Jeffrey Sprouse

5-HT1A receptor activation contributes to ziprasidone-induced dopamine release in the rat prefrontal cortex

Background: Ziprasidone (Zeldox) is a novel antipsychotic with a unique combination of antagonist activities at monoaminergic receptors and transporters and potent agonist activity at serotonin 5-HT1A receptors. 5-HT1A receptor agonism may be an important feature in ziprasidone’s clinical actions because 5-HT1A agonists increase cortical dopamine release, which may underlie efficacy against negative symptoms and reduce dopamine D2 antagonist-induced extrapyramidal side effects. This study investigated the in vivo 5-HT1A agonist activity of ziprasidone by measuring the contribution of 5-HT1A receptor activation to the ziprasidone-induced cortical dopamine release in rats. Methods: Effects on dopamine release were measured by microdialysis in prefrontal cortex and striatum. The role of 5-HT1A receptor activation was estimated by assessing the sensitivity of the response to pretreatment with the 5-HT1A antagonist, WAY-100635. For comparison, the D2/5-HT2A antagonists clozapine and olanzapine, the D2 antagonist haloperidol, the 5-HT2A antagonist MDL 100,907 and the 5-HT1A agonist 8-OHDPAT were included. Results: Low doses (<3.2 mg/kg) of ziprasidone, clozapine, and olanzapine increased dopamine release to approximately the same extent in prefrontal cortex as in striatum, but higher doses (≥3.2 mg/kg) resulted in an increasingly preferential effect on cortical dopamine release. The 5-HT1A agonist 8-OHDPAT produced a robust increase in cortical dopamine (DA) release without affecting striatal DA release. In contrast, the D2 antagonist haloperidol selectively increased striatal DA release, whereas the 5-HT2A antagonist MDL 100,907 had no effect on cortical or striatal DA release. Prior administration of WAY-100635 completely blocked the cortical DA increase produced by 8-OHDPAT and significantly attenuated the ziprasidone- and clozapine-induced cortical DA increase. WAY-100635 pretreatment had no effect on the olanzapine-induced DA increase. Conclusions: The preferential increase in DA release in rat prefrontal cortex produced by ziprasidone is mediated by 5-HT1A receptor activation. This result extends and confirms other in vitro and in vivo data suggesting that ziprasidone, like clozapine, acts as a 5-HT1A receptor agonist in vivo, which may contribute to its activity as an antipsychotic with efficacy against negative symptoms and a low extrapyramidal side effect liability.

8-OH-DPAT as a 5-HT7 agonist: phase shifts of the circadian biological clock through increases in cAMP production

Neurons in the suprachiasmatic nucleus (SCN), the site of the endogenous biological clock in mammals, fire spontaneously, peaking in firing rate near ZT6 or at the midpoint of the light phase in a 12:12 light-dark cycle. In rat hypothalamic slices, tissue incubations with drugs can produce a shift in this daily rhythm, the magnitude of which is dependent upon dose and the time of treatment. Previous work with 8-OH-DPAT had noted its ability to produce a phase advance, an earlier occurrence of the peak in neuronal firing, when applied at ZT6. Activation of 5-HT7 receptors was thought to be responsible for the shift, despite the clear preference of 8-OH-DPAT for 5-HT1A sites in terms of receptor binding affinity. In the present work, the actions of 8-OH-DPAT in SCN slices were confirmed and expanded to include additional dose-response and antagonist treatments. By itself, 8-OH-DPAT produced a concentration-dependent phase advance that was sensitive to co-application with 5-HT7 antagonists (ritanserin, mesulergine, SB-269970), but not to 5-HT1A antagonists (WAY-100,635, UH-301). Assignment of the receptor mechanisms for the antagonists employed was accomplished in experiments measuring binding affinities and the generation of cAMP, the latter monitored in a HEK-293 cell line expressing the r5-HT7 receptor and in tissue derived from rat SCN. The results indicate that the increases observed in cAMP levels are small but appear to be sufficient to produce a pharmacological resetting of the clock pacemaker. By aiding in the identification of the 5-HT receptor subtype responsible for the observed phase shifts and cAMP changes, 8-OH-DPAT represents an important pharmacological tool for 5-HT7 receptor activation, essentially broadening its role as the prototypical 5-HT1A agonist to one combining these two receptor activities.

Fluoxetine Modulates the Circadian Biological Clock via Phase Advances of Suprachiasmatic Nucleus Neuronal Firing

BACKGROUND The documented ability of serotonin (5-HT) to directly modulate circadian rhythms prompted interest in a similar role for therapeutic agents that readily enhance 5-HT neurotransmission, namely the selective serotonin reuptake inhibitors (SSRIs). METHODS Extracellular recordings of unit firing of suprachiasmatic nucleus (SCN) neurons maintained in slice culture enabled determinations of circadian rhythmicity. Shifts in the peak of activity were determined during the next circadian cycle following drug exposure. RESULTS Fluoxetine (10 microm, 60 minutes incubation) produced robust phase advances only in the presence of L-tryptophan (.5 microm), added to maintain serotonergic tone. CONCLUSIONS Actions of SSRIs at the level of the circadian biological clock add to the list of pharmacological effects for this drug class and encourage speculation as to their importance clinically.

An Inhibitor of Casein Kinase Iϵ Induces Phase Delays in Circadian Rhythms under Free-Running and Entrained Conditions

Casein kinase Iϵ (CKIϵ) is an essential component of the biological clock, phosphorylating PER proteins, and in doing so regulating their turnover and nuclear entry in oscillator cells of the suprachiasmatic nucleus (SCN). Although hereditary decreases in PER phosphorylation have been well characterized, little is known about the consequences of acute enzyme inhibition by pharmacological means. A novel reagent, 4-[3-cyclohexyl-5-(4-fluoro-phenyl)-3H-imidazol-4-yl]-pyrimidin-2-ylamine (PF-670462), proved to be both a potent (IC50 = 7.7 ± 2.2 nM) and selective (>30-fold with respect to 42 additional kinases) inhibitor of CKIϵ in isolated enzyme preparations; in transfected whole cell assays, it caused a concentration-related redistribution of nuclear versus cytosolic PER. When tested in free-running animals, 50 mg/kg s.c. PF-670462 produced robust phase delays when dosed at circadian time (CT)9 (–1.97 ± 0.17 h). Entrained rats dosed in normal light-dark (LD) and then released to constant darkness also experienced phase delays that were dose- and time of dosing-dependent. PF-670462 yielded only phase delays across the circadian cycle with the most sensitive time at CT12 when PER levels are near their peak in the SCN. Most importantly, these drug-induced phase delays persisted in animals entrained and maintained in LD throughout the entire experiment; re-entrainment to the prevailing LD required days in contrast to the rapid elimination of the drug (t1/2 = 0.46 ± 0.04 h). Together, these results suggest that inhibition of CKIϵ yields a perturbation of oscillator function that forestalls light as a zeitgeber, and they demonstrate that pharmacological tools such as PF-670462 may yield valuable insight into clock function.

Combined Administration of a 5-Hydroxytryptamine (5-HT)1D Antagonist and a 5-HT Reuptake Inhibitor Synergistically Increases 5-HT Release in Guinea Pig Hypothalamus In Vivo

Abstract: In vivo microdialysis in guinea pig hypothalamus was used to study the effect of serotonin [5‐hydroxytryptamine (5‐HT)] subtype 1D autoreceptor blockade on the increase in extracellular 5‐HT levels produced by a selective 5‐HT reuptake inhibitor (SSRI). Administration of the selective 5‐HT1D antagonist GR127935 at 0.3 mg/kg had no effect, but 5 mg/kg significantly increased extracellular levels of 5‐HT and 5‐hydroxyindoleacetic acid to 135% of basal values. Moreover, at these doses GR127935 significantly attenuated the decrease in extracellular 5‐HT levels following local perfusion with the selective 5‐HT1D agonist CP‐135,807. The SSRI sertraline at 2 mg/kg increased 5‐HT levels to 130% of basal levels. The combination of this low dose of sertraline with either dose of GR127935 resulted in a pronounced, long‐lasting increase in 5‐HT levels to 230% of basal values. These results indicate that the effects of an SSRI on terminal 5‐HT are significantly enhanced by coadministration of a 5‐HT1D antagonist and confirm that in addition to somatodendritic 5‐HT1A autoreceptors, terminal 5‐HT1D autoreceptors mitigate the effect of SSRIs on terminal 5‐HT. As such, antagonists of the 5‐HT1D autoreceptor could be useful as rapidly acting antidepressants and may shorten the onset of antidepressant action when combined with SSRIs.

Comparison of the novel antipsychotic ziprasidone with clozapine and olanzapine: inhibition of dorsal raphe cell firing and the role of 5-HT1A receptor activation.

Ziprasidone is a novel antipsychotic agent which binds with high affinity to 5-HT1A receptors (Ki = 3.4 nM), in addition to 5-HT1D, 5-HT2, and D2 sites. While it is an antagonist at these latter receptors, ziprasidone behaves as a 5-HT1A agonist in vitro in adenylate cyclase measurements. The goal of the present study was to examine the 5-HT1A properties of ziprasidone in vivo using as a marker of central 5-HT1A activity the inhibition of firing of serotonin-containing neurons in the dorsal raphe nucleus. In anesthetized rats, ziprasidone dose-dependently slowed raphe unit activity (ED50 = 300 μg/kg IV) as did the atypical antipsychotics clozapine (ED50 = 250 μg/kg IV) and olanzapine (ED50 = 1000 μg/kg IV). Pretreatment with the 5-HT1A antagonist WAY-100,635 (10 μg/kg IV) prevented the ziprasidone-induced inhibition; the same dose of WAY-100,635 had little effect on the inhibition produced by clozapine and olanzapine. Because all three agents also bind to α1 receptors, antagonists of which inhibit serotonin neuronal firing, this aspect of their pharmacology was assessed with desipramine (DMI), a NE re-uptake blocker previously shown to reverse the effects of α1 antagonists on raphe unit activity. DMI (5 mg/kg IV) failed to reverse the inhibitory effect of ziprasidone but produced nearly complete reversal of that of clozapine and olanzapine. These profiles suggest a mechanism of action for each agent, 5-HT1A agonism for ziprasidone and α1 antagonism for clozapine and olanzapine. The 5-HT1A agonist activity reported here clearly distinguishes ziprasidone from currently available antipsychotic agents and suggests that this property may play a significant role in its pharmacologic actions.

Comparison of the effects of sertraline and its metabolite desmethylsertraline on blockade of central 5-HT reuptake in vivo

N-demethylation of the selective serotonin reuptake inhibitor sertraline to desmethylsertraline yields a compound with 10- to 20-fold less potency at blocking serotonin (5-HT) reuptake as measured in vitro. In the present study desmethylsertraline (DMS) was examined in two in vivo models of reuptake inhibition—elevation of extracellular 5-HT in the corpus striatum as measured by microdialysis and inhibition of firing of serotonin-containing dorsal raphe neurons. Whereas sertraline (1, 3.2, and 10 mg/kg SC) produced a dose-dependent increase in extracellular 5-HT and a decrease in 5-HIAA in rat striatum, desmethylsertraline was without effect on either parameter. In similar fashion, desmethylsertraline had no effect on dorsal raphe cell firing at a dose (1,000 μg/kg IV) nearly 20-fold the ED50 for sertraline (52 μg/kg). Taken together, these data suggest that DMS does not contribute to the blockade of central 5-HT reuptake produced by sertraline in vivo and therefore would be expected to play a negligible role in its clinical activity.

Circadian Rhythm Phenotype of 5-HT₇ Receptor Knockout Mice: 5-HT and 8-OH-DPAT-Induced Phase Advances of SCN Neuronal Firing

In vitro neuronal recordings in the SCN have clearly documented shifts in the peak of unit activity following the application of serotonergic agents, and yet selectivity issues with these very tools have limited progress in establishing the precise receptor mechanisms. As an alternative strategy, mice werebred (C57BL/6J) lacking 1 serotonin receptor, the 5-HT7, to serve as a null background for this subtype; earlier work had documented the involvement of 5-HT7 receptors in the phase advances elicited by 8-OH-DPAT, a mixed 5-HT1A/7 agonist, in SCN slices prepared from rat donors. Single-unit recordings in sequential electrode passes revealed peaks of activity that occurred at nearly the same time in the knockout (KO; ZT4.2 ± 0.6) and wild-type animals (WT; ZT4.3 ± 0.1), where ZT0 marks the beginning of the light phase in a 12:12 LD cycle. Bath application of 8-OH-DPAT produced a phase advance in neuronal firing (2.1 ± 0.5 h) when applied 1 circadian cycle earlier at ZT6 (10 μM, 10 min), but surprisingly, the mean phase advance in slices prepared from KO mice (2.3 ± 0.1 h) was no different. Coapplication of 8-OH-DPAT with WAY-100,635 (10 μM), a highly selective 5-HT1A antagonist, significantly reduced the phase advance, both in experiments with WT and KO mice, suggesting the greater importance of this serotonin sub-type independent of genetic modification. 5-HT itself (0.5 ±M, 10 min) at ZT6 also yielded phase advances that were indistinguishable in slices prepared from WT and KO mice (1.8 ± 0.4 h and 2.1 ± 0.2 h, respectively) and that were also sensitive to WAY-100,635. Unlike the pattern with 8-OH-DPAT, however, 5-HT-induced phase advances, in both WT and KO mice, were blocked by ritanserin, in this paradigm useful as a 5-HT5A/7 antagonist (in addition to its more typical role as a 5-HT2A/2C antagonist). Serotonin antagonists when administered alone were without effect in slices from WT mice but produced significant phase shifts when administered to those from KO animals. Taken together, these results highlight the importance of the species used in establishing receptor mechanism. More provocatively, they support the involvement of multiple serotonin receptors in shifting the phase of circadian rhythms at ZT6.

2-aryloxy-4-alkylaminopyridines: discovery of novel corticotropin-releasing factor 1 antagonists.

An orally active clinical candidate of corticotropin-releasing factor 1 (CRF 1) antagonist 1 showed a significant positive food effect in dog and human after oral administration. Efforts to address the food effect issue led us to explore and discover compounds in series 2 as orally active CRF 1 receptor antagonists, in which some compounds showed improved physicochemical properties while retaining desired pharmacological properties. Compound 3a (CP-376395) was selected for further development, due not only to its reduced food effects but also its greater efficacy in CNS models. Compound 3a was advanced to the clinic. The synthesis of representative potential candidates and their in vitro, ex vivo, and in vivo data are described.

Small-molecule antagonists of melanopsin-mediated phototransduction

Melanopsin, expressed in a subset of retinal ganglion cells, mediates behavioral adaptation to ambient light and other non-image forming photic responses. This has raised the possibility that pharmacological manipulation of melanopsin can modulate several CNS responses including photophobia, sleep, circadian rhythms and neuroendocrine function. Here we describe the identification of a potent synthetic melanopsin antagonist with in vivo activity. Novel sulfonamide compounds inhibiting melanopsin (opsinamides) compete with retinal binding to melanopsin and inhibit its function without affecting rod/cone mediated responses. In vivo administration of opsinamides to mice specifically and reversibly modified melanopsin-dependent light responses including the pupillary light reflex and light aversion. The discovery of opsinamides raises the prospect of therapeutic control of the melanopsin phototransduction system to regulate light-dependent behavior and remediate pathological conditions.