Anne B. Need

Metabotropic glutamate 5 receptor antagonism is associated with antidepressant-like effects in mice

Antidepressant-like effects of metabotropic glutamate (mGlu)5 receptor antagonists have been reported previously. We now provide definitive identification of mGlu5 receptors as a target for these effects through the combined use of selective antagonists and mice with targeted deletion of the mGlu5 protein. In these experiments, the mGlu5 receptor antagonists 2-methyl-6-(phenylethynyl)-pyridine (MPEP) and the more selective and metabolically stable analog 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]-pyridine (MTEP) decreased immobility in the mouse forced swim test, a test predictive of antidepressant efficacy in humans. mGlu5 receptor knockout mice had a phenotype in the forced swim test that was congruent with the effects of receptor blockade; mGlu5 receptor knockout mice were significantly less immobile than their wild-type counterparts. Consistent with mGlu5 receptor mediation of the antidepressant-like effects of MPEP, the effects of MPEP were not observed in mGlu5 receptor knockout mice, whereas comparable effects of the tricyclic antidepressant imiprimine remained active in the mutant mice. MPEP and imiprimine resulted in a synergistic antidepressant-like effect in the forced swim test. The drug interaction was not likely because of increased levels of drugs in the brain, suggesting a pharmacodynamic interaction of mGlu5 and monoaminergic systems in this effect. Thus, the present findings substantiate the hypothesis that mGlu5 receptor antagonism is associated with antidepressant-like effects. This mechanism may not only provide a novel approach to the therapeutic management of depressive disorders but also may be useful in the augmentation of effects of traditional antidepressant agents.

Enhancement of antidepressant potency by a potentiator of AMPA receptors.

Abstract1. AMPA receptor potentiators (ARPs) exhibit antidepressant-like activity in preclinical tests (for example, the forced swim test) that are highly predictive of efficacy in humans. Unlike most currently used antidepressants, ARPs do not elevate extracellular levels of biogenic amines (e.g., 5HT, NE) in prefrontal cortex at doses that are active in the forced swim test.2. The present series of experiments examined the effects of combining the ARP, LY 392098, with biogenic amine-based antidepressants in the forced swim test. Male, NIH Swiss mice were placed in a cylinder of water and observed for attempted escape behaviors and immobility.3. LY 392098 dose-dependently decreased immobility as did a range of classical antidepressants. At doses of LY 392098 below those that decreased immobility, this compound significantly increased the potency with which fluoxetine and citalopram (SSRI antidepressants), imipramine (tricyclic antidepressant), duoxetine (norepinephrine/serotonin uptake blocker), nisoxetine (norepinephrine uptake inhibitor), and rolipram (PDE4 inhibitor) decreased immobility in the forced swim test with potency shifts upward of 5-fold (fluoxetine, imipramine, and rolipram). Likewise, ineffective doses of the traditional antidepressants potentiated the effects LY 392098 with shifts in the dose-effect functions that were 10-fold or more for citalopram, fluoxetine, imipramine, and duloxetine.4. Combined with other evidence for a role of AMPA receptors in the efficacy of antidepressants, the current data suggest that the addition of an ARP may augment the activity and perhaps the onset of the therapeutic effects of biogenic amine and second messenger-based antidepressants.

Analgesic effects of serotonergic, noradrenergic or dual reuptake inhibitors in the carrageenan test in rats: evidence for synergism between serotonergic and noradrenergic reuptake inhibition.

The efficacy of antidepressant drugs with serotonergic, noradrenergic, or dual reuptake inhibition was evaluated in reversing carrageenan-induced thermal hyperalgesia and mechanical allodynia in rats. Duloxetine (1-30mg/kg, i.p.), a balanced serotonergic-noradrenergic reuptake inhibitor (SNRI), was equiefficacious and more potent than the SNRI venlafaxine (3-100mg/kg, i.p.) in reversing both thermal hyperalgesia and mechanical allodynia induced by carrageenan. In addition, the selective noradrenergic reuptake inhibitors (NRIs) thionisoxetine (0.03-10mg/kg, i.p.) and desipramine (1-30mg/kg, i.p.) also produced complete reversals of carrageenan-induced thermal hyperalgesia. However, only thionisoxetine exhibited a greater than 80% reversal of the carrageenan-induced mechanical allodynia. In contrast, the selective serotonergic reuptake inhibitors (SSRIs) paroxetine, sertraline, and fluoxetine (0.3-10mg/kg i.p.) had little or no effect in the carrageenan model. In order to understand whether the observed enhanced effectiveness of the dual SNRIs was due to a possible synergism between serotonergic and noradrenergic reuptake inhibition, the effects of the NRI thionisoxetine alone and in combination with an inactive dose of the SSRI fluoxetine were determined. In the presence of fluoxetine, the potency of thionisoxetine in reversing carrageenan-induced hyperalgesia and allodynia was significantly increased by approximately 100-fold and brain concentrations of thionisoxetine were increased by 1.1- to 5-fold. The present data indicate fluoxetine pharmacodynamically potentiated the analgesic effects of thionisoxetine over and above a metabolic interaction between these two drugs. The present findings thus indicate that, in the carrageenan model, dual serotonergic-noradrenergic reuptake inhibition by dual SNRIs, or SSRI-NRI combinations, produces synergistic analgesic efficacy.

In vivo occupancy of dopamine D3 receptors by antagonists produces neurochemical and behavioral effects of potential relevance to attention-deficit-hyperactivity disorder.

Dopamine D3 receptors have eluded definitive linkage to neurologic and psychiatric disorders since their cloning over 20 years ago. We report a new method that does not employ a radiolabel for simultaneously defining in vivo receptor occupancy of D3 and D2 receptors in rat brain after systemic dosing using the tracer epidepride (N-[[(2S)-1-ethylpyrrolidin-2-yl]methyl]-5-iodo-2,3-dimethoxybenzamide). Decreases in epidepride binding in lobule 9 of cerebellum (rich in D3 receptors) were compared with nonspecific binding in the lateral cerebellum. The in vivo occupancy of the dopamine D3 receptors was dose dependently increased by SB-277011A (trans-N-[4-[2-(6-cyano-1,2,3,4-tetrahydroisoquinolin-2-yl)ethyl]cyclohexyl]-4-quinolinecarboxamide) and U99194 (2,3-dihydro-5,6-dimethoxy- N,N-dipropyl-1H-inden-2-amine). Both antagonists increased extracellular levels of acetylcholine (ACh) in the medial prefrontal cortex of rats and modified brain-tissue levels of ACh and choline. Consistent with these findings, the D3 receptor antagonists enhanced the acquisition of learning of rats either alone or in the presence of the norepinephrine uptake blocker reboxetine as with the attention-deficit–hyperactivity disorder (ADHD) drug methylphenidate. Like reboxetine, the D3 receptor antagonists also prevented deficits induced by scopolamine in object recognition memory of rats. Mice in which the dopamine transporter (DAT) has been deleted exhibit hyperactivity that is normalized by compounds that are effective in the treatment of ADHD. Both D3 receptor antagonists decreased the hyperactivity of DAT−/− mice without affecting the activity of wild type controls. The present findings indicate that dopamine D3 receptor antagonists engender cognition-enhancing and hyperactivity-dampening effects. Thus, D3 receptor blockade could be considered as a novel treatment approach for cognitive deficits and hyperactivity syndromes, including those observed in ADHD.

Discovery of the First α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptor Antagonist Dependent upon Transmembrane AMPA Receptor Regulatory Protein (TARP) γ-8

Transmembrane AMPA receptor regulatory proteins (TARPs) are a family of scaffolding proteins that regulate AMPA receptor trafficking and function. TARP γ-8 is one member of this family and is highly expressed within the hippocampus relative to the cerebellum. A selective TARP γ-8-dependent AMPA receptor antagonist (TDAA) is an innovative approach to modulate AMPA receptors in specific brain regions to potentially increase the therapeutic index relative to known non-TARP-dependent AMPA antagonists. We describe here, for the first time, the discovery of a noncompetitive AMPA receptor antagonist that is dependent on the presence of TARP γ-8. Three major iteration cycles were employed to improve upon potency, CYP1A2-dependent challenges, and in vivo clearance. An optimized molecule, compound (-)-25 (LY3130481), was fully protective against pentylenetetrazole-induced convulsions in rats without the motor impairment associated with non-TARP-dependent AMPA receptor antagonists. Compound (-)-25 could be utilized to provide proof of concept for antiepileptic efficacy with reduced motor side effects in patients.

A click-chemistry based strategy for labeling an anti-phosphatidylserine (PS) antibody with copper-64 via a cross-bridged tetraazamacrocyclic chelator scaffold

The κ-opioid receptors (KORs) are implicated in several neuropsychiatric diseases and addictive disorders. PET with radioligands provides a means to image the KOR in vivo and investigate its function in health and disease. The purpose of this study was to develop the selective KOR antagonist 11C-LY2459989 as a PET radioligand and characterize its imaging performance in nonhuman primates. Methods: LY2459989 was synthesized and assayed for in vitro binding to opioid receptors. Ex vivo studies in rodents were conducted to assess its potential as a tracer candidate. 11C-LY2459989 was synthesized by reaction of its iodophenyl precursor with 11C-cyanide, followed by partial hydrolysis of the resulting 11C-cyanophenyl intermediate. Imaging experiments with 11C-LY2459989 were performed in rhesus monkeys with arterial input function measurement. Imaging data were analyzed with kinetic models to derive in vivo binding parameters. Results: LY2459989 is a full antagonist with high binding affinity and selectivity for KOR (0.18, 7.68, and 91.3 nM, respectively, for κ, μ, and δ receptors). Ex vivo studies in rats indicated LY2459989 as an appropriate tracer candidate with high specific binding signals and confirmed its KOR binding selectivity in vivo. 11C-LY2459989 was synthesized in high radiochemical purity and good specific activity. In rhesus monkeys, 11C-LY2459989 displayed a fast rate of peripheral metabolism. Similarly, 11C-LY2459989 displayed fast uptake kinetics in the brain and an uptake pattern consistent with the distribution of KOR in primates. Pretreatment with naloxone (1 mg/kg, intravenously) resulted in a uniform distribution of radioactivity in the brain. Further, specific binding of 11C-LY2459989 was dose-dependently reduced by the selective KOR antagonist LY2456302 and the unlabeled LY2459989. Regional binding potential values derived from the multilinear analysis-1 (MA1) method, as a measure of in vivo specific binding signal, were 2.18, 1.39, 1.08, 1.04, 1.03, 0.59, 0.51, and 0.50, respectively, for the globus pallidus, cingulate cortex, insula, caudate, putamen, frontal cortex, temporal cortex, and thalamus. Conclusion: The novel PET radioligand 11C-LY2459989 displayed favorable pharmacokinetic properties, a specific and KOR-selective binding profile, and high specific binding signals in vivo, thus making it a promising PET imaging agent for KOR.

Identifying novel radiotracers for PET imaging of the brain: application of LC-MS/MS to tracer identification.

Nuclear medicine imaging biomarker applications are limited by the radiotracers available. Radiotracers enable the measurement of target engagement, or occupancy in relation to plasma exposure. These tracers can also be used as pharmacodynamic biomarkers to demonstrate functional consequences of binding a target. More recently, radiotracers have also been used for patient tailoring in Alzheimers disease seen with amyloid imaging. Radiotracers for the central nervous system (CNS) are challenging to identify, as they require a unique intersection of multiple properties. Recent advances in tangential technologies, along with the use of iterative learning for the purposes of deriving in silico models, have opened up additional opportunities to identify radiotracers. Mass spectral technologies and in silico modeling have made it possible to measure and predict in vivo characteristics of molecules to indicate potential tracer performance. By analyzing these data alongside other measures, it is possible to delineate guidelines to increase the likelihood of selecting compounds that can perform as radiotracers or serve as the best starting point to develop a radiotracer following additional structural modification. The application of mass spectrometry based technologies is an efficient way to evaluate compounds as tracers in vivo, but more importantly enables the testing of potential tracers that have either no label site or complex labeling chemistry which may deter assessment by traditional means; therefore, use of this technology allows for more rapid iterative learning. The ability to differentially distribute toward target rich tissues versus tissue with no/less target present is a unique defining feature of a tracer. By testing nonlabeled compounds in vivo and analyzing tissue levels by LC-MS/MS, rapid assessment of a compounds ability to differentially distribute in a manner consistent with target expression biology guides the focus of chemistry resources for both designing and labeling tracer candidates. LC-MS/MS has only recently been used for de novo tracer identification; however, this connection of mass spectral technology to imaging has initiated engagement from a wider community that brings diverse backgrounds into the tracer discovery arena.

Efficiency Gains in Tracer Identification for Nuclear Imaging: Can In Vivo LC-MS/MS Evaluation of Small Molecules Screen for Successful PET Tracers?

Positron emission tomography (PET) imaging has become a useful noninvasive technique to explore molecular biology within living systems; however, the utility of this method is limited by the availability of suitable radiotracers to probe specific targets and disease biology. Methods to identify potential areas of improvement in the ability to predict small molecule performance as tracers prior to radiolabeling would speed the discovery of novel tracers. In this retrospective analysis, we characterized the brain penetration or peak SUV (standardized uptake value), binding potential (BP), and brain exposure kinetics across a series of known, nonradiolabeled PET ligands using in vivo LC-MS/MS (liquid chromatography coupled to mass spectrometry) and correlated these parameters with the reported PET ligand performance in nonhuman primates and humans available in the literature. The PET tracers studied included those reported to label G protein-coupled receptors (GPCRs), intracellular enzymes, and transporters. Additionally, data for each tracer was obtained from a mouse brain uptake assay (MBUA), previously published, where blood-brain barrier (BBB) penetration and clearance parameters were assessed and compared against similar data collected on a broad compound set of central nervous system (CNS) therapeutic compounds. The BP and SUV identified via nonradiolabeled LC-MS/MS, while different from the published values observed in the literature PET tracer data, allowed for an identification of initial criteria values we sought to facilitate increased potential for success from our early discovery screening paradigm. Our analysis showed that successful, as well as novel, clinical PET tracers exhibited BP of greater than 1.5 and peak SUVs greater than approximately 150% at 5 min post dose in rodents. The brain kinetics appeared similar between both techniques despite differences in tracer dose, suggesting linearity across these dose ranges. The assessment of tracers in a CNS exposure model, the mouse brain uptake assessment (MBUA), showed that those compound with initial brain-to-plasma ratios >2 and unbound fraction in brain homogenate >0.01 were more likely to be clinically successful PET ligands. Taken together, early incorporation of a LC/MS/MS cold tracer discovery assay and a parallel MBUA can be an useful screening paradigm to prioritize and rank order potential novel PET radioligands during early tracer discovery efforts. Compounds considered for continued in vivo PET assessments can be identified quickly by leveraging in vitro affinity and selectivity measures, coupled with data from a MBUA, primarily the 5 min brain-to-plasma ratio and unbound fraction data. Coupled utilization of these data creates a strategy to efficiently screen for the identification of appropriate chemical space to invest in for radiotracer discovery.

Glucose Metabolic Trapping in Mouse Arteries: Nonradioactive Assay of Atherosclerotic Plaque Inflammation Applicable to Drug Discovery

Background 18F-Fluorodeoxyglucose (FDG)-positron emission tomography (PET) imaging of atherosclerosis in the clinic is based on preferential accumulation of radioactive glucose analog in atherosclerotic plaques. FDG-PET is challenging in mouse models due to limited resolution and high cost. We aimed to quantify accumulation of nonradioactive glucose metabolite, FDG-6-phosphate, in the mouse atherosclerotic plaques as a simple alternative to PET imaging. Methodology/Principal Findings Nonradioactive FDG was injected 30 minutes before euthanasia. Arteries were dissected, and lipids were extracted. The arteries were re-extracted with 50% acetonitrile-50% methanol-0.1% formic acid. A daughter ion of FDG-6-phosphate was quantified using liquid chromatography and mass spectrometry (LC/MS/MS). Thus, both traditional (cholesterol) and novel (FDG-6-phosphate) markers were assayed in the same tissue. FDG-6-phosphate was accumulated in atherosclerotic lesions associated with carotid ligation of the Western diet fed ApoE knockout mice (5.9 times increase compare to unligated carotids, p<0.001). Treatment with the liver X receptor agonist T0901317 significantly (2.1 times, p<0.01) reduced FDG-6-phosphate accumulation 2 weeks after surgery. Anti-atherosclerotic effects were independently confirmed by reduction in lesion size, macrophage number, cholesterol ester accumulation, and macrophage proteolytic activity. Conclusions/Significance Mass spectrometry of FDG-6-phosphate in experimental atherosclerosis is consistent with plaque inflammation and provides potential translational link to the clinical studies utilizing FDG-PET imaging.

Determining Pharmacological Selectivity of the Kappa Opioid Receptor Antagonist LY2456302 Using Pupillometry as a Translational Biomarker in Rat and Human

Background: Selective kappa opioid receptor antagonism is a promising experimental strategy for the treatment of depression. The kappa opioid receptor antagonist, LY2456302, exhibits ~30-fold higher affinity for kappa opioid receptors over mu opioid receptors, which is the next closest identified pharmacology. Methods: Here, we determined kappa opioid receptor pharmacological selectivity of LY2456302 by assessing mu opioid receptor antagonism using translational pupillometry in rats and humans. Results: In rats, morphine-induced mydriasis was completely blocked by the nonselective opioid receptor antagonist naloxone (3mg/kg, which produced 90% mu opioid receptor occupancy), while 100 and 300mg/kg LY2456302 (which produced 56% and 87% mu opioid receptor occupancy, respectively) only partially blocked morphine-induced mydriasis. In humans, fentanyl-induced miosis was completely blocked by 50mg naltrexone, and LY2456302 dose-dependently blocked miosis at 25 and 60mg (minimal-to-no blockade at 4–10mg). Conclusions: We demonstrate, for the first time, the use of translational pupillometry in the context of receptor occupancy to identify a clinical dose of LY2456302 achieving maximal kappa opioid receptor occupancy without evidence of significant mu receptor antagonism.