(2,8) = 0.8, P > .05 and (+)-S 14297, F(2,12) = 1.5, P > .05. Prolactin secretion: (+)-AJ 76, F(4,32) = 9.7, P < .05; GR 103,691, F(3,30) = 0.8, P > .05; haloperidol, F(5,47) = 18.7, P < .05; nafadotride,F(5,36) = 24.3, P < .05; (+)-UH 232,F(3,13) = 5.8, P < .05; U 99194, F(4,15) = 17.7, P < .05 and (+)-S 14297, F(4,28) = 0.5, P > .05. Striatal DA synthesis: GR 103,691, F(1,6) = 3.5, P > .05; nafadotride, F(4,19) = 90.5, P < .01; (+)-UH 232, F(3,12) = 34.5, P < .01; U 99194,F(3,17) = 108.2, P < .01 and S 14297,F(3,31) = 0.7, P > .05. (+)-AJ 76 and haloperidol data are from Gobert et al., (1995). * Significantly different from vehicle values in Dunnett’s test after ANOVA; * P < .05.
Discussion
Competition binding studies.
All antagonists displayed similar affinities at hD3 receptors irrespective of the radioligand used, though slightly higher affinities were generally observed with [3H]-(+)-PD 128,907. This observation may reflect the capacity of agonist radioligands to stabilize hD3 receptors in a high-affinity conformation, an interpretation consistent with its slow dissociation (“Results”) and implied by a ternary complex model of receptor coupling (Kenakin, 1996). In any case, a similar order of hD2/hD3selectivity was observed regardless of the D3 radioligand. With subnanomolar affinities, GR 103,691 and nafadotride were potent ligands at hD3 receptors (Murray et al., 1995;Sautel et al., 1996). The hD2/hD3selectivity ratio of nafadotride was, however, only modest, whereas GR 103,691 showed 60-fold selectivity. Because of a higher affinity at D2 sites in our hands, this value is about 2-fold less than that previously reported (table 1; Murray et al., 1995) but is still pronounced. (±)-S 11566 and its active eutomer (+)-S 14297 also exhibited marked selectivity ratios at D3vs. D2 sites, whereas a modest hD2/hD3 selectivity ratio was obtained for the less active distomer (−)-S 17777, as well as for U 99194 (Millanet al., 1994; 1995; Waters et al., 1993). Overall, the lowest D2/D3 ratios were observed for (+)-AJ 76 and (+)-UH 232 and the highest for (+)-S 14297 and GR 103,691 (table 1). However, GR 103,691 displayed high affinity at 5-HT1A receptors and alpha-1 adrenoceptors (table 2), in accordance with the findings of Murray et al.(1994). (+)-S 14297 also showed modest (about 30 fold-lower) activity at muscarinic hM1 receptors (Millan et al., 1995). Nevertheless, among the ligands tested, (+)-S 14297 presents a reasonable combination of marked hD3 affinity, pronounced hD3/hD2 selectivity and modest interactions at other receptor sites. Indeed, (+)-S 14297 shows ≥100-fold lower affinity at hD1 and hD5 receptors, multiple serotonergic and adrenergic receptors and all other sites (>50) as yet examined, with the exception of ς1 sites, for which it shows modest affinity (196 nM) (Millan et al., 1995).
Induction and blockade of hypothermia.
In a recent study, the comparative importance of D2 and D3 sites in mediating hypothermia was addressed in detail (Millan et al., 1994; 1995; Rivet et al., 1996). In a result consistent with a putative role of D3 receptors, (+)-PD 128,907 elicited hypothermia herein (fig. 2). Notwithstanding its superior affinity at D3 (and D2) receptors as compared with (+)-7-OH-DPAT (table 1; Akunne et al., 1995;Bristow et al., 1996), (+)-PD 128,907 was lesspotent in decreasing CT. We have also noted an inferior potency of (+)-PD 128,907 relative to (+)-7-OH-DPAT in a diversity of in vivo models in which pre- and/or postsynaptic D3and/or D2 receptors are implicated, including a reduction of locomotion, inhibition of the firing rate of ventral tegmental area-localized dopaminergic neurons and suppression of PRL secretion (Brocco et al., 1995; Gobert et al., 1995; Lejeune, F., unpublished observation). Collectively, these observations suggest that (+)-PD 128,907 is a less potent ligand than (+)-7-OH-DPAT in vivo, which probably reflects differential absorption, metabolism or access to the CNS.
(±)-S11566 and (+)-S 14297, but not (−)-S 17777, stereospecifically block the induction of hypothermia by (+)-7-OH-DPAT (table 3; fig. 3) (Millan et al., 1994; 1995). These data are extended herein to antagonism of the hypothermic actions of (+)-PD 128,907. The inability of GR 103,691 to block hypothermia, even at high doses, may reflect its poor in vivo bioavailability (see below). By contrast, the preferential D3 antagonists U 99194 and nafadotride both antagonized hypothermia elicited by (+)-7-OH-DPAT and (+)-PD 128,907 (table 3; fig. 3). The modestly preferential D3 antagonists (+)-UH 232 and (+)-AJ 76 likewise blocked the actions of (+)-7-OH-DPAT and (+)-PD 128,907, and, like all other antagonists, they did not modify basal CT. The lack of influence of (+)-UH 232 on basal CT is of note inasmuch as a recent study of transfected NG 10815 cells, employing stimulation of mitogenesis as a measure of coupling efficacy, suggested that (+)-UH 232 behaves as a weak partial agonist at D3 receptors (Griffon et al., 1995). However, it is difficult to compare efficacies at heterologously expressed receptors in cell lines to those at native receptors in situ. Further, the NG 10815/hD3receptor expression system possesses a high receptor density and is probably of greater sensitivity than the population of postsynaptic D3 receptors thought to mediate hypothermia (Lajinesset al., 1993; Millan et al., 1995; Sautelet al., 1995). Although the present data are consistent with a role of D3 receptors in the induction of hypothermia, a role of D2 sites should not be excluded, and the availability of more selective D2 and D3receptor antagonists, as well as studies in transgenic mice, will be necessary for a further evaluation of the respective roles of D3 and D2 receptors in controlling CT.
PRL secretion, DA synthesis and catalepsy: blockade of tonically active D2 receptors.
Tuberoinfundibular dopaminergic pathways exert a tonic, inhibitory control on the secretion of PRL (Ben-Jonathan et al., 1989; McDonald et al., 1984). Although multiple dopaminergic receptor types may indirectly modulate PRL secretion via these tuberoinfundibular neurons (Berry and Gudelsky, 1990), dopaminergic ligands modulate PRL secretion primarily via actions at dopaminergic receptors on lactotrophs in the adenohypophysis (McChesney et al., 1991). Neuroanatomical studies have indicated that these are of the D2 type (Levant et al., 1993; Lévesque, 1996). Pharmacological support for a predominant role of D2receptors has also been obtained. Thus the relative potencies of dopaminergic agonists and antagonists in decreasing and increasing PRL secretion, respectively, correlate with their affinity at D2 (but not D3) sites (Millan et al., 1995; Rivet et al., 1996). Indeed, compared with haloperidol, (+)-S 14297 modified PRL secretion very little (fig. 4). Further, U 99194, in line with its low D2 affinity, exerted only a weak influence on PRL levels. A previous study has also reported that (+)-AJ 76 weakly modifies PRL secretion in vivo(Eriksson et al., 1986). Although GR 103,691 was also inactive, this observation must be interpreted in the light of the following comments about its poor activity in vivo. Indeed, the affinity of GR 103,691 at hD2 receptors is virtually identical to that of (+)-UH 232, which potently elicited PRL secretion.
There is anatomical (Bouthenet et al., 1991; Diazet al., 1995; Gehlert et al., 1992; Herroelenet al., 1994; Larson et al., 1995; Lévesqueet al., 1992; Richtand et al., 1995;Meador-Woodruff et al., 1996), electrophysiological (Devoto et al., 1995; Lejeune and Millan, 1995), behavioral (Sanger et al., 1996) and biochemical (Gainetdinov et al., 1995; 1996; Gobert et al., 1995; 1996; O’Hara et al., 1996; Rivet et al., 1994; 1996; Tang et al., 1994) evidence that D3 receptors contribute to the phasic, inhibitory control of ascending dopaminergic pathways. However, D3 (auto)receptors do not appear to inhibittonically the activity of dopaminergic neurons (Koeltzow et al., 1998; Millan et al., 1995). Rather, this role is fulfilled by colocalized D2autoreceptors (Bowery et al., 1994; 1996; Meador-Woodruffet al., 1996; Mercuri et al., 1997; Millanet al., 1995; Momiyama et al., 1993; Pierceyet al., 1996; Seabrook et al., 1995). Correspondingly, the facilitation of DA synthesis and release in mesolimbic, mesocortical and nigrostriatal pathways reflects inactivation of D2 receptors, and this parameter was not significantly affected by (+)-S 14297. Thus the increase in DOPAC:DA levels provoked by (+)-UH 232 and (+)-AJ 76 supports previous studies performed with other procedures in suggesting that they accelerate DA turnover by blockade of D2 autoreceptors (Waters et al., 1994a). Furthermore, the induction of DA synthesis by nafadotride extends a previous study of Sautel et al. (1995)in which, over a similar dose range, nafadotride increased levels of DA metabolites in the striatum. The modest influence of U 99194 on DA turnover is also consistent with its weak affinity at these sites (Waters et al., 1993).
An enhancement in striatal DA turnover is generally correlated with the induction of catalepsy, a behavior attributed to blockade of postsynaptic dopaminergic receptors in the striatum and predictive of an extrapyramidal syndrome in the human (Casey, 1993). Support for an opposite control of motor behavior by D3vs.D2 receptors, and for a role of D2 receptors in mediating catalepsy, may be derived from several lines of study. First, knockout mice that lack D2 receptors display a catalepsy-like state, whereas mice with a null mutation for D3 receptors are hyperactive (Accili et al., 1996; Baik et al., 1995). Second, the preferential activation of postsynaptic D2 and of D3receptors enhances and depresses locomotor behavior, respectively (Bristow et al., 1996; Depoortere et al., 1996;Khroyan et al., 1995; Kling-Petersen et al., 1995; Sanger et al., 1996). Third, blockade of postsynaptic D2 and D3 receptors respectively suppresses and enhances (though more variably) locomotor activity (Millan et al., 1997; Sautel et al., 1996; Svensson et al., 1994; Waters et al., 1993; 1994b). Fourth, D2 and D3 receptors exert an opposite control of gene expression of neurotensin in the nucleus accumbens, a neuropeptide that modulates locomotor behavior (Diaz et al., 1994). Fifth, the potency of dopaminergic antagonists in eliciting catalepsy correlates closely with affinity at D2 receptors (Millan et al., 1997). Sixth, (+)-S 14297 inhibits the induction of catalepsy by haloperidol (Brocco et al., 1995;Millan et al., 1997). In analogy to the lack of cataleptogenic potential of (+)-S 14297, U 99194 failed to elicit catalepsy herein (fig. 4) and increases locomotor activity by an action at postsynaptic D3 receptors (Waters et al., 1994b). Similarly, low doses of nafadotride increase motor behavior (Sautel et al., 1995), whereas higher doses elicit catalepsy (Sautel et al., 1995) (fig. 4). The cataleptic actions of nafadotride, (+)-UH 232 and (+)-AJ 76 at high doses evidently reflect blockade of D2 receptors. In analogy to the other models of activity at D2 receptors, GR 103,691 was inactive.
Mechanisms underlying induction of catalepsy, PRL secretion and DA turnover.
The patterns of data obtained in the models of PRL secretion, induction of cerebral DA turnover and catalepsy were broadly similar. In contrast to the potent actions of haloperidol, (+)-S 14297 was inactive and U 99194 displayed modest activity. Nafadotride, (+)-UH 232 and (+)-AJ 76, which manifest only a mild preference for D3 receptors, displayed an intermediate pattern of behavior. Thus these in vivo findings correspond to the relative preference of these antagonists for D3vs. D2 sites in vitro. Interestingly, differences among the antagonists were apparent not only as concerns their relative potencies but also as regards their maximal effects. Thus haloperidol consistently produced the most marked actions, (+)-S 14297 was inactive, U 99194 was weakly active and the other antagonists provoked intermediate responses (tables 4 and 5; fig. 4). The question arises why maximal effects should differ. If antagonist actions simply reflected interruption of the activity of spontaneously released DA at D2 receptors, they should, in theory, all elicit the same maximal effect. There are several possible explanations. First, the non-D2 (or D3) receptor interactions of drugs may modify their respective maximal effects. Second, the dose ranges of drugs employed may have been insufficient to occupy D2receptors substantially. This may be the case for (+)-S 14297, but dose-response curves for other drugs were pursued up to doses at which D2 antagonist properties are clearly expressed (Broccoet al., 1995; Brocco, M., unpublished observation). Third, a functional interplay between postsynaptic D2 and D1 sites is well established (Creese and Fraser, 1987), and D2 and D3 receptors colocalized on individual neurons may mutually oppose each other’s activities (Surmeier et al., 1992; Le Moine and Bloch, 1996). Indeed, as mentioned above, the enhancement of motor behavior by D3 receptor blockade counters the motor-suppressive effects of D2 receptor antagonism (Millan et al., 1995; 1997). Thus, for the ligands tested herein, a progressive reduction in cataleptogenic potential paralleled a progressive reinforcement of D3vs. D2 antagonist preference. A fourth possibility is that haloperidol behaves as an inverse agonist at D2 receptors (Hall and Strange, 1997; Nilsson et al., 1996). A differential degree of inverse agonist activity at D2 sites could explain the contrasting maximal effects of antagonists on PRL secretion, etc. (fig. 4). However, the concept of inverse agonist actions cannot easily explain the occurrence of catalepsy in D2 knockout mice (Baik et al., 1995). Further, like haloperidol, clozapine is an inverse agonist at D2 receptors, but it does not evoke catalepsy (Hall and Strange, 1997). Irrespective of the reasons underlying the difference in maximal effects, the present data suggest that antagonists possessing a marked preference for D3 versus D2receptors may have a low extrapyramidal syndrome potential.
The inactivity of GR 103,691.
Pharmacokinetic factors also contribute to differential drug effects, and GR 103,691 was inactive, even at high doses relative to its in vitro affinities, in each of the in vivo paradigms employed herein. It was recently shown that, in contrast to haloperidol, GR 103,691 does not influence cerebral c-fos expression (Hurley et al., 1996). Indeed, the only in vivo effect documented to date for GR 103,691 is its ability to block the locomotion elicited by infusion of muscimol into the ventral tegmental area (Murray et al., 1995). In this model, GR 103,691 (0.3 mg/kg s.c.) was 6-fold less potent than haloperidol (0.05 mg/kg s.c.) despite its 5-fold higher affinity at D3 receptors, an observation in line with the present data indicating a lower bioavailability than anticipated. However, its low affinity at D3 receptors (Ki = 200 nM) notwithstanding, clozapine (0.005 mg/kg s.c.) was more potent than GR 103,691 in blocking the actions of muscimol. This observation suggests that activity in this model may reflect the involvement of receptors other than, or in addition to, D3 sites. Thus, to date, there are no unambiguous data for functional actions of GR 103,691 at D3 (or D2) sites in vivo. We have, moreover, found that high doses of GR 103,691 (10 mg/kg s.c.) are inactive in several other behavioral models of D2 receptor-mediated activity in rats, including inhibition of amphetamine-induced locomotion and reduction of conditioned avoidance responses (Brocco, M., unpublished observation) models, whereas haloperidol is active, with ID50 values of 0.04 and 0.05 mg/kg s.c., respectively. In addition, haloperidol abolishes inhibition of the firing of dopaminergic neurons in the ventrotegmental area by (+)-PD 128,905 with an ID50 of 0.003 mg/kg i.v. In contrast, even at an 80-fold higher dose of 0.25 mg/kg i.v., GR 103,691 only partially (about 40%) inhibits the action of (+)-PD 128,907 (Lejeune, F., unpublished observation). Herein, we also examined this issue by exploiting the marked affinity of GR 103,691 for 5-HT1A receptors and alpha-1 adrenoceptors. Agonists at 5-HT1A sites and antagonists atalpha-1 adrenoceptors both inhibit serotonergic neurons in the DRN (Hjorth and Sharp, 1990; Lejeune et al., 1994). However, GR 103,691, at doses up to 0.5 mg/kg i.v., neither modified basal firing rates of serotonergic neurons nor attenuated the inhibitory influence on these of the 5-HT1A agonist (±)-8-OH-DPAT (see “Results”). These observations suggest that a lack of in vivo activity is a general property of GR 103,691 irrespective of the receptor type concerned. This electrophysiological study was performed by the i.v. route in order to minimize problems of metabolism. Further, GR 103,691 does not modify PRL secretion even though the relevant D2 receptors are localized outside the blood-brain barrier. Thus it is not clear whether rapid metabolism, poor CNS penetration and/or other factors underlie the low activity of GR 103,691 in vivo.
General discussion.
Of the ligands examined, (+)-S 14297 and GR 103,691 appear the most appropriate ligands for the characterization and differentiation of activity at D3 as compared with D2 receptors in vitro. In this regard, GR 103,691 possesses an advantage in terms of its higher affinity and overall selectivity for D3vs. D2sites. However, GR 103,691 displays the disadvantage of marked affinity at both 5-HT1A receptors and alpha-1 adrenoceptors. Although these properties may not interfere with its use in transfected cell lines, they compromise its utility in functional studies of more complex systems. Furthermore, GR 103,691 appears to possess little bioavailability in vivo. In this respect, although its modest affinity at hM1 and ς1receptors must be mentioned (Millan et al., 1995), (+)-S 14297 appears to be a more suitable ligand for in vivostudies. In fact, few ligands have been reported that possess a marked D3vs. D2 preference comparable to that of (+)-S 14297 and GR 103,691 in vitro. Moreover,in vivo data are generally not available. Nevertheless, the recently described, 2-substituted 2-aminotetralin GR 218,231 [2-(R,S)-(dipropylamino)-6-(4-methoxyphenylsulfonylmethyl)-1,2,3,4-tetrahydronaphtalene] possesses >100-fold selectivity for D3vs.D2 sites, and its functional actions in vivowill be of interest to explore (Murray et al., 1996). Moreover, a modestly preferential (10–20-fold) antagonist, L 741,626, at D2 receptors has been documented (Bowery et al., 1996). As concerns the D2 receptor “family” in general, several potent and selective D4 receptor antagonists have now become available, including S 18126 ({2-[4-(2,3-dihydro benzo [1,4]dioxin-6-yl) piperazin-1-yl methyl] indan-2-yl}) and L 745,870 (3-(4-[4-chlorophenyl]piperazin-1-yl)methyl-1H-pyrrolo[2,3b]pyridine). Notably, both S 18126 and L 745,870 were inactive in the functional models employed herein, which indicates a lack of involvement of D4 receptors (Boyfield et al., 1996; Bristowet al., 1997; Kulagowski et al., 1996; Millanet al., 1996 and in press).
Conclusion.
In conclusion, the present data underpin the hypothesis that the selective blockade of D3 receptors does not provoke extrapyramidal side effects and fails to perturb dopaminergic transmission. On the other hand, whether an antagonist action at D3 receptors contributes to the therapeutic efficacy of antipsychotic drugs remains to be determined (Levant, 1997;Sokoloff and Schwartz, 1995). More generally, the potential significance of D3 receptors in depression, drug abuse and other psychiatric disorders requires further evaluation (Acri et al., 1995; Caine and Koob, 1993; Roberts and Ranaldi, 1995;Sokoloff and Schwartz, 1995; Spealman, 1996; Strange, 1993; Wallaceet al., 1996; Willner, 1983). To develop an improved understanding of the physiological and therapeutic significance of D3 (and D2) receptors, we need additional, chemically diverse, potent and selective D3 and D2 receptor antagonists.
Acknowledgments
We thank V. Pasteau, S. Aubry, C. Chaput, L. Verrièle, H. Gressier and S. Girardon for their excellent technical assistance.
Footnotes
-
Send reprint requests to: Dr. Mark J. Millan, Institut de Recherches Servier, Centre de Recherches de Croissy, Psychopharmacology Department, 125, Chemin de Ronde, 78290—Croissy-sur-Seine (Paris), France.
- Abbreviations:
- AD
- active dose
- (+)-AJ 76
- {(+)-(cis-(+)-5-methoxy-1-methyl-2-(n-propylamino)tetralin}
- ANOVA
- analysis of variance
- CHO
- Chinese hamster ovary
- CT
- core temperature
- DA
- dopamine
- DOPAC
- dihydroxyphenylacetic acid
- DRN
- dorsal raphe nucleus
- ID
- inhibitory dose
- GR 103
- 691, {4′-acetyl-N-{4-[(2-methoxy-phenyl)-piperazin-1-yl]-butyl}-biphenyl-4-carboxamide
- 5-HT
- serotonin
- MED
- minimal effective dose
- MOE
- maximal observed effect
- (+)-7-OH-DPAT
- {(+)-7-hydroxy-2-(di-n-propylamino)-tetralin)
- (±)-8-OH-DPAT
- {(±)-8-hydroxy-2-(di-n-propylamino)-tetralin)}
- (+)-PD 128
- 907, {(+)-(4aR,10bR)-3,4,4a,10b-tetrahydro-4-propyl-2H,5H-[1]-benzopyrano-[4,3-b]-1,4-oxazin-9-ol}
- PRL
- prolactin
- (+)-UH 232
- {cis-(+)-1S,2R-5-methoxy-1-methyl-2-(di-n-propylamino)tetralin}
- (±)-S 11566
- {(±)-[7-(N,N-dipropylamino)-5,6,7,8-tetrahydro-naphtho-(2,3b)-dihydro,2,3-furane]}
- (+)-S 14297
- {(+)-[7-(N,N-dipropylamino)-5,6,7,8-tetrahydro-naphtho-(2,3b)-dihydro,2,3-furane]}
- (−)-S 17777
- {(−)-[7-(N,N-dipropylamino)-5,6,7,8-tetrahydro-naphtho-(2,3b)-dihydro,2,3-furane]}
- U 99194
- {5,6-dimethoxy-indan-2-yl) dipropylamine}
- Received September 8, 1997.
- Accepted May 19, 1998.
- The American Society for Pharmacology and Experimental Therapeutics
References
In this issue
A Comparative In Vitro and In VivoPharmacological Characterization of the Novel Dopamine D3Receptor Antagonists (+)-S 14297, Nafadotride, GR 103,691 and U 99194
