Xavier Khawaja

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.

Proteomic analysis of protein changes developing in rat hippocampus after chronic antidepressant treatment: Implications for depressive disorders and future therapies

It is recognized that monoamine reuptake inhibitors (MARIs) exert beneficial effects in the treatment of major depression and general anxiety disorder. The aim of this study was to identify proteins regulated by this class of antidepressant using a proteome differential profiling approach. Either venlafaxine or fluoxetine was administered systemically to adult rats for 2 weeks, and protein patterns from rat hippocampal cytosolic extracts were compared by two‐dimensional gel electrophoresis. Silver‐stained protein spots displaying differential expression were identified by mass spectrometry. Thirty‐three protein spots were modulated by both drug treatments compared to controls. The classification of several proteins that were sorted by function suggested convergent pathway activities for both MARIs at the post‐receptor level. These included proteins associated with neurogenesis (insulin like growth factor 1 (IGF‐1), glia maturation factor [GMF]‐β), outgrowth/maintenance of neuronal processes (hippocampal cholinergic neurostimulating peptide [HCNP], PCTAIRE‐3), and with neural regeneration/axonal guidance collapsin response mediator protein (CRMP‐2) systems. Other modulated proteins indicated an increase in neuronal vesicular cell trafficking and synaptic plasticity (Ras‐related protein 4a (Rab4a), Ras‐related protein 1b (Rab1b), heat shock protein 10 [HSP10]), as well as neurosteroidogenic (hydroxysteroid sulfotransferase A) and possible anti‐apoptotic (dimethylargininase‐1 L‐N,N‐dimethylarginine dimethylaminohydrolase‐1 [DDAH‐1], pyruvate dehydrogenase‐E1 [PDH‐E1], antioxidant protein‐2 [AOP‐2]) pathway‐mediated regulatory events. Parallel studies to investigate further the effects of venlafaxine and fluoxetine on adult hippocampal neurogenesis in vivo by quantitative bromodeoxyuridine immunolabeling revealed a significant drug‐induced increase in the proliferation rate and long‐term survivability of progenitor stem cells located in the subgranular zone. These data suggest that MARIs share wide‐ranging proteome changes within the hippocampal formation, beyond 5‐HT/NE neurotransmission. This may reflect long‐term functional adaptations required for antidepressant activity.

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.

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.

Characterisation of the Binding of [3H]WAY-100635, a Novel 5-Hydroxytryptamine1A Receptor Antagonist, to Rat Brain

Abstract: The specific binding of [3H]WAY‐100635 {N‐[2‐[4‐(2‐[O‐methyl‐3H]methoxyphenyl)‐1‐piperazinyl]ethyl]‐N‐(2‐pyridinyl)cyclohexane carboxamide trihydrochloride} to rat hippocampal membrane preparations was time, temperature, and tissue concentration dependent. The rates of [3H]WAY‐100635 association (k+1 = 0.069 ± 0.015 nM−1 min−1) and dissociation (k−1 = 0.023 ± 0.001 min−1) followed monoexponential kinetics. Saturation binding isotherms of [3H]WAY‐100635 exhibited a single class of recognition site with an affinity of 0.37 ± 0.051 nM and a maximal binding capacity (Bmax) of 312 ± 12 fmol/mg of protein. The maximal number of binding sites labelled by [3H]WAY‐100635 was ∼36% higher compared with that of 8‐hydroxy‐2‐(di‐n‐[3H]‐propylamino)tetralin ([3H]8‐OH‐DPAT). The binding affinity of [3H]WAY‐100635 was significantly lowered by the divalent cations CaCl2 (2.5‐fold; p < 0.02) and MnCl2 (3.6‐fold; p < 0.05), with no effect on Bmax. Guanyl nucleotides failed to influence the KD and Bmax parameters of [3H]WAY‐100635 binding to 5‐HT1A receptors. The pharmacological binding profile of [3H]WAY‐100635 was closely correlated with that of [3H]8‐OH‐DPAT, which is consistent with the labelling of 5‐hydroxytryptamine1A (5‐HT1A) sites in rat hippocampus. [3H]WAY‐100635 competition curves with 5‐HT1A agonists and partial agonists were best resolved into high‐ and low‐affinity binding components, whereas antagonists were best described by a one‐site binding model. In the presence of 50 µM guanosine 5′‐O‐(3‐thiotriphosphate) (GTPγS), competition curves for the antagonists remained unaltered, whereas the agonist and partial agonist curves were shifted to the right, reflecting an influence of G protein coupling on agonist versus antagonist binding to the 5‐HT1A receptor. However, a residual (16 ± 2%) high‐affinity agonist binding component was still apparent in the presence of GTPγS, indicating the existence of GTP‐insensitive sites.

Regional distribution of regulators of G-protein signaling (RGS) 1, 2, 13, 14, 16, and GAIP messenger ribonucleic acids by in situ hybridization in rat brain

Regulators of G-protein signaling (RGS) proteins are a novel family of GTPase-activating proteins that interact with Galpha subunits of the Gi/o, Gz, Gq and G(12/13) subfamilies to dampen G-protein-coupled receptor (GPCR)-mediated signaling by accelerating intrinsic Galpha-GTPase activity. In the present study, we report on messenger ribonucleic acid (mRNA) localization in rat brain of six RGS genes by in situ hybridization. The distribution patterns of RGS2, RGS13, RGS14 and GAIP (Galpha interacting protein) overlapped in most brain regions examined. Highest regional expression was observed for RGS2 in the cerebral cortical layers, striatum, hippocampal formation, several thalamic and hypothalamic nuclei and hindbrain regions such as the pontine, interpeduncular and dorsal raphe nuclei. Levels of RGS14 mRNA closely paralleled those of RGS2 expression levels throughout most brain regions. RGS13 mRNA was enriched in the hippocampal formation, amygdala, mammillary nuclei as well as the pontine and interpeduncular nuclei. GAIP expression levels were highest in the hippocampal formation with moderate to low levels present in all other regions studied. Of the six RGS genes probed, RGS16 mRNA displayed a discrete localization predominantly in the thalamic midline/intralaminar and principal relay nuclei, and the hypothalamic suprachiasmatic nucleus. RGS1 mRNA signal was not detected in brain. In conclusion, the in situ hybridization studies for RGS2, RGS13, RGS14, RGS16 and GAIP mRNAs extend our knowledge of the distribution of RGS genes expressed in the rat central nervous system, and indicate overlapping RGS-enriched regions that may be indicative of functional diversification in GPCR signaling pathway modulation.

RGS7 Attenuates Signal Transduction Through the Gαq Family of Heterotrimeric G Proteins in Mammalian Cells

Abstract: The RGS proteins are a recently discovered family of G protein regulators that have been shown to act as GTPase‐activating proteins (GAPs) on the Gαi and Gαq subfamilies of the heterotrimeric G proteins. Here, we demonstrate that RGS7 is a potent GAP in vitro on Gαi1 and Gαo heterotrimeric proteins and that RGS7 acts to down‐regulate Gαq‐mediated calcium mobilization in a whole‐cell assay system using a transient expression protocol. This RGS protein and RGS4 are reported to be expressed predominantly in brain, and in situ hybridization studies have revealed similarities in the regional distribution of RGS and Gαq mRNA expression. Our findings provide further evidence to support a functional role for RGS4 and RGS7 in Gαq‐mediated signaling in the CNS.

Immunohistochemical localization of G protein β1, β2, β3, β4, β5, and γ3 subunits in the adult rat brain

Abstract: The regional distributions of the G protein β subunits (Gβ1–β5) and of the Gγ3 subunit were examined by immunohistochemical methods in the adult rat brain. In general, the Gβ and Gγ3 subunits were widely distributed throughout the brain, with most regions containing several Gβ subunits within their neuronal networks. The olfactory bulb, neocortex, hippocampus, striatum, thalamus, cerebellum, and brainstem exhibited light to intense Gβ immunostaining. Negative immunostaining was observed in cortical layer I for Gβ1 and layer IV for Gβ4. The hippocampal dentate granular and CA1–CA3 pyramidal cells displayed little or no positive immunostaining for Gβ2 or Gβ4. No anti‐Gβ4 immunostaining was observed in the pars compacta of the substantia nigra or in the cerebellar granule cell layer and Purkinje cells. Immunoreactivity for Gβ1 was absent from the cerebellar molecular layer, and Gβ2 was not detected in the Purkinje cells. No positive Gγ3 immunoreactivity was observed in the lateral habenula, lateral septal nucleus, or Purkinje cells. Double‐fluorescence immunostaining with anti‐Gγ3 antibody and individual anti‐Gβ1–β5 antibodies displayed regional selectivity with Gβ1 (cortical layers V–VI) and Gβ2 (cortical layer I). In conclusion, despite the widespread overlapping distributions of Gβ1–β5 with Gγ3, specific dimeric associations in situ were observed within discrete brain regions.

Antidepressant-like behavioral effects of IGF-I produced by enhanced serotonin transmission

Previous research has suggested that mobilization of neurotrophic factors, such as insulin-like growth factor I (IGF-I), can be involved in the effects of antidepressant treatments. The current experiments showed that IGF-I leads to antidepressant-like effects in the modified rat forced swim test when tested 3 days, but not 1 day, after i.c.v. administration. These effects were sustained longer than the antidepressants paroxetine and desipramine. In addition, blockade of the IGF-I receptor with the IGF-I antagonist JB1 30 min before IGF-I administration prevented the antidepressant-like effects of IGF-I. However, when JB1 was administered 3 days after IGF-I administration and 30 min prior to testing, the antidepressant-like effects of IGF-I were still present suggesting that IGF-1 produces a long-term activation of neural systems involved in the antidepressant response. Because the pattern of antidepressant-like effects of IGF-I resembled those of selective serotonin reuptake inhibitors, the role of serotonin in the behavioral effects of IGF-I was studied. Depletion of serotonin, by the tryptophan hydroxylase inhibitor para-chlorophenylalanine, blocked the antidepressant-like effects of IGF-I. Administration of IGF-I increased basal serotonin levels in the ventral hippocampus and altered the effects of acute citalopram. IGF-I administration did not change hippocampal cell proliferation at the 3-day timepoint when behavioral effects were seen. In addition, IGF-I did not alter the expression of mRNA levels of tryptophan hydroxylase or SERT in the brain stem, or [3H] citalopram binding in the hippocampus or cortex. Thus, IGF-I administration initiates a long-lasting cascade of neurochemical effects involving increased serotonin levels that results in antidepressant-like behavioral effects.

Immunohistochemical Distribution of RGS7 Protein and Cellular Selectivity in Colocalizing with Gαq Proteins in the Adult Rat Brain

Abstract : Regulators of G protein signaling (RGS) proteins serve as potent GTPase‐activating proteins for the heterotrimeric G proteins αi/o and αq/11. This study describes the immunohistochemical distribution of RGS7 throughout the adult rat brain and its cellular colocalization with Gαq/11, an important G protein‐coupled receptor signal transducer for phospholipase Cβ‐mediated activity. In general, both RGS7 and Gαq/11 displayed a heterogeneous and overlapping regional distribution. RGS7 immunoreactivity was observed in cortical layers I‐VI, being most intense in the neuropil of layer I. In the hippocampal formation, RGS7 immunoreactivity was concentrated in the strata oriens, strata radiatum, mossy fibers, and polymorphic cells, with faint to nondetectable immunolabeling within the dentate gyrus granule cells and CA1‐CA3 subfield pyramidal cells. Numerous diencephalic and brainstem nuclei also displayed dense RGS7 immunostaining. Dual immunofluorescence labeling studies with the two protein‐specific antibodies indicated a cellular selectivity in the colocalization between RGS7 and Gαq/11 within many discrete brain regions, such as the superficial cortical layer I, hilus area of the hippocampal formation, and cerebellar Golgi cells. To assess the ability of Gαq/11‐mediated signaling pathways to modulate dynamically RGS expression, primary cortical neuronal cultures were incubated with phorbol 12,13‐dibutyrate, a selective protein kinase C activator. A time‐dependent increase in levels of mRNA for RGS7, but not RGS4, was observed. Our results provide novel information on the region‐ and cell‐specific pattern of distribution of RGS7 with the transmembrane signal transducer, Gαq/11. We also describe a possible RGS7‐selective neuronal feedback adaptation on Gαq/11‐mediated pathway function, which may play an important role in signaling specificity in the brain.