A Comparative In Vitro and In Vivo Pharmacological Characterization of the Novel Dopamine D3 Receptor Antagonists (1)-S 14297, Nafadotride,

The benzofurane (1)-S 14297, the benzamide nafadotride, the aminoindane U 99194 and the arylpiperazine GR 103,691 have been proposed as “selective” antagonists at dopamine D3 vs. D2 receptors. Herein, we compared their in vitro affinities and in vivo actions to those of the aminotetralin D3 antagonists (1)-AJ 76 and (1)-UH 232. Affinities at recombinant, human (h)D3 and/or hD2 sites were determined by employing the mixed D2/D3 antagonist [ I]-iodosulpride and the preferential D3 ligands [H]-(1)-PD 128,907 and [H]-(1)-S 14297. [H]-(1)-PD 128,907, [H]-(1)-S 14297 and [I]-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. [ H]-(1)-PD 128,907) was GR 103,691 (0.4) ' nafadotride (0.5) . haloperidol (2) ' (1)-UH 232 (3) ' (1)-S 14297 (5) . (1)-AJ 76 (26) . U 99194 (160). The rank order of preference (Ki ratio, D2:D3) for D3 receptors (labeled by [ H]-PD 128,907) vs. D2 sites (labeled by [I]-iodosulpride) was (1)-S 14297 (61) ' GR 103,691 (60) . U 99194 (14) . nafadotride (9) ' (1)-UH 232 (8) ' (1)-AJ 76 (6) . haloperidol (0.2). (1)-S 14297 and GR 103,691 also showed greater than 100-fold selectivity at dopamine hD3 vs. 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 (1)-PD 128,907 (0.63 mg/kg s.c.) and a further preferential D3 agonist, (1)-7OH-DPAT (0.16 mg/kg s.c.). On the other hand, haloperidol, (1)-AJ 76, (1)-UH 232 and nafadotride all induced catalepsy in rats, whereas (1)-S 14297, U 99194 and GR 103,691 were inactive. Haloperidol, (1)-AJ 76, (1)-UH 232, nafadotride and (weakly) U 99194 also enhanced prolactin secretion and striatal dopamine synthesis, whereas (1)-S 14297 and GR 103,691 were inactive. However, despite its high affinity at 5-HT1A receptors and alpha-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 (6)-8OH-DPAT (0.005 mg/kg i.v.), a result that casts doubt on its activity in vivo. In conclusion, both (1)-S 14297 and GR 103,691 are markedly selective ligands that permit the characterization of actions at hD3 vs. hD2 receptors in vitro, but (1)-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 expressed via 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 Received for publication September 8, 1997. ABBREVIATIONS: AD, active dose; (1)-AJ 76, {(1)-(cis-(1)-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, {49-acetyl-N-{4-[(2-methoxy-phenyl)-piperazin-1-yl]-butyl}-biphenyl-4-carboxamide; 5-HT, serotonin; MED, minimal effective dose; MOE, maximal observed effect; (1)-7-OH-DPAT, {(1)-7-hydroxy-2-(di-n-propylamino)-tetralin); (6)-8-OH-DPAT, {(6)-8-hydroxy-2-(di-n-propylamino)-tetralin)}; (1)-PD 128,907, {(1)-(4aR,10bR)-3,4,4a,10b-tetrahydro-4-propyl-2H,5H-[1]-benzopyrano-[4,3-b]-1,4-oxazin-9-ol}; PRL, prolactin; (1)-UH 232, {cis-(1)-1S,2R5-methoxy-1-methyl-2-(di-n-propylamino)tetralin}; (6)-S 11566, {(6)-[7-(N,N-dipropylamino)-5,6,7,8-tetrahydro-naphtho-(2,3b)-dihydro,2,3-furane]}; (1)-S 14297, {(1)-[7-(N,N-dipropylamino)-5,6,7,8-tetrahydro-naphtho-(2,3b)-dihydro,2,3-furane]}; (2)-S 17777, {(2)-[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}. 0022-3565/98/2871-0187$03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 287, No. 1 Copyright © 1998 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 287:187–197, 1998 187 at A PE T Jornals on Jne 2, 2017 jpet.asjournals.org D ow nladed from 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; Bristow et al., 1997; Kulagowski et al., 1996; Merchant et 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; Lévesque et al., 1992). These approaches, together with studies of the preferential D3 agonists [H]-(1)-PD 128,907 and [H]-(1)-7-OH-DPAT (Hall et al., 1996; Herroelen et al., 1994), suggest that D3 sites 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 (Diaz et al., 1995; Gobert et al., 1995; Koeltzow et al., 1998; Meador-Woodruff et al., 1996). Despite the utility of gene knockout and antisense strategies (Accili et al., 1996; Baik et al., 1995; Carta et al., 1996, Tepper et al., 1997), ligands that interact selectively with D3 vs. 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 [I]-iodosulpride or [H]-spiperone that do not differentiate these sites (Chio et al., 1993; Millan et al., 1995; Sokoloff et al., 1992; Tang et al., 1994). On the basis of such studies, the aminotetralins (1)-AJ 76 and (1)-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 D3 vs. D2 sites have been identified: the aminoindane U 99194 (Waters et al., 1993), the benzofurane (6)-S 11566 and its active (1)-isomer (1)-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, (1)-S 14297 has been extensively employed in the functional evaluation of the potential significance of D3 receptors in vivo. It has been shown that (1)-S 14297 inhibits the hypothermia induced by dopaminergic agonists such as (1)-7-OH-DPAT and quinpirole in rodents, which suggests an involvement of D3 receptors in the control of CT (Millan et al., 1994; 1995; Rivet et al., 1996). At comparable doses, (1)-S 14297 neither modifies PRL secretion nor induces catalepsy in rats, two responses reflecting blockade of D2 receptors (Brocco et al., 1995; Millan et al., 1995; 1997). Interestingly, (1)-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, (1)-S 14297 attenuates the inhibitory influence of (1)-7-OH-DPAT and (1)-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 vivo functional characterization of actions mediated by D3 receptors. However, there has not yet been any comparative evaluation of the properties of these ligands. Thus, using the radioligands [H]-(1)-S 14297 and [H]-(1)-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 [I]-iodosulpride, the present study undertook a comparative evaluation of the binding profiles of (1)-S 14297, nafadotride, U 99194, GR 103,691, (1)-AJ 76, (1)-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 hD3 vs. 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 D3 receptors. 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 [I]-iodosulpride (0.1 and 0.2 nM for D2 and D3, respectively), [ H]-(1)-S 14297 (7 nM for D3) and [H]-(1)-PD 128,907 (2 nM for D3). Nonspecific binding was defined using 10 mM raclopride. Binding conditions for [I]-iodosulpride and [H]-(1)-S 14297 were as previously described (Newman-Tancredi et al., 1995; Sokoloff et al., 1992), and they were the same for [H]-(1)-PD 128,907 except that incubations were carried out for 90