[show abstract][hide abstract] ABSTRACT: Evidence suggests that 30-50% of patients suffering from major depressive disorder (MDD) are classified as suffering from treatment resistant depression (TRD) as they have an inadequate response to standard antidepressants. A key feature of this patient population is the increased incidence of co-morbid symptoms like anxiety and pain. Recognizing that current standards of care are largely focused on monoaminergic mechanisms of action (MOAs), innovative approaches to drug discovery for TRD are targeting glutamate hyperfunction. Here we describe the in vitro and in vivo profile of GRN-529, a novel negative allosteric modulator (NAM) of metabotropic glutamate receptor 5 (mGluR5). In cell based pharmacology assays, GRN-529 is a high affinity (Ki 5.4 nM), potent (IC50 3.1 nM) and selective (>1000-fold selective vs mGluR1) mGluR5 NAM. Acute administration of GRN-529 (0.1-30 mg/kg p.o.) had dose-dependent efficacy across a therapeutically relevant battery of animal models, comprising depression (decreased immobility time in tail suspension and forced swim tests) and 2 of the co-morbid symptoms overrepresented in TRD, namely anxiety (attenuation of stress-induced hyperthermia, and increased punished crossings in the four plate test) and pain (reversal of hyperalgesia due to sciatic nerve ligation or inflammation). The potential side effect liability of GRN-529 was also assessed using preclinical models: GRN-529 had no effect on rat sexual behavior or motor co-ordination (rotarod), however it impaired cognition in mice (social odor recognition). Efficacy and side effects of GRN-529 were compared to standard of care agents (antidepressant, anxiolytic or analgesics) and the tool mGluR5 NAM, MTEP. To assess the relationship between target occupancy and efficacy, ex vivo receptor occupancy was measured in parallel with efficacy testing. This revealed a strong correlation between target engagement, exposure and efficacy across behavioral endpoints, which supports the potential translational value of PET imaging to dose selection in patients. Collectively this broad spectrum profile of efficacy of GRN-529 supports our hypothesis that negative allosteric modulation of mGluR5 could represent an innovative therapeutic approach to the treatment of TRD. This article is part of a Special Issue entitled 'mGluR'.
[show abstract][hide abstract] ABSTRACT: Metabotropic glutamate receptor 7 (mGluR7) remains the most elusive of the eight known mGluRs primarily because of the limited availability of tool compounds to interrogate its potential therapeutic utility. The discovery of N,N'-dibenzhydrylethane-1,2-diamine dihydrochloride (AMN082) as the first orally active, brain-penetrable, mGluR7-selective allosteric agonist by Mitsukawa and colleagues (Proc Natl Acad Sci USA 102:18712-18717, 2005) provides a means to investigate this receptor system directly. AMN082 demonstrates mGluR7 agonist activity in vitro and interestingly has a behavioral profile that supports utility across a broad spectrum of psychiatric disorders including anxiety and depression. The present studies were conducted to extend the in vitro and in vivo characterization of AMN082 by evaluating its pharmacokinetic and metabolite profile. Profiling of AMN082 in rat liver microsomes revealed rapid metabolism (t(1/2) < 1 min) to a major metabolite, N-benzhydrylethane-1,2-diamine (Met-1). In vitro selectivity profiling of Met-1 demonstrated physiologically relevant transporter binding affinity at serotonin transporter (SERT), dopamine transporter (DAT), and norepinephrine transporter (NET) (323, 3020, and 3410 nM, respectively); whereas the parent compound AMN082 had appreciable affinity at NET (1385 nM). AMN082 produced antidepressant-like activity and receptor occupancy at SERT up to 4 h postdose, a time point at which AMN082 is significantly reduced in brain and plasma while the concentration of Met-1 continues to increase in brain. Acute Met-1 administration produced antidepressant-like activity as would be expected from its in vitro profile as a mixed SERT, NET, DAT inhibitor. Taken together, these data suggest that the reported in vivo actions of AMN082 should be interpreted with caution, because they may involve other mechanisms in addition to mGluR7.
Journal of Pharmacology and Experimental Therapeutics 07/2011; 338(1):345-52. · 3.89 Impact Factor
[show abstract][hide abstract] ABSTRACT: Biogenic amines such as norepinephrine, dopamine, and serotonin play a well-described role in the treatment of mood disorders and some types of pain. As alpha2A-adrenoceptors regulate the release of these neurotransmitters, we examined the therapeutic potential of BRL 44408, a potent (Ki=8.5 nM) and selective (>50-fold) alpha2A-adrenoceptor antagonist (K(B)=7.9 nM). In rats, BRL 44408 penetrated the central nervous system resulting in peak brain and plasma concentrations of 586 ng/g and 1124 ng/ml, respectively. In a pharmacodynamic assay, pretreatment with BRL 44408 to rats responding under a fixed-ratio 30 operant response paradigm resulted in a rightward shift of the clonidine dose-response curve, an effect indicative of alpha2-adrenoceptor antagonism in vivo. Consistent with presynaptic autoreceptor antagonism and tonic regulation of neurotransmitter release, acute administration of BRL 44408 elevated extracellular concentrations of norepinephrine and dopamine, but not serotonin, in the medial prefrontal cortex. Additionally, BRL 44408, probably by inhibiting alpha2A heteroceptors, produced a significant increase in cortical levels of acetylcholine. In the forced swim test and schedule-induced polydipsia assay, BRL 44408 produced an antidepressant-like response by dose-dependently decreasing immobility time and adjunctive water intake, respectively, while in a model of visceral pain, BRL 44408 exhibited analgesic activity by decreasing para-phenylquinone (PPQ)-induced abdominal stretching. Finally, BRL 44408 did not produce deficits in overall motor coordination nor alter general locomotor activity. This preclinical characterization of the neurochemical and behavioural profile of BRL 44408 suggests that selective antagonism of alpha2A-adrenoceptors may represent an effective treatment strategy for mood disorders and visceral pain.
The International Journal of Neuropsychopharmacology 10/2010; 13(9):1193-205. · 5.64 Impact Factor
[show abstract][hide abstract] ABSTRACT: The generation of new neurons occurs throughout adulthood in discrete brain regions, and may be regulated by neuropsychiatric diseases and therapeutic drug treatments. Most current methods that study this process measure the labeling of newborn cells by 5-bromo-2-deoxyuridine (BrdU) using immunohistochemical methods followed by the microscopic counting of BrdU positive cells. This method is time consuming and labor intensive, typically taking several weeks to analyze.
Therefore, we characterized a method to measure BrdU incorporation in the adult mouse hippocampus in vivo by using flow cytometry, which normally allows analysis of data within a single day.
The present study compared multiple BrdU dosing and loading protocols to determine a dosing strategy that produced the best signal to noise ratio. BrdU incorporation was also compared across different brain regions. The method was sensitive to a number of experimental disease manipulations. Induction of type-1 diabetes and depletion of norepinephrine reduced hippocampal cell proliferation. In contrast, chronic administration of electroconvulsive shock, a somatic treatment for depression, as well as chronic treatment with the antidepressant fluoxetine elevated hippocampal cell proliferation. This increase in cell proliferation with fluoxetine was detected as early as 14 days into treatment. Moreover, comparing measures of cell proliferation obtained by immunohistochemical and flow cytometric methods within the same animals were convergent and significantly correlated to each other. Flow cytometry was also sufficiently sensitive to quantify the survival of newly born cells.
These experiments validate the utility of flow cytometry in analyzing hippocampal cell proliferation and survival in a reliable and high-throughput fashion. The speedy analysis afforded by flow cytometry lends itself to be utilized in novel drug discovery and physiology.
Journal of pharmacological and toxicological methods 01/2009; 59(2):100-7. · 2.32 Impact Factor
[show abstract][hide abstract] ABSTRACT: Galanin's 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 galnon's 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 galnon's in vivo activity.