Pharmacological Modulation of GluK1 and GluK2 by NETO1, NETO2, and PSD95.

The association between the kainate receptors (KARs) GluK1 and GluK2 and the modifying proteins neuropilin- and tolloid-like 1 (NETO1), neuropilin- and tolloid-like 2 (NETO2), and postsynaptic density protein 95 (PSD95) is likely to produce distinct GluK1 and GluK2 pharmacology in postsynaptic neurons. However, little is known about their corresponding modulatory effects on GluK1 and GluK2 activity in high-throughput assays for cell-based drug discovery. Using heterologous cells that potentially mimic the response in native cells in a fluorescence imaging plate reader (FLIPR) assay, we have investigated assays that incorporate (1) coexpression of GluK1 or GluK2 with their modulatory proteins (NETO1, NETO2, PSD95) and/or (2) enablement of assays with physiological concentration of native GluK1 and GluK2 agonist (glutamate) in the absence of an artificial potentiator (e.g., concanavalin A [Con A]). We found that in the absence of Con A, both NETO1 and NETO2 accessory proteins are able to potentiate kainate- and glutamate-evoked GluK1-mediated Ca(2+) influx. We also noted the striking ability of PSD95 to enhance glutamate-stimulated potentiation effects of NETO2 on GluK1 without the need for Con A and with a robust signal that could be utilized for high-throughput FLIPR assays. These experiments demonstrate the utility of heterologous cells coexpressing PSD95/NETO2 with GluK1 or GluK2 in native cell-mimicking heterologous cell systems for high-throughput assays and represent new avenues into the discovery of KAR modulating therapies.

Phosphodiesterase 10A inhibitor, MP-10 (PF-2545920), produces greater induction of c-Fos in dopamine D2 neurons than in D1 neurons in the neostriatum.

Studies described here tested the hypothesis that phosphodiesterase 10A inhibition by a selective antagonist, MP-10, activates the dopamine D2 receptor expressing medium spiny neurons to a greater extent than the D1 receptor expressing neurons. We used regional pattern of c-Fos induction in the neostriatal subregions of rodents and direct assessment of D1-positive and -negative neurons in the DRd1a-tdTomato mice for the purpose. MP-10 (1, 3, 10 or 30 mg/kg, PO) dose-dependently increased c-Fos immunopositive nuclei in all regions of neostriatum. However, the effect was statistically greater in the dorsolateral striatum, a region known to be activated preferentially by the D2 antagonism, than the D1-activated dorsomedial striatum. The D2 antagonist, haloperidol (0.3, 1, or 3 mg/kg, PO) produced an identical, regional pattern of c-Fos induction favoring the dorsolateral striatum of the rat. In contrast, the D1 agonist, SKF82958 (0.5, 1, or 2 mg/kg, PO), induced greater expression of c-Fos in the dorsomedial striatum. The C57Bl/6 mouse also showed regionally preferential c-Fos activation by haloperidol (2 mg/kg, IP) and SKF82858 (3 mg/kg, IP). In the Drd1a-tdTomato mice, MP-10 (3 or 10 mg/kg, IP) increased c-Fos immunoreactivity in both types of neurons, the induction was greater in the D1-negative neurons. Taken together, both the regional pattern of c-Fos induction in the striatal sub-regions and the greater induction of c-Fos in the D1-negative neurons indicate that PDE10A inhibition produces a small but significantly greater activation of the D2-containing striatopallidal pathway.

GluK1 antagonists from 6-(carboxy)phenyl decahydroisoquinoline derivatives. SAR and evaluation of a prodrug strategy for oral efficacy in pain models.

The synthesis and structure-activity relationship of decahydroisoquinoline derivatives with various benzoic acid substitutions as GluK1 antagonists are described. Potent and selective antagonists were selected for a tailored prodrug approach in order to facilitate the evaluation of the new compounds in pain models after oral administration. Several diester prodrugs allowed for acceptable amino acid exposure and moderate efficacy in vivo.

GluK1 antagonists from 6-(tetrazolyl)phenyl decahydroisoquinoline derivatives: in vitro profile and in vivo analgesic efficacy.

We have explored the decahydroisoquinoline scaffold, bearing a phenyl tetrazole, as GluK1 antagonists with potential as oral analgesics. We have established the optimal linker atom between decahydroisoquinoline and phenyl rings and demonstrated an improvement of both the affinity for the GluK1 receptor and the selectivity against the related GluA2 receptor with proper phenyl substitution. In this Letter, we also disclose in vivo data that led to the discovery of LY545694·HCl, a compound with oral efficacy in two persistent pain models.

TRP channels as emerging targets for pain therapeutics.

BACKGROUND: The transient receptor potential (TRP) superfamily of ion channels are a large and diverse group that have received increased attention in recent years. The sub-family of thermo-TRPs which are regulated by temperature, among other physical and chemical stimuli, are of particular interest for the development of potential pain therapeutics. OBJECTIVE/METHODS: We review the advances in the field in recent years, focusing on a rationale for pain therapy and potential challenges associated with these targets. RESULTS/CONCLUSIONS: Vanilloid-type TRP 1 (TRPV1) is the most well studied and advanced member of the family, with selective agonists and antagonists already in clinical use or development, respectively. Among other thermo-TRPs (including TRPV2 - 4, Ankyrin type TRP 1 (TRPA1) and melastatin type TRP 8 (TRPM8)), TRPA1 and TRPM8 are emerging as promising novel pain targets.

Antiallodynic and antihyperalgesic effects of selective competitive GLUK5 (GluR5) ionotropic glutamate receptor antagonists in the capsaicin and carrageenan models in rats.

GLU(K5) kainate receptor subunits are abundant in pain pathways, including dorsal root ganglia and spinothalamic neurons, as well as in the thalamus and brain stem. A growing body of evidence indicates that the GLU(K5) kainate receptor subtype plays a prominent role in pain transmission, particularly in persistent pain. In the present studies, compounds from a novel series of amino acid GLU(K5) receptor antagonists were evaluated for their effectiveness in reversing capsaicin-induced mechanical allodynia as well as carrageenan-induced thermal hyperalgesia. In vitro, the amino acid compounds were efficacious in blocking glutamate-evoked calcium flux in cells expressing GLU(K5) but not GLU(K6) or GLU(A2), homomeric receptors. Electrophysiologically, the compounds exhibited selectivity for kainate receptors in dorsal root ganglion cells relative to alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid hydrobromide and N-methyl-d-aspartate receptors in hippocampal pyramidal neurons. The amino acid compounds were poorly efficacious in the pain tests after s.c. or p.o. administration. However, compounds were highly efficacious after central intracisternal administration, and the rank order of potencies correlated with their rank order of affinities at GLU(K5) receptors determined in vitro, indicating that the lack of activity after systemic administration was due to poor oral bioavailability. To increase oral bioavailability, isobutyl or 2-ethyl-butyl ester prodrugs of the parent amino acids were prepared. The prodrugs, which produced robust plasma levels of parent amino acids, were highly efficacious in the capsaicin and carrageenan tests. The present studies provide further evidence that selective Glu(K5) kainate receptor subtype antagonists can reverse allodynia and hyperalgesia, particularly in persistent pain states.

Pharmacological characterization of the competitive GLUK5 receptor antagonist decahydroisoquinoline LY466195 in vitro and in vivo.

The excitatory neurotransmitter glutamate has been implicated in both migraine and persistent pain. The identification of the kainate receptor GLU(K5) in dorsal root ganglia, the dorsal horn, and trigeminal ganglia makes it a target of interest for these indications. We examined the in vitro and in vivo pharmacology of the competitive GLU(K5)-selective kainate receptor antagonist LY466195 [(3S,4aR,6S,8aR)-6-[[(2S)-2-carboxy-4,4-difluoro-1-pyrrolidinyl]-methyl]decahydro-3-isoquinolinecarboxylic acid)], the most potent GLU(K5) antagonist described to date. Comparisons were made to the competitive GLU(K5)/alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor antagonist LY293558 [(3S,4aR,6R,8aR)-6-[2-(1(2)H-tetrazole-5-yl)ethyl]-decahydroisoquinoline-3-carboxylic acid], other decahydroisoquinoline GLU(K5) receptor antagonists, and the noncompetitive AMPA receptor antagonist LY300168 [1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodi-azepine]. When characterized electrophysiologically in rat dorsal root ganglion neurons, LY466195 antagonized kainate (30 microM)-induced currents with an IC50 value of 0.045 +/- 0.011 microM. In HEK293 cells transfected with GLU(K5), GLU(K2)/GLU(K5), or GLU(K5)/GLU(K6) receptors, LY466195 produced IC50 values of 0.08 +/- 0.02, 0.34 +/- 0.17, and 0.07 +/- 0.02 microM, respectively. LY466195 was efficacious in a dural plasma protein extravasation (PPE) model of migraine with an ID100 value of 100 microg/kg i.v. LY466195 was also efficacious in the c-fos migraine model, with a dose of 1 microg/kg i.v. significantly reducing the number of Fos-positive cells in the rat nucleus caudalis after electrical stimulation of the trigeminal ganglion. Furthermore, LY466195 showed no contractile activity in the rabbit saphenous vein in vitro. The diethyl ester prodrug of LY466195 was also efficacious in the same PPE and c-fos models after oral administration at doses of 10 and 100 microg/kg, respectively while having no N-methyl-D-aspartate antagonist-like behavioral effects at oral doses up to 100 mg/kg.

In vitro and in vivo studies in rats with LY293558 suggest AMPA/kainate receptor blockade as a novel potential mechanism for the therapeutic treatment of anxiety disorders.

RATIONALE: Although convergent evidence exists for a role of glutamate in the regulation of anxiety, the involvement of specific glutamate receptor subtypes has yet to be defined. OBJECTIVE: To evaluate the potential for blockade of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)/kainate receptors to produce anxioltyic-like effects with the AMPA/GLU(K5) (kainate) antagonist (3S,4aR,6R,8aR)-6-[2-(1(2)H-tetrazole-5yl)ethyl]decahydroisoquinoline-3carboxylic acid (LY293558) MATERIALS AND METHODS: Punished responding of rats was used to determine the efficacy of LY293558. Other in vivo and in vitro studies further characterized the specificity of LY293558 for AMPA/kainate receptors. RESULTS: LY293558 had a rank order of potency of GLU(K5) > or = GLU(K5/6) approximately GLU(A2i) approximately GLU(K2/5) approximately GLU(A1i) approximately GLU(A2o) approximately GLU(A3i) approximately GLU(A1o) > or = GLU(A3o) > or = GLU(A4i) approximately GLU(A4o) and >100 microM affinity for rat cortical GABA(A) receptors. Comparison of the blockade of AMPA- vs N-methyl-D-aspartate (NMDA)-induced inward currents demonstrated that LY293558 was five-fold more potent as an antagonist at AMPA vs NMDA receptors in vitro. In keeping with the low affinity of LY293558 for NMDA receptors, LY293558 was not effective in preventing NMDA-induced seizures in mice. LY293558 increased punished responding, a preclinical predictor of anxiolytic efficacy, at a dose that decreased unpunished responding (10 mg/kg, i.p.). Chlordiazepoxide produced comparable increases in both punished and unpunished responding. The NMDA antagonist dizocilpine [(+)-MK-801] also increased both punished and unpunished responding. CONCLUSIONS: These data along with those in the literature suggest that AMPA and/or kainate receptor blockade may be an important component to producing anxiolytic-like effects and may therefore be a target for compounds with efficacy in the therapeutic treatment of anxiety disorders.

Two prodrugs of potent and selective GluR5 kainate receptor antagonists actives in three animal models of pain.

Amino acids 5 and 7, two potent and selective competitive GluR5 KA receptor antagonists, exhibited high GluR5 receptor affinity over other glutamate receptors. Their ester prodrugs 6 and 8 were orally active in three models of pain: reversal of formalin-induced paw licking, carrageenan-induced thermal hyperalgesia, and capsaicin-induced mechanical hyperalgesia.

Ethyl (3S,4aR,6S,8aR)-6-(4-ethoxycar- bonylimidazol-1-ylmethyl)decahydroiso-quinoline-3-carboxylic ester: a prodrug of a GluR5 kainate receptor antagonist active in two animal models of acute migraine.

Amino diacid 3, a highly selective competitive GluR5 kainate receptor antagonist, exhibited high GluR5 receptor affinity and selectivity over other glutamate receptors. Its diethyl ester prodrug 4 was orally active in two models of migraine: the neurogenic dural plasma protein extravasation model and the nucleus caudalis c-fos expression model. These data suggest that a GluR5 kainate receptor antagonist might be an efficacious antimigraine therapy with a novel mechanism of action.