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 central nervous system responses, including photophobia, sleep, circadian rhythms and neuroendocrine function. Here we describe the identification of a potent synthetic melanopsin antagonist with in vivo activity. New sulfonamide compounds inhibiting melanopsin (opsinamides) compete with retinal binding to melanopsin and inhibit its function without affecting rod- and 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.

5-MeO-DMT modifies innate behaviors and promotes structural neural plasticity in mice.

Serotonergic psychedelics are gaining increasing interest as potential therapeutics for a range of mental illnesses. Compounds with short-lived subjective effects may be clinically useful because dosing time would be reduced, which may improve patient access. One short-acting psychedelic is 5-MeO-DMT, which has been associated with improvement in depression and anxiety symptoms in early phase clinical studies. However, relatively little is known about the behavioral and neural mechanisms of 5-MeO-DMT, particularly the durability of its long-term effects. Here we characterized the effects of 5-MeO-DMT on innate behaviors and dendritic architecture in mice. We showed that 5-MeO-DMT induces a dose-dependent increase in head-twitch response that is shorter in duration than that induced by psilocybin at all doses tested. 5-MeO-DMT also substantially suppresses social ultrasonic vocalizations produced during mating behavior. 5-MeO-DMT produces long-lasting increases in dendritic spine density in the mouse medial frontal cortex that are driven by an elevated rate of spine formation. However, unlike psilocybin, 5-MeO-DMT did not affect the size of dendritic spines. These data provide insights into the behavioral and neural consequences underlying the action of 5-MeO-DMT and highlight similarities and differences with those of psilocybin.

Effects of the sigma-1 receptor agonist blarcamesine in a murine model of fragile X syndrome: neurobehavioral phenotypes and receptor occupancy.

Fragile X syndrome (FXS), a disorder of synaptic development and function, is the most prevalent genetic form of intellectual disability and autism spectrum disorder. FXS mouse models display clinically-relevant phenotypes, such as increased anxiety and hyperactivity. Despite their availability, so far advances in drug development have not yielded new treatments. Therefore, testing novel drugs that can ameliorate FXS' cognitive and behavioral impairments is imperative. ANAVEX2-73 (blarcamesine) is a sigma-1 receptor (S1R) agonist with a strong safety record and preliminary efficacy evidence in patients with Alzheimer's disease and Rett syndrome, other synaptic neurodegenerative and neurodevelopmental disorders. S1R's role in calcium homeostasis and mitochondrial function, cellular functions related to synaptic function, makes blarcamesine a potential drug candidate for FXS. Administration of blarcamesine in 2-month-old FXS and wild type mice for 2 weeks led to normalization in two key neurobehavioral phenotypes: open field test (hyperactivity) and contextual fear conditioning (associative learning). Furthermore, there was improvement in marble-burying (anxiety, perseverative behavior). It also restored levels of BDNF, a converging point of many synaptic regulators, in the hippocampus. Positron emission tomography (PET) and ex vivo autoradiographic studies, using the highly selective S1R PET ligand [18F]FTC-146, demonstrated the drug's dose-dependent receptor occupancy. Subsequent analyses also showed a wide but variable brain regional distribution of S1Rs, which was preserved in FXS mice. Altogether, these neurobehavioral, biochemical, and imaging data demonstrates doses that yield measurable receptor occupancy are effective for improving the synaptic and behavioral phenotype in FXS mice. The present findings support the viability of S1R as a therapeutic target in FXS, and the clinical potential of blarcamesine in FXS and other neurodevelopmental disorders.

Antagonism of 5-HT(1A) receptors uncovers an excitatory effect of SSRIs on 5-HT neuronal activity, an action probably mediated by 5-HT(7) receptors.

Both microdialysis and electrophysiology were used to investigate whether another serotonin (5-HT) receptor subtype next to the 5-HT(1A) autoreceptor is involved in the acute effects of a selective serotonin reuptake inhibitor on 5-HT neuronal activity. On the basis of a previous study, we decided to investigate the involvement of the 5-HT(7) receptors. Experiments were performed with the specific 5-HT(7) antagonist SB 258741 and the putative 5-HT(7) agonist AS19. In this study WAY 100.635 was used to block 5-HT(1A) receptors. Systemic administration of SB 258741 significantly reduced the effect of combined selective serotonin reuptake inhibitor and WAY 100.635 administration on extracellular 5-HT in the ventral hippocampus as well as 5-HT neuronal firing in the dorsal raphe nucleus. In the microdialysis study, co-administration of AS19 and WAY 100.635 showed a biphasic effect on extracellular 5-HT in ventral hippocampus, hinting at opposed 5-HT(7) receptor mediated effects. In the electrophysiological experiments, systemic administration of AS19 alone displayed a bell-shaped dose-effect curve: moderately increasing 5-HT neuronal firing at lower doses while decreasing it at higher doses. SB 258741 was capable of blocking the effect of AS19 at a low dose. This is consistent with the pharmacological profile of AS19, displaying high affinity for 5-HT(7) receptors and moderate affinity for 5-HT(1A) receptors. The data are in support of an excitatory effect of selective serotonin reuptake inhibitors on 5-HT neuronal activity mediated by 5-HT(7) receptors. It can be speculated, that the restoration of 5-HT neuronal firing upon chronic antidepressant treatment, which is generally attributed to desensitization of 5-HT(1A) receptors alone, in fact results from a shift in balance between 5-HT(1A) and 5-HT(7) receptor function.

ANAVEX®2-73 (blarcamesine), a Sigma-1 receptor agonist, ameliorates neurologic impairments in a mouse model of Rett syndrome.

Rett syndrome (RTT) is a severe neurodevelopmental disorder that is associated in most cases with mutations in the transcriptional regulator MECP2. At present, there are no effective treatments for the disorder. Despite recent advances in RTT genetics and neurobiology, most drug development programs have focused on compounds targeting the IGF-1 pathway and no pivotal trial has been completed as yet. Thus, testing novel drugs that can ameliorate RTT's clinical manifestations is a high priority. ANAVEX2-73 (blarcamesine) is a Sigma-1 receptor agonist and muscarinic receptor modulator with a strong safety record and preliminary evidence of efficacy in patients with Alzheimer's disease. Its role in calcium homeostasis and mitochondrial function, cellular functions that underlie pathological processes and compensatory mechanisms in RTT, makes blarcamesine an intriguing drug candidate for this disorder. Mice deficient in MeCP2 have a range of physiological and neurological abnormalities that mimic the human syndrome. We tested blarcamesine in female heterozygous mice carrying one null allele of Mecp2 (HET) using a two-tier approach, with age-appropriate tests. Administration of the drug to younger and older adult mice resulted in improvement in multiple motor, sensory, and autonomic phenotypes of relevance to RTT. The latter included motor coordination and balance, acoustic and visual responses, hindlimb clasping, and apnea in expiration. In line with previous animal and human studies, blarcamesine also showed a good safety profile in this mouse model of RTT. Clinical studies in RTT with blarcamesine are ongoing.

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.

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.

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 were bred (C57BL/6J) lacking 1 serotonin receptor, the 5-HT(7), to serve as a null background for this subtype; earlier work had documented the involvement of 5-HT(7) receptors in the phase advances elicited by 8-OH-DPAT, a mixed 5-HT(1A/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 microM, 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 microM), a highly selective 5-HT(1A) 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-HT(5A/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.

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.

Characterization of the 5-HT(7) receptor. Determination of the pharmacophore for 5-HT(7) receptor agonism and CoMFA-based modeling of the agonist binding site.

On the basis of a set of 20 diverse 5-HT(7) receptor agonists, the pharmacophore for 5-HT(7) receptor agonism was determined. Additionally two CoMFA models were developed, based on different alignments of the agonists. Both models show good correlations between experimental and predictive pK(i) values and show a high degree of similarity. The CoMFA fields were subsequently used to map the agonist binding site of the model of the 5-HT(7) receptor. Important roles in ligand binding are attributed to Asp162 of TM3 (interaction with a protonated nitrogen), and Thr244 of TM5 (interaction with a substituent at an aromatic moiety). Amino acid residues of the aromatic cluster of TM6 are hypothesized to play an important role in ligand binding as pi-pi stacking moieties. Agonists missing a hydrogen-bond-accepting moiety, but possessing an aromatic substituent instead, seem to bind the receptor with high affinity as well by occupying a lipophilic pocket hosted by residues of TM5 and TM6.

Novel 5-HT7 receptor inverse agonists. Synthesis and molecular modeling of arylpiperazine- and 1,2,3,4-tetrahydroisoquinoline-based arylsulfonamides.

A series of arylpiperazine- and 1,2,3,4-tetrahydroisoquinoline-based arylsulfonamides was synthesized and evaluated for their interactions with the constitutively active 5-HT7 receptor. Effects on basal adenylate cyclase activity were measured using HEK-293 cells expressing the rat 5-HT7. All ligands produced a decrease of adenylate cyclase activity, indicative of their inverse agonism. Additionally, computational studies with a set of 22 inverse agonists, including these novel inverse agonists and inverse agonists known from literature, resulted in a pharmacophore model and a CoMFA model (R2 = 0.97, SE = 0.18). Docking of inverse agonists at the binding site of a model of the helical parts of the 5-HT7 receptor, based on the alpha carbon template for 7-TM GPCRs, revealed interesting molecular interactions and a possible explanation for observed structure-activity relationships.

The effects of combining serotonin reuptake inhibition and 5-HT7 receptor blockade on circadian rhythm regulation in rodents.

Disruption of circadian rhythms may lead to mood disorders. The present study investigated the potential therapeutic utility of combining a 5-HT7 antagonist with a selective serotonin (5-HT) reuptake inhibitor (SSRI), the standard of care in depression, on circadian rhythm regulation. In tissue explants of the suprachiasmatic nucleus (SCN) from PER2::LUC mice genetically modified to report changes in the expression of a key clock protein, the period length of PER2 bioluminescence was shortened in the presence of AS19, a 5-HT7 partial agonist. This reduction was blocked by SB269970, a selective 5-HT7 antagonist. The SSRI, escitalopram, had no effect alone on period length, but a combination with SB269970, yielded significant increases. Dosed in vivo, escitalopram had little impact on the occurrence of activity onsets in rats given access to running wheels, whether the drug was given acutely or sub-chronically. However, preceding the escitalopram treatment with a single acute dose of SB269970 produced robust phase delays, in keeping with the in vitro explant data. Taken together, these findings suggest that the combination of an SSRI and a 5-HT7 receptor antagonist has a greater impact on circadian rhythms than that observed with either agent alone, and that such a multimodal approach may be of therapeutic value in treating patients with poor clock function.

Circadian rhythm dysregulation in bipolar disorder.

When circadian rhythms - the daily oscillations of various physiological and behavioral events that are controlled by a central timekeeping mechanism - become desynchronized with the prevailing light:dark cycle, a maladaptative response can result. Animal data based primarily on genetic manipulations and clinical data from biomarker and gene expression studies support the notion that circadian abnormalities underlie certain psychiatric disorders. In particular, bipolar disorder has an interesting link to rhythm-related disease biology; other mood disturbances, such as major depressive disorder, seasonal affective disorder and the agitation and aggression accompanying severe dementia (sundowning), are also linked to changes in circadian rhythm function. Possibilities for pharmacological intervention derive most readily from the molecular oscillator, the cellular machinery that drives daily rhythms.

Chronic treatment with a selective inhibitor of casein kinase I delta/epsilon yields cumulative phase delays in circadian rhythms.

INTRODUCTION: Casein kinase I epsilon/delta phosphorylates certain clock-related proteins as part of a complex arrangement of transcriptional/translational feedback loops that comprise the circadian oscillator in mammals. Pharmacologic inhibition leads to a delay of the oscillations with the magnitude of this effect dependent upon the timing of drug administration. OBJECTIVE: Earlier studies by our lab described the actions of a selective CKI epsilon/delta inhibitor, PF-670462, on circadian behavior following acute dosing; the present work extended these studies to chronic once-daily treatment. METHODS: Gross motor activity was used to estimate the circadian rhythms of rats maintained under a 12 L:12 D cycle. PF-670462, 10 or 30 mg/kg/day s.c., was administered once daily for 20 days either at ZT6 or ZT11 (i.e., 6 or 11 h after light onset). RESULTS: Chronic administration of PF-670462, performed at a fixed time of day, produced delays in the activity onsets of rats that cumulated with the duration of dosing. Dosing at ZT11 yielded more robust delays than dosing at ZT6 in keeping with earlier phase-response analyses with this agent. CONCLUSIONS: The magnitude of the shifts in activity onsets achieved with chronic dosing of PF-670462 appears to be a function of the dose and the previously established phase relationship. Its cumulative effect further suggests that the pharmacodynamic t (1/2) of the drug greatly exceeds its pharmacokinetic one. Most importantly, these changes in circadian behavior occurred in the presence of a fixed L:D cycle, confirming the drug to be a robust modulator of circadian phase in the presence of the natural zeitgeber.

Inhibition of casein kinase I epsilon/delta produces phase shifts in the circadian rhythms of Cynomolgus monkeys.

INTRODUCTION: Circadian rhythms in mammals depend upon the cyclic oscillations of transcriptional/translational feedback loops in pacemaker cells of the suprachiasmatic nucleus. The rise and fall of clock-related proteins is a function of synthesis and degradation, the latter involving phosphorylation by casein kinase Iepsilon and delta. OBJECTIVE: Earlier studies by our lab described the actions of a selective CKIepsilon/delta inhibitor, PF-670462, on circadian behavior in rats; the present work extended these studies to a diurnal species, Cynomolgus monkeys. MATERIALS AND METHODS: General cage activity was used to estimate the circadian rhythms of eight telemeterized monkeys under baseline conditions and following s.c. doses of PF-670462. RESULTS AND DISCUSSION: Consolidated bouts of activity were noted during periods of light with a repeating period length of roughly 24 h based on their onset. Reassessment in constant dim light (42 vs. 450 lx) again yielded period lengths of 24 h, in this instance revealing the animals' endogenous rhythm. PF-670462 (10-100 mg/kg s.c.) produced a dose-dependent phase delay in much the same manner as that observed previously in rats. Dosing occurred 1.5 h prior to lights-off, roughly coincident with peaking levels of PER protein, a primary substrate of CKIepsilon/delta. CONCLUSIONS: These findings suggest that the time of dosing, when held constant in both the monkey and rat studies, produced nearly identical effects despite the subjects' diurnal or nocturnal preference. Importantly, these changes in rhythm occurred in the presence of light, revealing the drug as a powerful zeitgeber in a non-human primate and, by extension, in man.

An inhibitor of casein kinase I epsilon induces phase delays in circadian rhythms under free-running and entrained conditions.

Casein kinase Iepsilon (CKIepsilon) 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 (IC(50) = 7.7 +/- 2.2 nM) and selective (>30-fold with respect to 42 additional kinases) inhibitor of CKIepsilon 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 (t(1/2) = 0.46 +/- 0.04 h). Together, these results suggest that inhibition of CKIepsilon 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.

Pharmacological modulation of circadian rhythms: a new drug target in psychotherapeutics.

Daily variation in an organism's physiology and behaviour is regulated by the synchrony that is achieved between the internal timing mechanisms - the circadian rhythms of the biological clock - and the prevailing environmental cues. Proper synchrony constitutes an adaptive response; improper or lost synchrony may well yield maladaptation and, in the case of humans, a psychiatric disorder. On a basic level, the circadian system is comprised of three parts: a central oscillator, its various neuronal inputs and its outputs. For all three of these parts, the dissemination of new information is moving at an unprecedented pace, and the number of molecular targets for the opportunistic pharmacologist is growing in step. Monoamines, neuropeptides, kinases - sorting through all these, much less developing one into a drug discovery programme, may be the biggest challenge. However, the potential benefits in targeting a basic flaw in a fundamental biological system may be enormous.

Serotonin-induced phase advances of SCN neuronal firing in vitro: a possible role for 5-HT5A receptors?

Spontaneous firing rates of neurons in the suprachiasmatic nuclei (SCN) follow a consistent pattern, peaking near the midpoint of the light phase in a 12:12 light/dark schedule, and repeating this brief period of increased activity in subsequent circadian cycles. These carefully timed fluctuations reflect the output signal of the SCN, long recognized as the site of the endogenous biological clock in mammals. In rat hypothalamic slices, bath incubations of 8-OH-DPAT had previously been shown to elicit phase advances when applied at ZT6 (or 6 h following the onset of light), an action that could readily be attributed to 5-HT7 receptor activation. The present studies set out with the simple goal of establishing that the same receptor mechanism was responsible for the phase-shifting actions of 5-HT itself. Surprisingly, the phase advances elicited by 5-HT (0.5 microM, 1 h) at ZT6 were reduced by one 5-HT7 antagonist, ritanserin (10 microM), but not by another, mesulergine (10 microM). Receptor binding studies demonstrated a 25-fold greater affinity of ritanserin for h5-HT5A sites compared to mesulergine (Ki = 71 nM vs. 1,800 nM), an observation suggestive of a 5-HT5A mechanism for 5-HT and consistent with earlier observations of robust labeling of 5-HT5A sites in the SCN. 5-HT generated by the addition of L-tryptophan (10 microM, 1 h) to the slices displayed the same pattern of sensitivity, that is, blockade by ritanserin but not by mesulergine. Rp-cAMPS, a cAMP antagonist, failed to block the phase shifts elicited by 5-HT at a concentration (1 microM) previously shown to be effective against 8-OH-DPAT-induced phase shifts, in keeping with the proposed negative coupling of 5-HT5A receptors to cAMP production. Taken together, these results suggest that activation of both 5-HT5A and 5-HT7 receptors can produce phase advances of the circadian clock in vitro when they occur during mid-subjective day.