Kevin Matthew Gardinier

Tachykinin receptor antagonists

The present invention relates to selective NK-1 receptor antagonists of Formula (I); or a pharmaceutically acceptable salt thereof, for the treatment of disorders associated with an excess of tachykinins

Perampanel, an Antagonist of α-Amino-3-Hydroxy-5-Methyl-4-Isoxazolepropionic Acid Receptors, for the Treatment of Epilepsy: Studies in Human Epileptic Brain and Nonepileptic Brain and in Rodent Models

Perampanel [Fycompa, 2-(2-oxo-1-phenyl-5-pyridin-2-yl-1,2-dihydropyridin-3-yl)benzonitrile hydrate 4:3; Eisai Inc., Woodcliff Lake, NJ] is an AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptor antagonist used as an adjunctive treatment of partial-onset seizures. We asked whether perampanel has AMPA receptor antagonist activity in both the cerebral cortex and hippocampus associated with antiepileptic efficacy and also in the cerebellum associated with motor side effects in rodent and human brains. We also asked whether epileptic or nonepileptic human cortex is similarly responsive to AMPA receptor antagonism by perampanel. In rodent models, perampanel decreased epileptic-like activity in multiple seizure models. However, doses of perampanel that had anticonvulsant effects were within the same range as those engendering motor side effects. Perampanel inhibited native rat and human AMPA receptors from the hippocampus as well as the cerebellum that were reconstituted into Xenopus oocytes. In addition, with the same technique, we found that perampanel inhibited AMPA receptors from hippocampal tissue that had been removed from a patient who underwent surgical resection for refractory epilepsy. Perampanel inhibited AMPA receptor–mediated ion currents from all the tissues investigated with similar potency (IC50 values ranging from 2.6 to 7.0 μM). Cortical slices from the left temporal lobe derived from the same patient were studied in a 60-microelectrode array. Large field potentials were evoked on at least 45 channels of the array, and 10 μM perampanel decreased their amplitude and firing rate. Perampanel also produced a 33% reduction in the branching parameter, demonstrating the effects of perampanel at the network level. These data suggest that perampanel blocks AMPA receptors globally across the brain to account for both its antiepileptic and side-effect profile in rodents and epileptic patients.

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.

Forebrain-selective AMPA-receptor antagonism guided by TARP γ-8 as an antiepileptic mechanism

Pharmacological manipulation of specific neural circuits to optimize therapeutic index is an unrealized goal in neurology and psychiatry. AMPA receptors are important for excitatory synaptic transmission, and their antagonists are antiepileptic. Although efficacious, AMPA-receptor antagonists, including perampanel (Fycompa), the only approved antagonist for epilepsy, induce dizziness and motor impairment. We hypothesized that blockade of forebrain AMPA receptors without blocking cerebellar AMPA receptors would be antiepileptic and devoid of motor impairment. Taking advantage of an AMPA receptor auxiliary protein, TARP γ-8, which is selectively expressed in the forebrain and modulates the pharmacological properties of AMPA receptors, we discovered that LY3130481 selectively antagonized recombinant and native AMPA receptors containing γ-8, but not γ-2 (cerebellum) or other TARP members. Two amino acid residues unique to γ-8 determined this selectivity. We also observed antagonism of AMPA receptors expressed in hippocampal, but not cerebellar, tissue from an patient with epilepsy. Corresponding to this selective activity, LY3130481 prevented multiple seizure types in rats and mice and without motor side effects. These findings demonstrate the first rationally discovered molecule targeting specific neural circuitries for therapeutic advantage.

Pyridine metallations in the synthesis of triazole based NK-1 antagonists

Regioselective pyridine metallation chemistry was used to produce N-(3-chloropyridin-4-ylmethyl)-N-methyl-1-(3,5-bis-trifluoromethylbenzyl)-5-phenyl-1H-[1,2,3]triazole-4-carboxamide (9a) and [l-(3,5-bis-trifluoromethyl-benzyl)-5-morpholin-4-yl-1H-[1,2,3]triazol-4-yl]-[3-(3-chloropyridin-4-yl)-5-hydroxymethylisoxazol-4-yl]methanone (16a), which exhibit NK-1 antagonist activity.

Structural Determinants of the γ-8 TARP Dependent AMPA Receptor Antagonist

The forebrain specific AMPA receptor antagonist, LY3130481/CERC-611, which selectively antagonizes the AMPA receptors associated with TARP γ-8, an auxiliary subunit enriched in the forebrain, has potent antiepileptic activities without motor side effects. We designated the compounds with such activities as γ-8 TARP dependent AMPA receptor antagonists (γ-8 TDAAs). In this work, we further investigated the mechanisms of action using a radiolabeled γ-8 TDAA and ternary structural modeling with mutational validations to characterize the LY3130481 binding to γ-8. The radioligand binding to the cells heterologously expressing GluA1 and/or γ-8 revealed that γ-8 TDAAs binds to γ-8 alone without AMPA receptors. Homology modeling of γ-8, based on the crystal structures of a distant TARP homologue, murine claudin 19, in conjunction with knowledge of two γ-8 residues previously identified as critical for the LY3130481 TARP-dependent selectivity provided the basis for a binding mode prediction. This allowed further rational mutational studies for characterization of the structural determinants in TARP γ-8 for LY3130481 activities, both thermodynamically as well as kinetically.

Electroencephalographic, cognitive, and neurochemical effects of LY3130481 (CERC-611), a selective antagonist of TARP-γ8-associated AMPA receptors

&NA; 6‐[(1S)‐1‐[1‐[5‐(2‐hydroxyethoxy)‐2‐pyridyl]pyrazol‐3‐yl]ethyl]‐3H‐1,3‐benzothiazol‐2‐one (LY3130481 or CERC‐611) is a selective antagonist of AMPA receptors containing transmembrane AMPA receptor regulatory protein (TARP) &ggr;−8. This molecule has been characterized as a potent and efficacious anticonvulsant in an array of acute and chronic epilepsy models in rodents. The present set of experiments was designed to assess the effects of LY3130481 on the electroencephelogram (EEG), cognitive function, and neurochemical outflow. LY3130481 disrupted food‐maintained responding in rats and spontaneous alternation in a Y‐maze in mice. In rat fear conditioning, LY3130481 caused a deficit in trace (hippocampal‐dependent), but not in delay fear conditioning. Although these effects on cognitive performances were observed, the known cognitive‐impairing anticonvulsant, topiramate, did not always produce deficits under these assay conditions. LY3130481 produced modest increases in wake times in rats. In addition, LY3130481 was able to attenuate some impairing effects of standard antiepileptic drugs. The motor‐impairing effects of the lacosamide were attenuated by LY3130481 as was the decrease in non‐rapid‐eye movement sleep induced by carbamazepine. Evaluation of the effect of LY3130481 on neurotransmitter and metabolite efflux in the rat medial prefrontal cortex, using in vivo microdialysis, revealed significant increases in the pro‐cognitive and wake‐promoting neurotransmitters, histamine and acetylcholine, as well as in serotonin, telemethylhistamine, 5‐HIAA, HVA and MHPG. LY3130481 thus presents a novel behavioral profile that will have to be evaluated in patients to fully appreciate its implications for therapeutics. LY3130481 is currently under clinical development as CERC‐611 as an antiepileptic. HighlightsLY3130481 (CERC‐611) is a selective antagonist of AMPA receptors TARP &ggr;−8 protein.LY3130481 is currently under clinical development as CERC‐611 as an antiepileptic.LY3130481 produced mixed results in rodent cognition assessments.LY3130481 produced modest increases in wake times in rats and increased wake and cognition‐associated neurotransmitters.LY3130481 attenuated some impairing effects of standard antiepileptic drugs.

A Comment on “Discovery and Characterization of AMPA Receptor Modulators Selective for TARP-γ8”

We would like to make the readers of JPET aware of literature precedents for the very exciting work recently published by [Maher et al. (2016)][1] (“Discovery and Characterization of AMPA Receptor Modulators Selective for TARP- γ 8”) in JPET . In this article, [Maher et al. (2016)][1] describe

Reconstitution of synaptic Ion channels from rodent and human brain in Xenopus oocytes: a biochemical and electrophysiological characterization

Disruption in the expression and function of synaptic proteins, and ion channels in particular, is critical in the pathophysiology of human neuropsychiatric and neurodegenerative diseases. However, very little is known regarding the functional and pharmacological properties of native synaptic human ion channels, and their potential changes in pathological conditions. Recently, an electrophysiological technique has been enabled for studying the functional and pharmacological properties of ion channels present in crude membrane preparation obtained from post‐mortem frozen brains. We here extend these studies by showing that human synaptic ion channels also can be studied in this way. Synaptosomes purified from different regions of rodent and human brain (control and Alzheimers) were characterized biochemically for enrichment of synaptic proteins, and expression of ion channel subunits. The same synaptosomes were also reconstituted in Xenopus oocytes, in which the functional and pharmacological properties of the native synaptic ion channels were characterized using the voltage clamp technique. We show that we can detect GABA, (RS)‐α‐Amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid, and NMDA receptors, and modulate them pharmacologically with selective agonists, antagonists, and allosteric modulators. Furthermore, changes in ion channel expression and function were detected in synaptic membranes from Alzheimers brains. Our present results demonstrate the possibility to investigate synaptic ion channels from healthy and pathological brains. This method of synaptosomes preparation and injection into oocytes is a significant improvement over the earlier method. It opens the way to directly testing, on native ion channels, the effects of novel drugs aimed at modulating important classes of synaptic targets.

Targeted Blockade of TARP-γ8-Associated AMPA Receptors: Anticonvulsant Activity with the Selective Antagonist LY3130481 (CERC-611)

BACKGROUND & OBJECTIVE 6-[(1S)-1-[1-[5-(2-hydroxyethoxy)-2-pyridyl]pyrazol-3-yl]ethyl]- 3H-1,3-benzothiazol-2-one (LY3130481 or CERC-611) is a selective antagonist of AMPA receptors containing transmembrane AMPA receptor regulatory protein (TARP) γ-8 that is under development for epilepsy. The present study provided a broad inquiry into its anticonvulsant properties. LY3130481 was anticonvulsant in multiple acute seizure provocation models in mice and rats. In addition, LY3130481 was effective against absence seizures in the GAERS genetic model and in the Frings mouse model. Likewise, LY3130481 attenuated convulsions in mice and rats with long-term induction of seizures (e.g., corneal, pentylenetetrazole, hippocampal, and amygdala kindled seizures). In slices of epileptic human cortex, LY3130481 significantly decreased neuronal firing frequencies. LY3130481 displaced from rat brain a radioligand specific for AMPA receptors associated with TARP γ-8 whereas non-TARP-selective molecules did not. Binding was also observed in hippocampus freshly transected from a patient. RESULTS & CONCLUSION Taken as a whole, the findings reported here establish the broad anticonvulsant efficacy of LY3130481 indicating that blockade of AMPA receptors associated with TARP γ-8 is sufficient for these protective effects.