S 15535, A Novel Benzodioxopiperazine Ligand of Serotonin (5-HT)1A Receptors: I. Interaction with Cloned Human (h)5- HT1A, Dopamine hD2/hD3 and ha2A-Adrenergic Receptors in Relation to Modulation of Cortical Monoamine Release and Activity in Models of Potential Antidepressant Activity

The novel, potential anxiolytic, S 15535 (4-(benzodioxan-5-yl)1(indan-2-yl)piperazine), is an agonist and antagonist (weak partial agonist) at preand postsynaptic serotonin (5-HT)1A receptors, respectively. Herein, we characterized its influence on dialysate levels of 5-HT, dopamine (DA) and NAD simultaneously determined in single samples of the frontal cortex (FCX) of freely moving rats, and compared its activity in several other models of potential antidepressant (AD) properties with those of the 5-HT reuptake inhibitor (SSRI), fluoxetine. S 15535 displayed high affinity at cloned human (h) 5-HT1A receptors (Ki 5 0.7 nM) and .250-fold lower affinity at cloned hD2 (400 nM), hD3 (248 nM) and ha2A-adrenergic (AR) (190 nM) receptors. S 15535 (0.08–5.0 mg/kg s.c.) markedly and dose-dependently suppressed dialysate levels of 5-HT in the FCX, nucleus accumbens and striatum of freely moving rats, whereas fluoxetine (10.0 mg/kg s.c.) elevated levels of 5-HT in each structure. In contrast to 5-HT, dialysate levels of DA and NAD in the FCX were dose-dependently increased by S 15535, and this effect was mimicked by fluoxetine. The influence of S 15535 and fluoxetine on FCX levels of DA was regionally specific inasmuch as dialysate levels of DA in the accumbens and striatum were not modified. The selective 5-HT1A antagonist, WAY 100,635 (N-{2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl}-N-(2-pyridinyl) cyclohexanecarboxamide (0.16) transiently elicited a slight increase in cortical levels of 5-HT, an action opposite to that of S 15535. Further, in the presence of WAY 100,635 (0.16), the influence of S 15535 (0.63) on cortical levels of 5-HT, DA and NAD was markedly attenuated. Upon chronic administration of S 15535 or fluoxetine (10.0 mg/kg s.c. daily for 14 days, in each case), there was no significant alteration in the density of b-AR receptors in the FCX. However, in contrast to fluoxetine, S 15535 elicited a significant (25%) decrease in the density (Bmax) of 5-HT2A receptors labeled by [ H]ketanserin in the cortex; there was no alteration in Kd. In a learned helplessness paradigm in rats, S 15535 (0.63–40.0 mg/kg p.o.) markedly reduced escape deficits on each of three consecutive days of testing. Fluoxetine (2.0–8.0 mg/kg i.p.) was also active in each session, but presented a biphasic dose-response curve. Finally, under the conditions used, neither S 15535 (0.63–10.0) nor fluoxetine (0.63–10.0) decreased immobility time in the forced swim test. In conclusion, S 15535 is a selective ligand of cloned, h5-HT1A receptors. Its agonist actions at 5-HT1A autoreceptors underlie its ability to decrease extracellular levels of 5-HT in the FCX, and likely contribute to the increase in extracellular levels of DA and NAD evoked by S 15535 in this structure. Further, S 15535 is active in several other, although not all, models of potential AD activity. Thus, although S 15535 is under development as an anxiolytic agent, a further characterization of its putative AD actions would be of interest. A dysregulation of activity at 5-HT1A receptors is implicated in the pathophysiology of anxiety, depression and other psychiatric disorders (see Broekkamp et al., 1995; Coplan et al., 1995; Maes and Meltzer, 1995; Roth and Meltzer, 1995). Further, chemically diverse 5-HT1A receptor ligands exert anxiolytic and AD properties both in experimental models and in man; for example, the azaspirone, buspirone, the benzodioxane, flesinoxan, the naphthylpiperazine, S 14671 and the aminotetralin, 8-OH-DPAT (Fabre, 1990; Lucki et al., 1994; Schreiber et al., 1994; see Broekkamp et al., 1995; Received for publication March 19, 1996. ABBREVIATIONS: AD, antidepressant; AR, adrenergic; CHO, Chinese hamster ovary; DA, dopamine; LH, learned helplessness; NAD, noradrenaline; 5-HT, serotonin; SSRI, serotonin reuptake inhibitor; 5-HIAA, 5-hydroxyindole acetic acid; DOPAC, dihydroxyphenylacetic acid; DRN, dorsal raphe nucleus. 0022-3565/97/2821-0132$03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 282, No. 1 Copyright © 1997 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 282:132–147, 1997 132 at A PE T Jornals on M ay 3, 2017 jpet.asjournals.org D ow nladed from Coplan et al., 1995; Maes and Meltzer, 1995; Roth and Meltzer, 1995). There is also evidence that 5-HT1A receptors are involved in the expression of the AD properties of fluoxetine and other SSRIs (Detke et al., 1995; Gambarana et al., 1995). Actions at both preand postsynaptic 5-HT1A receptors appear to play a role in these therapeutic actions of 5-HT1A receptor ligands, although their relative contribution remains to be clarified (see Coplan et al., 1995; Lucki et al., 1994; Maes and Meltzer, 1995; Roth and Meltzer, 1995; Thiébot and Martin, 1991). In light of the above, 5-HT1A receptor ligands offer an attractive target for the development of novel treatments for anxiety, depression and related psychiatric disorders. We have recently discovered a chemically novel benzodioxopiperazine, S 15535 (under clinical development for the treatment of anxiety), which displays exceptional selectivity for 5-HT1A receptors in rodents (Millan et al., 1994b). This is of importance because, with the exception of the phenylpiperazine antagonist, WAY 100,635 (Pike et al., 1995), virtually all 5-HT1A ligands (or their metabolites) exhibit marked antagonist properties at other receptor types in vivo; in particular, DA D2 receptors (e.g., binospirone, zalospirone and buspirone), a1-AR receptors (flesinoxan and NAN-190), a2-AR receptors (NAN-190, buspirone and ipsapirone) and b-AR receptors ((2)-pindolol and other alkylarylamines) (Millan et al., 1994b; Van Wijngaarden et al., 1990). Further, S 15535 behaves in vivo as an agonist at 5-HT1A autoreceptors and as an antagonist (or weak partial agonist) at postsynaptic 5-HT1A receptors (Newman-Tancredi et al., 1996a; Peglion et al., 1995). This pattern of activity of S 15535 may be compared with high-efficacy agonists at preand postsynaptic 5-HT1A receptors, such as 8-OH-DPAT, S 14671 and flesinoxan (Millan et al., 1992); ligands acting as full/partial agonists at pre/postsynaptic 5-HT1A receptors, such as buspirone, ipsapirone and zalospirone (Glennon and Dukat, 1995; Gobert et al., 1995a; Millan et al., 1992; Thiébot and Martin, 1991) and ligands which behave as antagonists at both preand postsynaptic 5-HT1A receptors, such as WAY 100,635, (2)-alprenolol and (2)-tertatolol (Millan et al., 1994b; Pike et al., 1995). The combination of marked selectivity for 5-HT1A receptors and differential efficacy at pre (agonist)and post (antagonist)-synaptic populations appears to impart anxiolytic properties of S 15535 in the relative absence of the disruptive motor, endocrine and amnesic actions provoked by the activation of postsynaptic sites (Millan et al., 1997, accompanying paper). The purpose of the studies reported in the present paper was to evaluate the activity of S 15535 in several models of potential AD properties. Complementary AD activity would be of particular interest for an anxiolytic agent in the light of the increasing diagnosis of co-morbid anxious and depressive states (Coplan et al., 1995). S 15535 possesses high affinity at rat 5-HT1A versus DA D2 and a2D-AR receptors (Millan et al., 1994b) and, herein, we examine its interaction with recombinant h5-HT1A, hD2 and hD3 and ha2A-AR (the human homolog of rat a2D) receptors. Serotonin1A, D2/D3 and a2A-AR sites are present as autoreceptors on serotoninergic, dopaminergic and adrenergic pathways, respectively (Gobert et al., 1995a, b; Millan et al., 1995). In this respect, we focused on a possible modulation by S 15535 of the activity of dopaminergic and adrenergic projections to the FCX. This is of interest inasmuch as a deficit in prefrontal cortical dopaminergic transmission may be a common feature of depressive states (Carlson et al., 1993; Karoum et al., 1994; Zacharko and Anisman, 1991). Indeed, mechanistically diverse AD drugs, such as SSRIs, tricyclics and the 5-HT1A partial agonist, buspirone, enhance dialysate levels of DA in rat FCX (Arborelius et al., 1993; Jordan et al., 1994; Tanda et al., 1994; Wong et al., 1995). Moreover, adrenergic pathways innervating the cortex play a facilitatory role in cognito-attentional processes, which may be compromised in depressive states, and an enhancement in their activity may be associated with AD effects (Aston-Jones et al., 1991b; Broekkamp et al., 1995). Thus, we examined the influence of S 15535 on dialysate levels of DA, NAD and 5-HT in the FCX of freely moving rats and compared these actions with those in two further structures in which dopaminergic pathways are implicated in the control of mood and cognition: the nucleus accumbens and the striatum (Willner, 1995). As a second and related approach, we examined the influence of long-term administration of S 15535 on the density of b-AR and 5-HT2A receptors in the cortex. There is evidence implicating 5-HT2A receptors in the control of mood, and their activation may be related to the pathophysiology of depressive, as well as anxiolytic and psychotic, states (see Coplan et al., 1995; Maes and Meltzer, 1995; Roth and Meltzer, 1995). As a third model of potential AD activity, the LH procedure was selected inasmuch as the escape deficits evoked by inescapable shock are considered to reflect a state of decreased motivation, resignation and psychomotor retardation, key elements of depressive conditions in man (Thiébot and Martin, 1991). Further, this model is responsive to both SSRIs, such as fluoxetine, and to agonists at 5-HT1A receptors, such as 8-OH-DPAT and buspirone (Martin et al., 1990a, b, 1991). Finally, we also examined the influence of S 15535 on the behavior of rats in a forced swim paradigm (Porsolt et al., 1979). This model displays certain similarities to the LH paradigm and is sensitive to high-efficacy 5-HT1A agonists, although it is poorly responsive to low-efficacy 5-HT1A agonists and SSRIs (Lucki et al., 1994; Schreiber and De Vry, 1993; Schreiber et al., 1994).