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Many patients with nervous system disorders have considerable unmet clinical needs or suffer debilitating drug side effects. A major limitation of exiting treatment approaches is that traditional small molecule pharmacotherapy lacks sufficient specificity to effectively treat many neurological diseases. Chemogenetics is a new gene therapy technology that targets an engineered receptor to cell types involved in nervous system dysfunction, enabling highly selective drug-controlled neuromodulation. Here, we discuss chemogenetic platforms and considerations for their potential application as human nervous system therapies.

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.

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.

Pharmacological magnetic resonance imaging (phMRI) is one method by which a drug's pharmacodynamic effects in the brain can be assessed. Although phMRI has been frequently used in preclinical and clinical settings, the extent to which a phMRI signature for a compound translates between rodents and humans has not been systematically examined. In the current investigation, we aimed to build on recent clinical work in which the functional response to 0.1 and 0.2 mg/70 kg i.v. buprenorphine (partial µ-opioid receptor agonist) was measured in healthy humans. Here, we measured the phMRI response to 0.04 and 0.1 mg/kg i.v. buprenorphine in conscious, naive rats to establish the parallelism of the phMRI signature of buprenorphine across species. PhMRI of 0.04 and 0.1 mg/kg i.v. buprenorphine yielded dose-dependent activation in a brain network composed of the somatosensory cortex, cingulate, insula, striatum, thalamus, periaqueductal gray, and cerebellum. Similar dose-dependent phMRI activation was observed in the human phMRI studies. These observations indicate an overall preservation of pharmacodynamic responses to buprenorphine between conscious, naive rodents and healthy human subjects, particularly in brain regions implicated in pain and analgesia. This investigation further demonstrates the usefulness of phMRI as a translational tool in neuroscience research that can provide mechanistic insight and guide dose selection in drug development.

Substance P (SP) and neurokinin-1 receptors (NK-1R) are localized within central and peripheral sensory pain pathways. The roles of SP and NK-1R in pain processing, the anatomical distribution of NK-1R and efficacy observed in preclinical pain studies involving pain and sensory sensitization models, suggested that NK-1R antagonists (NK-1RAs) would relieve pain in patient populations. Despite positive data available in preclinical tests for a role of NK-1RAs in pain, clinical studies across several pain conditions have been negative. In this review, we discuss how functional imaging-derived information on activity in pain-processing brain regions could have predicted that NK-1RAs would have a low probability of success in this therapeutic domain.

The behavioral response to pain is driven by sensory and affective components, each of which is mediated by the CNS. Subjective pain ratings are used as readouts when appraising potential analgesics; however, pain ratings alone cannot enable a characterization of CNS pain circuitry during pain processing or how this circuitry is modulated pharmacologically. Having a more objective readout of potential analgesic effects may allow improved understanding and detection of pharmacological efficacy for pain. The pharmacological/functional magnetic resonance imaging (phMRI/fMRI) methodology can be used to objectively evaluate drug action on the CNS. In this context, we aimed to evaluate two drugs that had been developed as analgesics: one that is efficacious for pain (buprenorphine (BUP)) and one that failed as an analgesic in clinical trials aprepitant (APREP). Using phMRI, we observed that activation induced solely by BUP was present in regions with μ-opioid receptors, whereas APREP-induced activation was seen in regions expressing NK(1) receptors. However, significant pharmacological modulation of functional connectivity in pain-processing pathways was only observed following BUP administration. By implementing an evoked pain fMRI paradigm, these drugs could also be differentiated by comparing the respective fMRI signals in CNS circuits mediating sensory and affective components of pain. We report a correlation of functional connectivity and evoked pain fMRI measures with pain ratings as well as peak drug concentration. This investigation demonstrates how CNS-acting drugs can be compared, and how the phMRI/fMRI methodology may be used with conventional measures to better evaluate candidate analgesics in small subject cohorts.

Functional magnetic resonance imaging (fMRI) experiments are more complex compared with standard radiological imaging, involving additional data streams and hardware along with complex analysis methods. Here, we propose guidelines based around mitigating risks associated with the complexities of the technique at the level of the individual imaging protocol, including workable and effective quality assurance/quality control procedures and rigorous, predefined, analysis pipelines. Our aim is to provide a framework for 'good imaging practice' (GIP), enabling these requirements to be addressed at an appropriate level of detail. The development of a procedural framework for GIP in pharmaceutical fMRI studies could lead to greater acceptance of the method within industry and facilitate validation and, eventually, qualification of the technique as an imaging biomarker.

There is an increasing body of evidence implicating a role for alpha-amino-3-hydroxy-5-methyl-4 isoxazoleproprionic acid (AMPA) receptors in major depression and in the actions of antidepressant drugs. Alterations in AMPA receptors and other ionotropic glutamate receptors have been reported in depression, and following antidepressant treatment. Compounds which augment signaling through AMPA receptors (AMPA receptor potentiators) exhibit antidepressant-like behavioral effects in animal models, and produce neuronal effects similar to those produced by currently available antidepressants, including neurotrophin induction and increases in hippocampal progenitor cell proliferation. Additionally, the antidepressant fluoxetine has been found to alter AMPA receptor phosphorylation in a manner that is expected to increase AMPA receptor signaling. Data from mutant mice suggest that AMPA receptors may regulate the expression of brain-derived neurotrophic factor, a neurotrophin which has been implicated in the actions of antidepressant therapies. Combined, these data suggest that AMPA receptors may be in a key position to regulate mood disorders, and that compounds which target AMPA receptors may prove useful in the clinical management of depression.

To date, the blood oxygenated-level dependent (BOLD) functional magnetic resonance imaging (fMRI) technique has enabled an objective and deeper understanding of pain processing mechanisms embedded within the human central nervous system (CNS). In order to further comprehend the benefits and limitations of BOLD fMRI in the context of pain as well as the corresponding subjective pain ratings, we evaluated the univariate response, test-retest reliability and confidence intervals (CIs) at the 95% level of both data types collected during evoked stimulation of 40°C (non-noxious), 44°C (mildly noxious) and a subject-specific temperature eliciting a 7/10 pain rating. The test-retest reliability between two scanning sessions was determined by calculating group-level interclass correlation coefficients (ICCs) and at the single-subject level. Across the three stimuli, we initially observed a graded response of increasing magnitude for both VAS (visual analog score) pain ratings and fMRI data. Test-retest reliability was observed to be highest for VAS pain ratings obtained during the 7/10 pain stimulation (ICC=0.938), while ICC values of pain fMRI data for a distribution of CNS structures ranged from 0.5 to 0.859 (p

Pain is an important survival and protection mechanism for animals. However, chronic/persistent pain may be differentiated from normal physiological pain in that it confers no obvious advantage. An accumulating body of pharmacological, electrophysiological, and behavioral evidence is emerging in support of the notion that glutamate receptors play a crucial role in pain pathways and that modulation of glutamate receptors may have potential for therapeutic utility in several categories of persistent pain, including neuropathic pain resulting from injury and/or disease of central (e.g., spinal cord injury) or peripheral nerves (e.g., diabetic neuropathy, radiculopathy) and inflammatory or joint-related pain (e.g., rheumatoid arthritis, osteoarthritis). This review focuses on the role of glutamate receptors, including both ionotropic (AMPA, NMDA and kainate) and metabotropic (mGlu1-8) receptors in persistent pain states with particular emphasis on their expression patterns in nociceptive pathways and their potential as targets for pharmacological intervention strategies.

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.

Depression affects a large percentage of the general population and can produce devastating consequences to affected individuals, families and society. Although the treatment of depression has been advanced by traditional antidepressants, improvements are needed across several dimensions (e.g., overall treatment efficacy, therapeutic onset time, and side effect profile). The alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor has an allosteric modulatory site(s) for which potent positive modulators (potentiators) have been designed. These compounds produce antidepressant-like effects in animal models, increase levels of brain-derived neurotrophic factor (BDNF) and engender neurogenesis in vivo. Although these effects are also produced by traditional antidepressants, AMPA receptor potentiators appear to produce their effects through a novel mechanism.

Glutamatergic hyperactivity is implicated migraine pathogenesis. Also, LY293558, an α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate (KA) receptor antagonist, is effective in preclinical models of migraine. We therefore tested LY293558 in acute migraine. We conducted a randomized, triple-blind, parallel-group, double-dummy, multicentre trial of 1.2 mg/kg intravenous (IV) LY293558, 6 mg subcutaneous (SC) sumatriptan, or placebo in the treatment of acute migraine. The primary efficacy variable was the headache response rate, i.e. headache score improvement from moderate/severe at baseline to mild/none at 2 h. Of 45 enrolled patients, 44 patients (20M:24F; mean age ± SD = 40 ± 9 years) completed the study. Response rates were 69% for LY293558 ( P = 0.017 vs. placebo), 86% for sumatriptan ( P < 0.01 vs. placebo) and 25% for placebo. LY293558 and sumatriptan were superior to placebo ( P < 0.01 for all comparisons) on all other measures of improvement in pain and migraine associated symptoms. Fifteen percent of patients who took LY293558 reported adverse events (AEs) ( n = 2; one mild, one severe). Fifty-three percent of patients who took sumatriptan ( n = 8; seven mild, one moderate) and 31% of those who received placebo reported AEs ( n = 5; four mild, one severe). The efficacy and safety results of LY293558 in this small migraine proof of concept trial, together with supportive preclinical data, provide evidence for a potential role of nonvasoactive AMPA/KA antagonists in treating migraine. Larger trials are needed to further test the hypothesis.

An increasing body of evidence suggests that native kainate receptors form ion channels from homomeric and heteromeric combinations of five receptor subunits: GluR5, GluR6, GluR7, KA1 and KA2. We have examined the activity of agonists and antagonists at recombinant human kainate receptors expressed in HEK293 cells, using both whole-cell electrophysiological recording and 96-well plate fluo-3 based calcium microfluorimetry (FLIPR). Both homomeric (GluR5 and GluR6) and heteromeric (GluR5/6, GluR5/KA2 and GluR6/KA2) receptors were examined. Heteromeric receptor assemblies showed electrophysiological and pharmacological profiles which were distinct from homomeric channels. Several agonists, including AMPA, ATPA and (S)-5-iodowillardiine, and antagonists, including gamma-D-glutamylaminomethylsulphonic acid (GAMS) and the decahydroisoquinoline compounds LY293558, LY377770 and LY382884, were found to act at GluR5-containing channels while having no effect at GluR6 homomers. AMPA, ATPA and (S)-5-iodowillardiine did activate GluR6/KA2 heteromers, but only as partial agonists. Additionally, ATPA was shown to act as an antagonist at homomeric GluR6 receptors at high concentrations (IC50 approximately 2 mM). Kynurenic acid was also found to differentiate between GluR6 and GluR6/KA2 receptors, antagonizing glutamate at GluR6 (IC50 = 0.4 mM), while having no effect at GluR6/KA2 channels. The results of the current study provide a broad pharmacological characterization of both homomeric and heteromeric recombinant human kainate receptors, and identify which compounds are likely to be useful tools for studying these various receptor subtypes.

Glutamate is the major excitatory transmitter in the brain. Recent developments in the molecular biology and pharmacology of the α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) subtype of glutamate receptors have led to the discovery of selective, potent, and systemically active AMPA receptor potentiators. These molecules enhance synaptic transmission and play important roles in plasticity and cognitive processes. In the present study, we first characterized a novel AMPA receptor potentiator, (R)-4′-[1-fluoro-1-methyl-2-(propane-2-sulfonylamino)-ethyl]-biphenyl-4-carboxylic acid methylamide (LY503430), on recombinant human GLUA1–4 and native preparations in vitro and then evaluated the potential neuroprotective effects of the molecule in rodent models of Parkinson9s disease. Results indicated that submicromolar concentrations of LY503430 selectively enhanced glutamate-induced calcium influx into human embryonic kidney 293 cells transfected with human GLUA1, GLUA2, GLUA3, or GLUA4 AMPA receptors. The molecule also potentiated AMPA-mediated responses in native cortical, hippocampal, and substantia nigra neurons. We also report here that LY503430 provided dose-dependent functional and histological protection in animal models of Parkinson9s disease. The neurotoxicity after unilateral infusion of 6-hydroxydopamine into either the substantia nigra or the striatum of rats and that after systemic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in mice were reduced. Interestingly, LY503430 also had neurotrophic actions on functional and histological outcomes when treatment was delayed until well after (6 or 14 days) the lesion was established. LY503430 also produced some increase in brain-derived neurotrophic factor in the substantia nigra and a dose-dependent increases in growth associated protein-43 (GAP-43) expression in the striatum. Therefore, we propose that AMPA receptor potentiators offer the potential of a new disease modifying therapy for Parkinson9s disease.

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.

Developments in the molecular biology and pharmacology of GLU(K5), a subtype of the kainate class of ionotropic glutamate receptors, have enabled insights into the roles of this subunit in synaptic transmission and plasticity. However, little is known about the possible functions of GLU(K5)-containing kainate receptors in pathological conditions. We report here that, in hippocampal slices, selective antagonists of GLU(K5)-containing kainate receptors prevented development of epileptiform activity--evoked by the muscarinic agonist, pilocarpine--and inhibited the activity when it was pre-established. In conscious rats, these GLU(K5) antagonists prevented and interrupted limbic seizures induced by intra-hippocampal pilocarpine perfusion, and attenuated accompanying rises in extracellular L-glutamate and GABA. This anticonvulsant activity occurred without overt side effects. GLU(K5) antagonism also prevented epileptiform activity induced by electrical stimulation, both in vitro and in vivo. Therefore, we propose that subtype-selective GLU(K5) kainate receptor antagonists offer a potential new therapy for epilepsy.

The mechanisms involved in mossy fiber LTP in the hippocampus are not well established. In the present study, we show that the kainate receptor antagonist LY382884 (10 μM) is selective for presynaptic kainate receptors in the CA3 region of the hippocampus. At a concentration at which it blocks mossy fiber LTP, LY382884 selectively blocks the synaptic activation of a presynaptic kainate receptor that facilitates AMPA receptor-mediated synaptic transmission. Following the induction of mossy fiber LTP, there is a complete loss of the presynaptic kainate receptor-mediated facilitation of synaptic transmission. These results identify a central role for the presynaptic kainate receptor in the induction of mossy fiber LTP. In addition, these results suggest that the pathway by which kainate receptors facilitate glutamate release is utilized for the expression of mossy fiber LTP.

Enantiomerically pure (2S,4R)-4-substituted glutamic acids were prepared and tested for homomeric GluR5 and GluR6 kainate subtype receptor affinity. Some of the 4-cinnamyl analogues showed high selectivity and potency (K(i)25 nM) for the GluR5 receptors. The greatest selectivity and potency were achieved with the 3-(2-naphthyl)prop-2-enyl compound. This compound, LY339434, has negligible activity at the AMPA and kainate receptors GluR1, -2, -4 and -6. Although, LY339434 shows agonist activity at NMDA receptors in cultural hippocampal neurons (approximate EC(50) of 2.5 microM), we consider that LY339434 should be a useful pharmacological tool for the investigation of the functional role of GluR5 kainate receptors.

We have examined the pharmacology of kainate receptors in cultured hippocampal neurons (6-8 days in vitro (DIV)) from embryonic rats (E17). Cultured neurons were pre-treated with concanavalin A to remove kainate receptor desensitization and whole-cell voltage clamp electrophysiology employed to record inward currents in response to glutamatergic agonists and antagonists. N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA) receptor responses were blocked using MK801 (3 microM) and the 2,3-benzodiazepine, LY300168 (GYKI53655, 50 microM), respectively. Inward currents were recorded in hippocampal neurons upon application of kainate and the 2S,4R isomer of 4-methyl glutamic acid (SYM2081) with EC50 values of 3.4 +/- 0.4 microM and 1.6 +/- 0.5 microM, respectively (n = 6 cells). The GluR5 selective agonists, LY339434 (100 microM) and (RS)-2-amino-3-(3-hydroxy-5-tert-butyl-4-isoxazolyl) propionic acid (ATPA) (100 microM), did not evoke detectable inward currents in any cell responding to kainate. LY293558 and the selective GluR5 antagonist, LY382884, had weak antagonist effects on responses evoked by either kainate or (2S,4R)-4-methyl glutamate (IC50 > 300 microM). The quinoxalinedione, 2,3-dihyro-6-nitro-7-sulfamoyl-benzo(f)quinoxaline (NBQX), blocked both kainate and (2S,4R)-4-methyl glutamate-activated currents at much lower concentrations (IC50 approximately 10 microM). These results provide pharmacological evidence that ion channels comprised of GluR6 kainate receptor subunits mediate kainate receptor responses in hippocampal neurons cultured 6-8 DIV.

The effects of a novel synthetic macrocyclic polyamine (LY310315) were investigated on recombinant human N-type Ca2+ channels stabley expressed in HEK293 cells. LY310315 proved to be a potent and reversible N-type Ca2+ channel antagonist. Inhibition by this compound was dose-dependent with an IC50 of approximately 0.4 microM at pH 7.35. LY310315 blocked very rapidly at all concentrations tested. Upon washout, recovery of the Ca2+ current developed with a time constant of approximately 30 s. Use-dependence in the development of block indicated that voltage-dependent transitions in the channel protein were required to permit significant inhibition. Application of100 times the IC50 dose of LY310315 to the interior of the cell produced no detectable Ca2+ current inhibition. LY310315 had no effects on the kinetics of channel activation or deactivation but did slightly slow the rate of macroscopic inactivation observed during a 300 ms test depolarisation. In the presence of LY310315 the activation curve was significantly shallower. This resulted in a shift in the activation midpoint voltage to a more depolarised levels. LY310315-induced inhibition of human N-type channels was strongly dependent on the extracellular pH, with increased potency seen upon extracellular acidification. Although most effective against N-type Ca2+ channels, LY310315 was also found to inhibit both P-type and L-type Ca2+ channels. LY310315 proved to be a weak blocker of Na+ currents, but produced approximately 50% of the K+ currents of AtT20 cells at a concentration of 0.5 microM.

The pharmacological properties of voltage-dependent calcium channel (VDCC) subtypes appear mainly to be determined by the α 1 pore-forming subunit but, whether P-and Q-type VDCCs are encoded by the same α 1 gene presently is unresolved. To investigate this, we used IgG antibodies to presynaptic VDCCs at motor nerve terminals that underlie muscle weakness in the autoimmune Lambert–Eaton myasthenic syndrome (LEMS). We first studied their action on changes in intracellular free Ca 2+ concentration [Ca 2+ ] i in human embryonic kidney (HEK293) cell lines expressing different combinations of human recombinant VDCC subunits. Incubation for 18 h with LEMS IgG (2 mg/ml) caused a significant dose-dependent reduction in the K + -stimulated [Ca 2+ ] i increase in the α 1A cell line but not in the α 1B , α 1C , α 1D , and α 1E cell lines, establishing the α 1A subunit as the target for these autoantibodies. Exploiting this specificity, we incubated cultured rat cerebellar neurones with LEMS IgG and observed a reduction in P-type current in Purkinje cells and both P- and Q-type currents in granule cells. These data are consistent with the hypothesis that the α 1A gene encodes for the pore-forming subunit of both P-type and Q-type VDCCs.

In a cell line (C2D7) stably expressing the human N-type calcium channel encoded by the subunits α1B-a, β1b, α2bδ, we have analysed the Ca2+ currents produced by a range of action potential-like voltage protocols (APVPs). Such protocols consistently produced robust inward currents that could be eliminated by co-application of the Ca2+ channel blocking ions Cd2+ and La3+. The amplitude, latency to peak and area of the current produced by APVPs was dependent on the precise waveform of voltage protocol employed and the temperature. Short bursts of APVPs applied at 100 Hz produced a depression of the Ca2+ current amplitude which was dependent on the half-width of the APVP employed. In contrast, no frequency-dependent changes in the evoked current kinetics were detected. The amount of current depression seen during an 100 Hz 8 APVP burst was greatly enhanced by increasing the temperature from 22 to 37°C. Alterations to the intracellular Ca2+ buffering capacity suggested that the Ca2+ current depression produced during an APVP train arose, at least in part, from a Ca2+-dependent inactivation of the human N-type Ca2+ channel.

The functional expression of the kainate subtype of glutamate receptor (GluR) has been investigated in cultured rat cerebellar granule cells using single cell intracellular calcium ([Ca 2+ ] i ) measurements. Both AMPA- and kainate-induced [Ca 2+ ] i increases could be blocked completely by the AMPA receptor-selective antagonist LY300168 (50 μM). However, following treatment with concanavalin A, an inhibitor of kainate receptor desensitisation, 30% of cells showed a kainate-induced [Ca 2+ ] i rise of >100 nM in the presence of LY300168. Responses to 30 μM kainate in the presence of LY300168 were virtually abolished by the AMPA and GluR5 kainate receptor competitive antagonist LY293558 (100 μM). These results demonstrate the presence of functional kainate receptors on cultured cerebellar granule cells, and suggest that the GluR5 subtype of kainate receptor plays a significant role in kainate receptor-mediated [Ca 2+ ] i increases.

In the present study we have examined the effects of the small organic molecules: NNC 09-0026 ((-)-trans-1-butyl-4-(4-dimethylaminophenyl)-3-[(4-trifluoromethyl-ph eno xy) methyl] piperidine dihydrochloride); SB 201823-A (4-[2-(3,4-dichlorophenoxy)ethyl]-1-pentyl piperidine hydrochloride); NS 649 (2-amino-1-(2,5-dimethoxyphenyl)-5-trifluoromethyl benzimidazole); CNS 1237 (N-acenaphthyl-N'-4-methoxynaphth-1-yl guanidine) and riluzole on human omega-conotoxin sensitive N-type voltage-dependent Ca2+ channel currents (ICa) expressed in HEK293 cells, on Na+ channel currents (INa) in acutely isolated cerebellar Purkinje neurones in vitro and in the gerbil model of global cerebral ischaemia in vivo. Estimated IC50 values for steady-state inhibition of ICa were as follows; NNC 09-0026, 1.1 microM; CNS 1237, 4.2 microM; SB 201823-A, 11.2 microM; NS 649, 45.7 microM and riluzole, 233 microM. Estimated IC50 values for steady-state inhibition of Na+ channel currents were as follows: NNC 09-0026, 9.8 microM; CNS 1237, 2.5 microM; SB 201823-A, 4.6 microM; NS 649, 36.7 microM and riluzole, 9.4 microM. In the gerbil model of global cerebral ischaemia the number of viable cells (mean +/- S.E.M.) per 1 mm of the CA1 was 215 +/- 7 (sham operated), 10 +/- 2 (ischaemic control), 44 +/- 15 (NNC 09-0026 30 mg/kg i.p.), 49 +/- 19 (CNS 1237 30 mg/kg i.p.), 11 +/- 2 (SB 201823-A 10 mg/kg i.p.), 17 +/- 4 (NS 649 50 mg/kg i.p.) and 48 +/- 18 (riluzole 10 mg/kg i.p.). Thus NNC 09-0026, CNS 1237 and riluzole provided significant neuroprotection when administered prior to occlusion while SB 201823-A and NS 649 failed to protect. These results indicate that the Ca2+ channel antagonists studied not only inhibited human N-type voltage-dependent Ca2+ channels but were also effective blockers of rat Na+ channels. Both NNC 09-0026 and CNS 1237 showed good activity at both Ca2+ and Na+ channels and this may contribute to the observed neuroprotection.

Treatment of cerebellar neurones in culture with an antisense oligonucleotide (ODN) against α1A, reduced the whole-cell P-type calcium channel current relative to mismatch ODN treated controls (p < 0.001). Therefore, AgaIVA (50 nM) reduced whole-cell calcium current in mismatch and antisense treated cells by 70 ± 4 and 19 ± 3%, respectively. © 1997 Elsevier Science Ltd. All rights reserved.

The novel compound LY354740 is a conformationally constrained analog of glutamate, which was designed for interaction at metabotropic glutamate (mGlu) receptors. In this paper the selectivity of LY354740 for recombinant human mGlu receptor subtypes expressed in non-neuronal (RGT) cells is described. At human mGlu2 receptors, LY354740 produced >90% suppression of forskolin-stimulated cAMP formation with an ec50 of 5.1 ± 0.3 nM. LY354740 was six-fold less potent in activating human mGlu3 receptors (ec50 = 24.3 ± 0.5 nM). LY354740 inhibition of forskolin-stimulated cAMP formation in human mGlu2 receptor-expressing cells was blocked by competitive mGlu receptor antagonists, including (+)-α-methyl-4-carboxyphenylglycine (MCPG) and LY307452 ((2S,4S0-2-amino-4-(4,4-diphenylbut-1-yl)-pentane-1,5-dioic acid). LY354740 had no agonist or antagonist activities at cells expressing human mGlu4 or mGlu7 (group III mGlu receptors) (ec50s > 100 000 nM). When tested at group I phosphoinositide-coupled human mGlu receptors (mGlu1a and mGlu5a), LY354740 did not activate or inhibit mGlu receptor agonist-evoked phosphoinositide hydrolysis at up to 100 000 nM. Electrophysiological experiments also demonstrated that LY354740 also had no appreciable activity in cells expressing human recombinant AMPA (GluR4) and kainate (GluR6) receptors. Thus, LY354740 is a highly potent, efficacious and selective group II (mGlu2/3) receptor agonist, useful to explore the functions of these receptors in situ. © 1997 Elsevier Science Ltd. All rights reserved.

The whole cell variant of the patch-clamp technique was used to investigate the actions of polyamine spider toxins and their analogues on high voltage-activated Ca 2+ currents. The actions of synthesised FTX (putative natural toxin from the American funnel web spider), sFTX-3.3, Orn-FTX-3.3 and Lys-FTX-3.3 (synthetic analogues of FTX) were studied using cultured dorsal root ganglion neurones from neonatal rats, C2D7 cells (HEK293 cells stably coexpressing recombinant human N-type voltage-activated Ca 2+ channel, α 1B-1 - α 2b δβ 1b subunits) and freshly isolated cerebellar Purkinje neurones. In dorsal root ganglion neurones, sFTX-3.3 (10 μ M) inhibited high voltage-activated Ca 2+ currents evoked by depolarisations to 0 mV from a holding potential of −90 mV. Partial overlap in Ca 2+ current sensitivity to the polyamine sFTX-3.3 and the peptide spider toxin ω -Aga IVA was observed. However, evidence also suggests sFTX-3.3 and ω -Aga IVA do not show complete pharmacological overlap and that distinct parts of the Ca 2+ current are sensitive to one of two inhibitors. The arginine group on sFTX-3.3 appears to be important for its inhibitory action on Ca 2+ currents, because analogues where this amino acid was replaced with either ornithine (Orn-FTX-3.3) or lysine (Lys-FTX-3.3) were relatively inactive at concentrations below 1 mM. Synthesised FTX (100 μ M) was inactive as an inhibitor of Ca 2+ currents recorded from dorsal root ganglion and only produced modest effects in Purkinje neurones and C2D7 cells. At a concentration of 1 mM, nonselective actions were observed that indicated that synthesised FTX and sFTX-3.3 could reversibly inhibit both N- and P-type Ca 2+ channels equally well. In conclusion, the potency of polyamines as nonselective inhibitors of Ca 2+ channels is in part determined by the presence of a terminal arginine, and this may involve an interaction between terminal guanidino groups with Ca 2+ binding sites.

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.

The activity and selectivity of the glutamate receptor antagonists belonging to the 2,3-benzodiazepine class of compounds have been examined at recombinant human non-NMDA glutamate receptors expressed in HEK293 cells and on native rat NMDA and non-NMDA receptors in vitro. The racemic 2,3-benzodiazepines GYKI52466, LY293606 (GYKI53405) and LY300168 (GYKI53655) inhibited AMPA (10 microM)-mediated responses in recombinant human GluR1 receptors expressed in HEK293 cells with approximate IC50 values of 18 microM, 24 microM and 6 microM, respectively and AMPA (10 microM) responses in recombinant human GluR4 expressing HEK293 cells with approximate IC50 values of 22 microM, 28 microM and 5 microM, respectively. GYKI 52466, LY293606 and LY300168 were non-competitive antagonists of AMPA receptor-mediated responses in acutely isolated rat cerebellar Purkinje neurons with approximate IC50 values of 10 microM, 8 microM and 1.5 microM, respectively. The activity of racemic compounds LY293606 and LY300168 was established to reside in the (-) isomer of each compound. At a concentration of 100 microM, GYKI52466, LY293606 and LY300168 produced30% inhibition of kainate-activated currents evoked in HEK293 cells expressing either human homomeric GluR5 or GluR6 receptors or heteromeric GluR6+KA2 kainate receptors. The activity of the 2,3-benzodiazepines at 100 microM was weak at kainate receptors, but was stereoselective. Similar levels of inhibition were observed for kainate-induced currents in dorsal root ganglion neurons. Intact tissue preparations were also used to examine the stereoselective actions of the 2,3-benzodiazepines. In the cortical wedge preparation, the active isomer of LY300168, LY303070, produced a non-competitive antagonism of AMPA-evoked depolarizations with smaller changes in depolarizations induced by kainate and no effect on NMDA-dependent depolarizations. LY303070 was also effective in preventing 30 microM AMPA-induced depolarizations in isolated spinal cord dorsal roots with an approximate IC50 value of 1 microM. Synaptic transmission in the hemisected spinal cord preparation was stereoselectively antagonized by the active isomers of LY300168 and LY293606. In summary, these results indicate that 2,3-benzodiazepines are potent, selective and stereospecific antagonists of the AMPA subtype of the non-NMDA glutamate receptor.

Ifenprodil and eliprodil are both non-competitive NMDA receptor antagonists which have been shown to inhibit neuronal Ca2+ channel currents. We have examined the effects of these agents on two defined subtypes of voltage-dependent Ca2+ channels and in the gerbil model of global cerebral ischaemia. Recombinantly expressed human alpha 1B-1 alpha 2b beta 1-3 Ca2+ subunits in HEK293 cells, which results in an omega-conotoxin-sensitive neuronal N-type voltage-dependent Ca2+ channel and omega-Aga IVA sensitive Ca2+ channels (P-type) in acutely isolated cerebellar Purkinje neurones were reversibly inhibited by ifenprodil and eliprodil. Human N-type Ca2+ channel currents were inhibited by ifenprodil and eliprodil with IC50 values of 50 microM and 10 microM respectively whereas P-type Ca2+ channel currents were inhibited reversibly by ifenprodil and eliprodil with approximate IC50 values of 60 microM and 9 microM respectively. Maximum current block observed for both channel subtypes was approximately 80% for both ifenprodil and eliprodil. For neuroprotection studies, animals were subjected to 5 min bilateral carotid artery occlusion with or without administration of either ifenprodil or eliprodil (5, 10 or 20 mg/kg i.p.) immediately after surgery followed by two further doses (2.5, 5 or 10 mg/kg, respectively) at 3 and 6 h post-occlusion. Both compounds provided significant protective effects against ischaemia-induced neurodegeneration in the CA1 region of the hippocampus. These results indicate that both ifenprodil and eliprodil protect against ischaemia-induced neurodegeneration when administered post-occlusion and that they also block N and P-type voltage-dependent Ca2+ channels.

The synthesis and pharmacological characterization of a novel series of 4-aryl-substituted kainic acid analogs are described. Receptor affinities were determined on recombinantly expressed humGluR6 kainate receptors and on [3H]kainate binding to rat forebrain kainate receptors. Functional agonist potencies were assessed using whole cell voltage clamp recordings in cells expressing humGluR6 receptors. Substitution of phenyl for the methyl at the C-4 position of kainic acid produced 11 which has high affinity and agonist potency at the GluR6 receptor. Substitution on phenyl led to a series of compounds with varying affinity for this kainate receptor. Agonist potency correlated with receptor affinity and with no derivative could antagonist activity be identified. Affinities for the humGluR6 kainate receptor were approximately 10-50 less than the observed affinities at rat forebrain kainate receptors. Furthermore, within the series of 4-aryl-substituted kainic acid analogs, there was a high degree of correlation between binding affinities for humGluR6 receptors and competition with kainate binding to rat forebrain kainate receptors.

The in vitro and in vivo pharmacology of a structurally novel competitive antagonist for the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) subtype of excitatory amino acid receptors is described. LY215490, (+/-)(6-(2-(1-H-tetrazol-5-yl)ethyl) decahydroisoquinoline-3-carboxylic acid), was shown to displace selectively 3H-AMPA and 3H-6-cyano-7-nitro- quinoxaline-2,3-dione (3H-CNQX) binding to rat brain membranes. LY215490 potently antagonized quisqualate-and AMPA-induced depolarizations of rat cortical slices in a competitive manner, while requiring higher concentrations to antagonize the effects of N-methyl-D-aspartate (NMDA) and kainate. In slices of rat hippocampus, LY215490 also selectively antagonized AMPA-evoked release of 3H-norepinephrine. These AMPA receptor activities were due to the (-) isomer of the compound. (3S,4aR,6R, 8aR)-6-[2-(1(2)H-tetrazole-5-yl)ethyl] decahydroisoquinoline-3-carboxylic acid (LY293558). LY215490 was centrally active following parenteral administration in mice as demonstrated by protection versus maximal electroshock seizures and decreases in spontaneous motor activity. LY215490 (its active isomer being LY293558) represents a novel pharmacological agent for in vitro and in vivo studies of AMPA receptor function in the CNS.

We examined the mechanism by which capsaicin produces its toxic effects on cultures of rat sensory neurons. Capsaicin caused a robust increase in [Ca2+]i in a subpopulation of cultured rat dorsal root ganglion neurons. Similarly, a brief exposure to capsaicin resulted in delayed degeneration of a subpopulation of the cells. This subpopulation (about 35% of the cells present) was characterized by a capsaicin-induced uptake of Co2+, which could be detected cytochemically. Both capsaicin-induced Co2+ uptake and capsaicin-induced cell death were blocked by the capsaicin antagonist Ruthenium Red. Cell death was also prevented by removal of external calcium or by inhibiting calcium-activated proteases such as calpain. Evidence that calpain activity was increased was provided by examining the amount of degradation of the preferred calpain substrate alpha-spectrin. Capsaicin treatment produced a significant increase in the levels of the 150,000 molecular weight spectrin breakdown product. Furthermore, applying the protease inhibitors E64 or MDL 28,170 reduced capsaicin-mediated cell death. It is concluded that capsaicin kills a subpopulation of sensory neurons by activating a receptor-operated channel. The consequent Ca2+ ion influx causes large increases in [Ca2+]i and subsequent activation of Ca(2+)-sensitive proteases. This model provides support for the role of [Ca2+]i as the orchestrator of delayed neuronal degeneration.

Neuropeptide Y, one of the scions of the pancreatic polypeptide family, is found throughout the nervous system. Based on its abundance alone, one would expect neuropeptide Y to play an important role in the regulation of neuronal activity, and indeed many pharmacological studies have demonstrated neuromodulatory effects of neuropeptide Y. Here, William F. Colmers and David Bleakman review the known actions of neuropeptide Y on the electrical properties of nerve cells. Neuropeptide Y inhibits Ca2+ currents, and modulates transmitter release in a highly selective manner. Neuropeptide Y might be quite important in the regulation of neuronal state, as exemplified by its actions in the hippocampus and the dorsal raphé nucleus.

Buprenorphine (BUP) is a partial agonist at μ-, δ- and ORL1 (opioid receptor-like)/nociceptin receptors and antagonist at the κ-opioid receptor site. BUP is known to have both analgesic as well as antihyperalgesic effects via its central activity, and is used in the treatment of moderate to severe chronic pain conditions. Recently, it was shown that intravenous (IV) administration of 0.2mg/70 kg BUP modulates the blood oxygenation level-dependent (BOLD) functional magnetic resonance imaging (fMRI) response to acute noxious stimuli in healthy human subjects. The present study extends these observations by investigating the effects of BUP dose and route of administration on central nervous system (CNS) pain circuitry. Specifically, the modulation of evoked pain BOLD responses and resting state functional connectivity was measured following IV (0.1 and 0.2mg/70 kg) and sublingual (SL) (2mg) BUP administration in healthy human subjects. While 0.1mg/70 kg IV BUP is sub-analgesic, both 0.2mg/70 kg IV BUP and 2.0mg SL BUP are analgesic doses of the drug. Evoked BOLD responses were clearly modulated in a dose-dependent manner. The analgesic doses of BUP by both routes of administration yielded a potentiation in limbic/mesolimbic circuitry and attenuation in sensorimotor/sensory-discriminative circuitry. In addition, robust decreases in functional connectivity between the putamen and the sensorimotor/sensory-discriminative structures were observed at the two analgesic doses subsequent to measuring the maximum plasma BUP concentrations (C(max)). The decreases in functional connectivity within the sensorimotor/sensory-discriminative circuitry were also observed to be dose-dependent in the IV administration cohorts. These reproducible and consistent functional CNS measures at clinically effective doses of BUP demonstrate the potential of evoked pain fMRI and resting-state functional connectivity as objective tools that can inform the process of dose selection. Such methods may be useful during early clinical phase evaluation of potential analgesics in drug development.

Calcium influx through voltage-sensitive Ca2+ channels is the normal physiological stimulus for the activity-dependent release of neurotransmitters at synaptic contacts. It has been postulated that presynaptic inhibition of transmitter release is due to a reduction in Ca2+ influx at the nerve terminal, which could result from the direct inhibition of Ca2+ channels. Neuropeptide Y and noradrenaline act as cotransmitters at many sympathetic synapses. Both of these substances produce presynaptic inhibition and can inhibit Ca2+ currents in the soma of sympathetic neurons. Here we provide direct evidence that presynaptic inhibition produced by neuropeptide Y at sympathetic nerve terminals is associated with a reduction in Ca2+ influx and that this is due to the selective inhibition of neuronal N-type Ca2+ channels.

Septal neurons from embryonic rats were grown in tissue culture. Microfluorimetric and electrophysiological techniques were used to study Ca2+ homeostasis in these neurons. The estimated basal intracellular free ionized calcium concentration ([Ca2+]i) in the neurons was low (50-100 nM). Depolarization of the neurons with 50 mM K+ resulted in rapid elevation of [Ca2+]i to 500-1,000 nM showing recovery to baseline [Ca2+]i over several minutes. The increases in [Ca2+]i caused by K+ depolarization were completely abolished by the removal of extracellular Ca2+, and were reduced by approximately 80% by the 'L-type' Ca2+ channel blocker, nimodipine (1 microM). [Ca2+]i was also increased by the excitatory amino acid L-glutamate, quisqualate, AMPA and kainate. Responses to AMPA and kainate were blocked by CNQX and DNQX. In the absence of extracellular Mg2+, large fluctuations in [Ca2+]i were observed that were blocked by removal of extracellular Ca2+, by tetrodotoxin (TTX), or by antagonists of N-methyl D-aspartate (NMDA) such as 2-amino 5-phosphonovalerate (APV). In zero Mg2+ and TTX, NMDA caused dose-dependent increases in [Ca2+]i that were blocked by APV. Caffeine (10 mM) caused transient increases in [Ca2+]i in the absence of extracellular Ca2+, which were prevented by thapsigargin, suggesting the existence of caffeine-sensitive ATP-dependent intracellular Ca2+ stores. Thapsigargin (2 microM) had little effect on [Ca2+]i, or on the recovery from K+ depolarization. Removal of extracellular Na+ had little effect on basal [Ca2+]i or on responses to high K+, suggesting that Na+/Ca2+ exchange mechanisms do not play a significant role in the short-term control of [Ca2+]i in septal neurons. The mitochondrial uncoupler, CCCP, caused a slowly developing increase in basal [Ca2+]i; however, [Ca2+]i recovered as normal from high K+ stimulation in the presence of CCCP, which suggests that the mitochondria are not involved in the rapid buffering of moderate increases in [Ca2+]i. In simultaneous electrophysiological and microfluorimetric recordings, the increase in [Ca2+]i associated with action potential activity was measured. The amplitude of the [Ca2+]i increase induced by a train of action potentials increased with the duration of the train, and with the frequency of firing, over a range of frequencies between 5 and 200 Hz. Recovery of [Ca2+]i from the modest Ca2+ loads imposed on the neuron by action potential trains follows a simple exponential decay (tau = 3-5 s).

There is increasing interest in the application of quantitative magnetic resonance imaging (MRI) methods to drug development, but as yet little standardization or best practice guidelines for its use in this context. Pharmaceutical trials are subject to regulatory constraints and sponsor company processes, including site qualification and expectations around study oversight, blinding, quality assurance and quality control (QA/QC), analysis and reporting of results. In this article, we review the processes on the sponsor side and also the procedures involved in data acquisition at the imaging site. We then propose summary recommendations to help guide appropriate imaging site qualification, as part of a framework of 'good imaging practice' for functional (f)MRI studies applied to drug development.

1. We have examined the effects of neuropeptide Y (NPY) on synaptic transmission and [Ca2+]i signals in rat hippocampal neurones grown in culture. [Ca2+]i in individual neurones displayed frequent spontaneous fluctuations often resulting in an elevated plateau [Ca2+]i. These fluctuations were reduced by tetrodotoxin (1 microM) or combinations of the excitatory amino acid antagonists 6-cyano-7-dinitro-quinoxaline (CNQX) (10 microM) and aminophosphonovalerate (APV) (50 microM), indicating that they were the result of glutamatergic transmission occurring between hippocampal neurones. 2. [Ca2+]i fluctuations were also prevented by Ni2+ (200 microM), by the GABAB receptor agonist, baclofen (10 microM) and by NPY (100 nM) or Y2 receptor-selective NPY agonists. Following treatment of cells with pertussis toxin, NPY produced only a brief decrease in [Ca2+]i fluctuations which rapidly recovered. 3. Perfusion of hippocampal neurones with 50 mM K+ produced a large rapid increase in [Ca2+]i. This increase was slightly reduced by NPY or by a combination of CNQX and APV. The effects of CNQX/APV occluded those of NPY. NPY had no effect on Ba2+ currents measured in hippocampal neurones under whole cell voltage-clamp even in the presence of intracellular GTP-gamma-S. On the other hand, Ba2+ currents were reduced by both Cd2+ (200 microM) and baclofen (10 microM). 4. Current clamp recordings from hippocampal neurones demonstrated the occurrence of spontaneous e.p.s.ps and action potential firing which were accompanied by increases in [Ca2+]i. This spontaneous activity and the accompanying [Ca2+]i signals were prevented by application of NPY (100 nM). When hippocampal neurones were induced to fire trains of action potentials in the absence of synaptic transmission, these were accompanied by an increase in cell soma [Ca2+]j. NPY (100 nM) had no effect on these cell soma [Ca2+], signals. NPY (100 nM) also had no effect on inward currents generated in hippocampal neurones by micropipette application of glutamate (50 microM).5. Thus, NPY is able to abolish excitatory neurotransmission in hippocampal cultures through a pertussis toxin-sensitive mechanism. However, no effect of NPY on Ca2+ influx into the cell soma of these hippocampal neurones could be discerned. These results are consistent with a localized presynaptic inhibitory effect of NPY on glutamate release in hippocampal neurones in culture.

Kainate receptors (KARs) are involved in both NMDA receptor-independent long-term potentiation (LTP) and synaptic facilitation at mossy fibre synapses in the CA3 region of the hippocampus. However, the identity of the KAR subtypes involved remains controversial. Here we used a highly potent and selective GluK1 (formerly GluR5) antagonist (ACET) to elucidate roles of GluK1-containing KARs in these synaptic processes. We confirmed that ACET is an extremely potent GluK1 antagonist, with a Kb value of 1.4+/-0.2 nM. In contrast, ACET was ineffective at GluK2 (formerly GluR6) receptors at all concentrations tested (up to 100 microM) and had no effect at GluK3 (formerly GluR7) when tested at 1 microM. The X-ray crystal structure of ACET bound to the ligand binding core of GluK1 was similar to the UBP310-GluK1 complex. In the CA1 region of hippocampal slices, ACET was effective at blocking the depression of both fEPSPs and monosynaptically evoked GABAergic transmission induced by ATPA, a GluK1 selective agonist. In the CA3 region of the hippocampus, ACET blocked the induction of NMDA receptor-independent mossy fibre LTP. To directly investigate the role of pre-synaptic GluK1-containing KARs we combined patch-clamp electrophysiology and 2-photon microscopy to image Ca2+ dynamics in individual giant mossy fibre boutons. ACET consistently reduced short-term facilitation of pre-synaptic calcium transients induced by 5 action potentials evoked at 20-25Hz. Taken together our data provide further evidence for a physiological role of GluK1-containing KARs in synaptic facilitation and LTP induction at mossy fibre-CA3 synapses.

1. The identity of the neuropeptide Y (NPY) receptor associated with the observed inhibition of neuronal Ca2+ currents (ICa) in rat dorsal root ganglion (DRG) cells has been established on the basis of agonist responses to analogues and carboxy terminal (C-terminal) fragments of the NPY molecule. 2. Whole cell barium currents (IBa) in DRG cells were reversibly inhibited by 100 nM NPY, 100 nM PYY and C-terminal fragments of NPY in a manner that correlated with the length of the NPY fragments (for inhibition of the IBa NPY = PYY greater than NPY2-36 greater than NPY13-36 greater than NPY16-36 greater than NPY18-36 much greater than NPY25-36). 3. C-terminal fragments of NPY were also effective in reversibly reducing the ICa, the associated increase in the intracellular Ca2+ concentration [( Ca2+]i) and the increased [Ca2+]i produced by evoked action potentials in the DRG cells. In addition, a Ca(2+)-activated Cl- conductance was also reversibly reduced by NPY fragments only when accompanied by a reduction in Ca2+ entry. 4. We conclude that the Y2 receptor for neuropeptide Y is coupled to inhibition of Ca2+ influx via voltage-sensitive calcium channels in DRG cells.

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.

Some N3-substituted analogues of willardiine such as 11 and 13 are selective kainate receptor antagonists. In an attempt to improve the potency and selectivity for kainate receptors, a range of analogues of 11 and 13 were synthesized with 5-substituents on the uracil ring. An X-ray crystal structure of the 5-methyl analogue of 13 bound to GLUK5 revealed that there was allowed volume around the 4- and 5-positions of the thiophene ring, and therefore the 4,5-dibromo and 5-phenyl (67) analogues were synthesized. Compound 67 (ACET) demonstrated low nanomolar antagonist potency on native and recombinant GLUK5-containing kainate receptors (KB values of 7 +/- 1 and 5 +/- 1 nM for antagonism of recombinant human GLUK5 and GLUK5/GLUK2, respectively) but displayed IC50 values100 microM for antagonism of GLUA2, GLUK6, or GLUK6/GLUK2.

Since their discovery in the 1950s, ionotropic glutamate receptors represent a class of receptor proteins that mediate fast excitatory transmission in the central nervous system. In this chapter, we describe the extensive studies of agents that act at the various subtypes of ionotropic glutamate receptor and how these agents have been used to delineate the physiological and pathophysiological roles of these receptors. Keywords: N-methyl-D-aspartate; kainate; a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; AMPA; othosteric; allosteric

The hypothesis that kainate receptor blockade would be associated with anxiolytic-like effects was tested with a selective ligand, 3S,4aR,6S,8aR-6-((4-carboxyphenyl)methyl)-1,2,3,4,4a,5,6,7,8,8a-decahydroisoquinoline-3-carboxylic acid (LY382884). LY382884 selectively binds the GLU(K5) kainate receptor subunit (K(b)=0.6 microM) and has 30 microM or greater affinity for cloned human AMPA receptor subtypes. The anxiolytic potential of LY382884 was tested in rats responding under a Vogel conflict procedure, a pharmacologically validated model for the prediction of antianxiety efficacy in humans. Both the benzodiazepine anxiolytic chlordiazepoxide and LY382884 increased suppressed licking without affecting rates of non-suppressed licking. In contrast, an AMPA receptor selective antagonist, 7H-1,3-dioxolo[4,5-h][2,3]benzodiazepine-7-carboxamide, 5-(4-aminophenyl)-8,9-dihydro-N,8-dimethyl-, monohydrochloride (9CI) (GYKI53655), did not increase suppressed responding. The finding that a selective GLU(K5) receptor antagonist produced anxiolytic-like effects in an animal model predictive of efficacy in humans combined with data in the literature on glutamatergic modulation of anxiety suggests that kainate receptor sensitivity to glutamate might be an important mediating event in the pathophysiological expression of anxiety states. The selective targeting of kainate receptors with an antagonist could therefore be a novel pharmacological mechanism to treat anxiety disorders.