Chad E. Beyer

Innovative Approaches for the Development of Antidepressant Drugs: Current and Future Strategies

SummaryDepression is a highly debilitating disorder that has been estimated to affect up to 21% of the world population. Despite the advances in the treatment of depression with selective serotonin reuptake inhibitors (SSRIs) and serotonin and norepinephrine reuptake inhibitors (SNRIs), there continue to be many unmet clinical needs with respect to both efficacy and side effects. These needs range from efficacy in treatment resistant patients, to improved onset, to reductions in side effects such as emesis or sexual dysfunction. To address these needs, there are numerous combination therapies and novel targets that have been identified that may demonstrate improvements in one or more areas. There is tremendous diversity in the types of targets and approaches being taken. At one end of a spectrum is combination therapies that maintain the benefits associated with SSRIs but attempt to either improve efficacy or reduce side effects by adding additional mechanisms (5-HT1A, 5-HT1B, 5-HT1D, 5-HT2C, α-2A). At the other end of the spectrum are more novel targets, such as neurotrophins (BDNF, IGF), based on recent findings that antidepressants induce neurogenesis. In between, there are many approaches that range from directly targeting serotonin receptors (5-HT2C, 5-HT6) to targeting the multiplicity of potential mechanisms associated with excitatory (glutamate, NMDA, mGluR2, mGluR5) or inhibitory amino acid systems (GABA) or peptidergic systems (neurokinin 1, corticotropin-releasing factor 1, melanin-concentrating hormone 1, V1b). The present review addresses the most exciting approaches and reviews the localization, neurochemical and behavioral data that provide the supporting rationale for each of these targets or target combinations.

ADX47273 [S-(4-Fluoro-phenyl)-{3-[3-(4-fluoro-phenyl)-[1,2,4]-oxadiazol-5-yl]-piperidin-1-yl}-methanone]: A Novel Metabotropic Glutamate Receptor 5-Selective Positive Allosteric Modulator with Preclinical Antipsychotic-Like and Procognitive Activities

Positive allosteric modulators (PAMs) of metabotropic glutamate receptor subtype 5 (mGlu5) enhance N-methyl-d-aspartate receptor function and may represent a novel approach for the treatment of schizophrenia. ADX47273 [S-(4-fluoro-phenyl)-{3-[3-(4-fluoro-phenyl)-[1,2,4]oxadiazol-5-yl]-piperidin-1-yl}-methanone], a recently identified potent and selective mGlu5 PAM, increased (9-fold) the response to threshold concentration of glutamate (50 nM) in fluorometric Ca2+ assays (EC50 = 170 nM) in human embryonic kidney 293 cells expressing rat mGlu5. In the same system, ADX47273 dose-dependently shifted mGlu5 receptor glutamate response curve to the left (9-fold at 1 μM) and competed for binding of [3H]2-methyl-6-(phenylethynyl)pyridine (Ki = 4.3 μM), but not [3H]quisqualate. In vivo, ADX47273 increased extracellular signal-regulated kinase and cAMP-responsive element-binding protein phosphorylation in hippocampus and prefrontal cortex, both of which are critical for glutamate-mediated signal transduction mechanisms. In models sensitive to antipsychotic drug treatment, ADX47273 reduced rat-conditioned avoidance responding [minimal effective dose (MED) = 30 mg/kg i.p.] and decreased mouse apomorphine-induced climbing (MED = 100 mg/kg i.p.), with little effect on stereotypy or catalepsy. Furthermore, ADX47273 blocked phencyclidine, apomorphine, and amphetamine-induced locomotor activities (MED = 100 mg/kg i.p.) in mice and decreased extracellular levels of dopamine in the nucleus accumbens, but not in the striatum, in rats. In cognition models, ADX47273 increased novel object recognition (MED = 1 mg/kg i.p.) and reduced impulsivity in the five-choice serial reaction time test (MED = 10 mg/kg i.p.) in rats. Taken together, these effects are consistent with the hypothesis that allosteric potentiation of mGlu5 may provide a novel approach for development of antipsychotic and procognitive agents.

Neuropharmacological profile of novel and selective 5-HT6 receptor agonists: WAY-181187 and WAY-208466.

One of the most recently identified serotonin (5-hydroxytryptamine (5-HT)) receptor subtypes is the 5-HT6 receptor. Although in-depth localization studies reveal an exclusive distribution of 5-HT6 mRNA in the central nervous system, the precise biological role of this receptor still remains unknown. In the present series of experiments, we report the pharmacological and neurochemical characterization of two novel and selective 5-HT6 receptor agonists. WAY-181187 and WAY-208466 possess high affinity binding (2.2 and 4.8 nM, respectively) at the human 5-HT6 receptor and profile as full receptor agonists (WAY-181187: EC50=6.6 nM, Emax=93%; WAY-208466: EC50=7.3 nM; Emax=100%). In the rat frontal cortex, acute administration of WAY-181187 (3–30 mg/kg, subcutaneous (s.c.)) significantly increased extracellular GABA concentrations without altering the levels of glutamate or norepinephrine. Additionally, WAY-181187 (30 mg/kg, s.c.) produced modest yet significant decreases in cortical dopamine and 5-HT levels. Subsequent studies showed that the neurochemical effects of WAY-181187 in the frontal cortex could be blocked by pretreatment with the 5-HT6 antagonist, SB-271046 (10 mg/kg, s.c.), implicating 5-HT6 receptor mechanisms in mediating these responses. Moreover, the effects of WAY-181187 on catecholamines were attenuated by an intracortical infusion of the GABAA receptor antagonist, bicuculline (10 μM), confirming a local relationship between 5-HT6 receptors and GABAergic systems in the frontal cortex. In the dorsal hippocampus, striatum, and amygdala, WAY-181187 (10–30 mg/kg, s.c.) elicited robust elevations in extracellular levels of GABA without producing similar effects on concentrations of norepinephrine, serotonin, dopamine, or glutamate. In contrast to these brain regions, WAY-181187 had no effect on the extracellular levels of GABA in the nucleus accumbens or thalamus. Additional studies showed that WAY-208466 (10 mg/kg, s.c.) preferentially elevated cortical GABA levels following both acute and chronic (14 day) administration, indicating that neurochemical tolerance does not develop following repeated 5-HT6 receptor stimulation. In hippocampal slice preparations (in vitro), 5-HT6 receptor agonism attenuated stimulated glutamate levels elicited by sodium azide and high KCl treatment. Furthermore, in the rat schedule-induced polydipsia model of obsessive compulsive disorder (OCD), acute administration of WAY-181187 (56–178 mg/kg, po) decreased adjunctive drinking behavior in a dose-dependent manner. In summary, WAY-181187 and WAY-208466 are novel, selective, and potent 5-HT6 receptor agonists displaying a unique neurochemical signature in vivo. Moreover, these data highlight a previously undescribed role for 5-HT6 receptors to modulate basal GABA and stimulated glutamate transmission, as well as reveal a potential therapeutic role for this receptor in the treatment of some types of anxiety-related disorders (eg OCD).

ADX47273: A Novel Metabotropic Glutamate Receptor 5 Selective Positive Allosteric Modulator with Preclinical Antipsychotic-Like and Pro-cognitive Activities

Positive allosteric modulators (PAMs) of metabotropic glutamate receptor subtype 5 (mGlu5) enhance N-methyl-d-aspartate receptor function and may represent a novel approach for the treatment of schizophrenia. ADX47273 [S-(4-fluoro-phenyl)-{3-[3-(4-fluoro-phenyl)-[1,2,4]oxadiazol-5-yl]-piperidin-1-yl}-methanone], a recently identified potent and selective mGlu5 PAM, increased (9-fold) the response to threshold concentration of glutamate (50 nM) in fluorometric Ca2+ assays (EC50 = 170 nM) in human embryonic kidney 293 cells expressing rat mGlu5. In the same system, ADX47273 dose-dependently shifted mGlu5 receptor glutamate response curve to the left (9-fold at 1 μM) and competed for binding of [3H]2-methyl-6-(phenylethynyl)pyridine (Ki = 4.3 μM), but not [3H]quisqualate. In vivo, ADX47273 increased extracellular signal-regulated kinase and cAMP-responsive element-binding protein phosphorylation in hippocampus and prefrontal cortex, both of which are critical for glutamate-mediated signal transduction mechanisms. In models sensitive to antipsychotic drug treatment, ADX47273 reduced rat-conditioned avoidance responding [minimal effective dose (MED) = 30 mg/kg i.p.] and decreased mouse apomorphine-induced climbing (MED = 100 mg/kg i.p.), with little effect on stereotypy or catalepsy. Furthermore, ADX47273 blocked phencyclidine, apomorphine, and amphetamine-induced locomotor activities (MED = 100 mg/kg i.p.) in mice and decreased extracellular levels of dopamine in the nucleus accumbens, but not in the striatum, in rats. In cognition models, ADX47273 increased novel object recognition (MED = 1 mg/kg i.p.) and reduced impulsivity in the five-choice serial reaction time test (MED = 10 mg/kg i.p.) in rats. Taken together, these effects are consistent with the hypothesis that allosteric potentiation of mGlu5 may provide a novel approach for development of antipsychotic and procognitive agents.

Comparison of the effects of antidepressants on norepinephrine and serotonin concentrations in the rat frontal cortex: an in-vivo microdialysis study.

The present study employed in-vivo microdialysis techniques in the freely moving rat to systematically compare the neurochemical effects of various antidepressant agents on extracellular concentrations of norepinephrine (NE) and serotonin (5-HT) in the frontal cortex. We found that acute administration of the tricyclic antidepressant, desipramine (3–30 mg/kg, s.c.) and the dual serotonin/norepinephrine reuptake inhibitor, venlafaxine (3–30 mg/kg, s.c.), produced dose-dependent and robust increases in cortical NE concentrations (498% and 403%, respectively). Conversely, acute injection of the selective serotonin reuptake inhibitors, fluoxetine (30 mg/kg, s.c.) and paroxetine (1–10 mg/kg, s.c.), did not alter forebrain NE concentrations. However, paroxetine did produce a significant increase in cortical NE concentrations (164%) when administered at 30 mg/kg. These changes in NE were not paralleled by 5-HT, which showed no increase following administration of desipramine, venlafaxine, paroxetine or fluoxetine. Combination treatment with the 5-HT1A receptor antagonist, WAY-100635 (0.3 mg/kg, s.c.), significantly enhanced extracellular 5-HT concentrations following venlafaxine (10 and 30 mg/kg), fluoxetine (30 mg/kg) and paroxetine (3–30 mg/kg). Alternatively, WAY-100635 produced no augmentation of the antidepressant induced changes in extracellular NE. Collectively, these studies show that paroxetine, at low to intermediate doses, and fluoxetine are selective for 5-HT versus NE systems, whereas venlafaxine produces similar effects on both 5-HT and NE levels at the effective doses tested.

Receptor and behavioral pharmacology of WAY-267464, a non-peptide oxytocin receptor agonist.

The widely reported effects of oxytocin (OT) on CNS function has generated considerable interest in the therapeutic potential for targeting this system for a variety of human psychiatric diseases, including anxiety disorders, autism, schizophrenia, and depression. The utility of synthetic OT, as both a research tool and neurotherapeutic, is limited by the physiochemical properties inherent in most neuropeptides, notably its short half-life and poor blood brain barrier penetration. Subsequently, the discovery and development of non-peptide molecules that act as selective agonists of the oxytocin receptor (OTR) has been an important goal of the field. In this study, we report the receptor and behavioral pharmacology of WAY-267464, a first generation small-molecule OTR agonist. WAY-267464 is a high-affinity, potent, and selective (vs. V1a, V2, V1b) agonist of the OTR. In assays measuring both behavioral (four-plate test, elevated zero maze) and autonomic (stress-induced hyperthermia) parameters of the anxiety response, WAY-267464 exhibits an anxiolytic-like profile similar to OT. We have demonstrated that the anxiolytic-like profile of WAY-267464 is mediated through central sites of action. WAY-267464 also significantly reverses disruption in prepulse inhibition of the acoustic startle reflex induced by either MK-801 or amphetamine, similar to the antipsychotic-like effects previously reported for OT. Interestingly, in the mouse tail suspension test, WAY-267464 failed to produce changes in immobility that are seen with OT, raising the question of whether the antidepressant-like activity of OT may be working independently of the OTR. A selective OTR antagonist also failed to block the effects of OT on immobility in the TST. The significance of these findings for shaping the clinical development of OTR agonists is discussed.

Depression-like phenotype following chronic CB1 receptor antagonism

Rimonabant was the first clinically marketed cannabinoid (CB)(1) receptor antagonist developed to treat obesity. Unfortunately, CB(1) receptor antagonism produced adverse psychiatric events in patients. To determine whether this occurs pre-clinically, we investigated the effects of rimonabant in rodent models of mood disorders. Chronic treatment with rimonabant increased immobility time in the rat forced swim test and reduced the consumption of sucrose-sweetened water in an assay postulated to model anhedonia. These responses were similar to the effects elicited by chronic mild stress in these behavioral models, which, taken together, are indicative of a depression-like phenotype. Additionally, chronic treatment with rimonabant produced decreases in frontal cortex serotonin levels, marked reductions in hippocampal cell proliferation, survival, and BDNF levels, and elevations in the concentrations of pro-inflammatory cytokines including interferon gamma and TNF alpha. These preclinical findings mimic clinical reports and implicate possible mechanisms responsible for the unfavorable psychiatric events reported following chronic rimonabant use.

Amiloride is neuroprotective in an MPTP model of Parkinson's disease.

The diuretic amiloride has recently proven neuroprotective in models of cerebral ischemia, a property attributable to the drugs inhibition of central acid-sensing ion channels (ASICs). Given that Parkinsons disease (PD), like ischemia, is associated with cerebral lactic acidosis, we tested amiloride in the MPTP-treated mouse, a model of PD also manifesting lactic acidosis. Amiloride was found to protect substantia nigra (SNc) neurons from MPTP-induced degeneration, as determined by attenuated reductions in striatal tyrosine hydroxylase (TH) and dopamine transporter (DAT) immunohistochemistry, as well as smaller declines in striatal DAT radioligand binding and dopamine levels. More significantly, amiloride also preserved dopaminergic cell bodies in the SNc. Administration of psalmotoxin venom (PcTX), an ASIC1a blocker, resulted in a much more modest effect, attenuating only the deficits in striatal DAT binding and dopamine. These findings represent the first experimental evidence of a potential role for ASICs in the pathogenesis of Parkinsons disease.

Do selective serotonin reuptake inhibitors acutely increase frontal cortex levels of serotonin

Selective serotonin uptake inhibitors (SSRIs) exert their effects by inhibiting serotonin (5-HT) re-uptake. Although blockade occurs almost immediately, the neurochemical effects on 5-HT, as measured by in vivo microdialysis, have been a matter of considerable debate. In particular, literature reports yield conflicting neurochemical results in the rat frontal cortex. Thus, while some groups consistently find increases in extracellular 5-HT levels following acute SSRI administration, others reproducibly report an absence of these acute serotonergic effects. In an attempt to unravel this apparent discrepancy, we combined published literature with in-house microdialysis experiments. When we plotted the lateral stereotaxic coordinate of the dialysis probe against published reports on the acute effects of fluoxetine a clear correlation was revealed. Whereas pronounced increases in SSRI-induced 5-HT were observed when the dialysis probe was placed 0 to 1 mm from the midline, effects diminished when the lateral probe placement was greater than 3 mm from the midline. In-house microdialysis studies corroborated these reports. Overall, these results illustrate - for the first time - that the midline stereotaxic coordinate is critical for interpreting the acute serotonergic effects of SSRIs within the frontal cortex. Moreover, the common observation that the clinical efficacy of SSRIs is not evident following acute administration complements preclinical microdialysis results in the lateral frontal cortex. The significance of this observation, along with potential explanations for the disparate neurochemical findings in the medial versus lateral cortices, will be discussed.

Anxiolytic-like activity of the non-selective galanin receptor agonist, galnon

Galanins influence on monoaminergic neurotransmission, together with its discrete CNS distribution in corticolimbic brain areas, points to a potential role for this neuropeptide in mediating anxiety- and depression-like responses. To evaluate this hypothesis, the non-selective galanin receptor agonist, galnon, was tested in multiple preclinical models of anxiolytic- and antidepressive-like activity. Acute administration of galnon (0.03-1mg/kg, i.p.) dose-dependently increased punished crossings in the four plate test, with magnitude similar to the effects of the endogenous ligand, galanin (0.1-1.0 microg, i.c.v.). Moreover, the effects of galnon and galanin were blocked by central administration of the non-selective galanin receptor antagonist, M35 (10 microg, i.c.v.). Interestingly, the benzodiazepine receptor antagonist, flumazenil (1mg/kg, i.p.), reversed galnons effect in the four plate test, implicating GABAergic neurotransmission as a potential mechanism underlying this anxiolytic-like response. In the elevated zero maze, galnon (0.3-3.0mg/kg, i.p.) and galanin (0.03-0.3 microg, i.c.v.) increased the time spent in the open arms, while in the stress-induced hyperthermia model, galnon (0.3-30 mg/kg, i.p.) attenuated stress-induced changes in body temperature. Consistent with these anxiolytic-like effects, in vivo microdialysis showed that acute galnon (3mg/kg, i.p.) treatment preferentially elevated levels of GABA in the rat amygdala, a brain area linked to fear and anxiety behaviors. In contrast to the effects in anxiety models, neither galnon (1-5.6 mg/kg, i.p.) nor galanin (0.3-3.0 microg, i.c.v.) demonstrated antidepressant-like effects in the mouse tail suspension test. Galnon (1-10mg/kg, i.p.) also failed to reduce immobility time in the rat forced swim test. In vitro, galnon and galanin showed affinity for human galanin receptors expressed in Bowes melanoma cells (K(i)=5.5 microM and 0.2 nM, respectively). Galanin displayed high affinity and functional potency for membranes expressing rat GALR1 receptors (K(i)=0.85 nM; EC(50)=0.6 nM), while galnon (10 microM) failed to displace radiolabeled galanin or inhibit cAMP production in the same GALR1 cell line. Galnon (10 microM) showed affinity for NPY1, NK2, M5, and somatostatin receptors but no affinity for galanin receptors expressed in rat hippocampal membranes. Taken together, the present series of studies demonstrate novel effects of galnon in various preclinical models of anxiety and highlight the galaninergic system as a novel therapeutic target for the treatment of anxiety-related disorders. Moreover, these data indicate rodent GALR1 receptors do not mediate galnons in vivo activity.