Chemically Modified, α-Amino-3-hydroxy-5-methyl-4-isoxazole (AMPA) Receptor RNA Aptamers Designed for in Vivo Use.

Glutamate ion channels have three subtypes, that is, α-amino-3-hydroxy-5-methyl-4-isoxazole (AMPA), kainate, and N-methyl-d-aspartate (NMDA) receptors. Excessive activity of these receptor subtypes either individually or collectively is involved in various neurological disorders. RNA aptamers as antagonists of these receptors are potential therapeutics. For developing aptamer therapeutics, the RNA aptamers must be chemically modified to become ribonuclease-resistant or stable in biological fluids. Using systematic evolution of ligands by exponential enrichment (SELEX) and a chemically modified library, prepared enzymatically (i.e., the library contains RNAs with 2'-fluoro modified nucleoside triphosphates or ATPs, CTPs and UTPs, but regular GTPs), we have isolated an aptamer. The short aptamer (69 nucleotides) FN1040s selectively inhibits the GluA1 and GluA2Qflip AMPA receptor subunits, whereas the full-length aptamer (101 nucleotides) FN1040 additionally inhibits GluK1, but not GluK2, kainate receptor, and GluN1a/2A and GluN1a/2B, the two major native NMDA receptors. The two aptamers show similar potency (2-4 µM) and are stable with a half-life of at least 2 days in serum-containing medium or cerebrospinal fluid. Therefore, these two aptamers are amenable for in vivo use.

AMPA/kainate glutamate receptors contribute to inflammation, degeneration and pain related behaviour in inflammatory stages of arthritis.

OBJECTIVES: Synovial fluid glutamate concentrations increase in arthritis. Activation of kainate (KA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptors (GluRs) increase interleukin-6 (IL-6) release and cause arthritic pain, respectively. We hypothesised that AMPA and KA GluRs are expressed in human arthritis, and that intra-articular NBQX (AMPA/KA GluR antagonist) prevents pain and pathology in antigen-induced arthritis (AIA). METHODS: GluR immunohistochemistry was related to synovial inflammation and degradation in osteoarthritis (OA) and rheumatoid arthritis (RA). A single intra-articular NBQX injection was given at induction, and knee swelling and gait of AIA and AIA+NBQX rats compared over 21 days, before imaging, RT-qPCR, histology and immunohistochemistry of joints. Effects of NBQX on human primary osteoblast (HOB) activity were determined. RESULTS: AMPAR2 and KA1 immunolocalised to remodelling bone, cartilage and synovial cells in human OA and RA, and rat AIA. All arthritic tissues showed degradation and synovial inflammation. NBQX reduced GluR abundance, knee swelling (p<0.001, days 1-21), gait abnormalities (days 1-2), end-stage joint destruction (p<0.001), synovial inflammation (p<0.001), and messenger RNA expression of meniscal IL-6 (p<0.05) and whole joint cathepsin K (p<0.01). X-ray and MRI revealed fewer cartilage and bone erosions, and less inflammation after NBQX treatment. NBQX reduced HOB number and prevented mineralisation. CONCLUSIONS: AMPA/KA GluRs are expressed in human OA and RA, and in AIA, where a single intra-articular injection of NBQX reduced swelling by 33%, and inflammation and degeneration scores by 34% and 27%, respectively, exceeding the efficacy of approved drugs in the same model. AMPA/KA GluR antagonists represent a potential treatment for arthritis.

NGX426, an oral AMPA-kainate antagonist, is effective in human capsaicin-induced pain and hyperalgesia.

BACKGROUND: Non-N-methyl-D-aspartate receptor subtypes modulate neurotransmitter release and mediate fast excitatory postsynaptic potentials. This study evaluated the effects of an oral prodrug to tezampanel, a selective α-amino-3-hydroxy-5-methly-4-isoxazole-proprionic acid/kainate receptor antagonist, on intradermal capsaicin-induced pain and hyperalgesia. METHODS: This was a randomized, double blind, crossover, placebo-controlled study. Eighteen subjects received 150 or 90 mg NGX426, or placebo, separated by a washout of 6 ± 2 days. In each treatment period, two intradermal injections of capsaicin were given in the volar region of alternate forearms at 30- and 120-minute drug/placebo administration. Spontaneous pain, elicited pain, and area of hyperalgesia were determined at certain time points after each injection. Subjects were asked to rate the painfulness of a 1-minute long 45°C heat stimulus (brief thermal stimulation [BTS]) applied to the anterior thigh at 4 hours and 30 minutes following drug administration, then every 30 minutes through 6 hours following drug administration. RESULTS: The 150-mg dose produced a statistically definitive reduction in spontaneous pain for all time points relative to placebo. The 90-mg dose produced a statistically significant reduction for the early time point and the entire time interval. Both doses significantly reduced elicited pain at all time points. For the BTS, the 150-mg group reached statistical significance compared with placebo at the 270-minute time point only. CONCLUSIONS: This study demonstrated that NGX426 reduces capsaicin-induced pain and hyperalgesia in human volunteers with low incidence of side effects that suggests that this class of drug may be effective in the treatment of clinical pain.

[Effects of ionotropic glutamate receptor channel blockers on the development of audiogenic seizures in Krushinski-Molodkina rats].

The action of noncompetitive blockers of glutamate receptors has been investigated on Krushinski-Molodkina rats genetically-prone to audiogenic seizures. The selective blockers of NMDA receptor channels, memantine and IEM-1921, and their dicationic homologues, IEM-1925 and IEM-1754, capable of blocking in varying degrees both NMDA and Ca-permeable AMPA receptor channels, were studied. The drugs were injected intramuscularly to rats with the different time intervals (30 min, 1, 2 or 3 hours) before sound signal. The effects of the drugs on latent period of initial locomotor activity provoked by audio stimulation (8 kHz sine-wave tone, 90 dB volume), the appearance of clonic convulsions of different intensities, and, finally, tonic convulsions with limb and tail extension were evaluated. Within 30 min after injection IEM-1921 at a dose of 5 mg/kg, 33% of rats manifested a complete absence of convulsive reactions to sound, and in 59% of rats audiogenic seizures occured only in the form of motor excitation without a generalized clonic-tonic convulsions. Memantine at a dose of 5 mg/kg did not cause a complete blockade of seizures, but after 1 h of injection in 50% of the rats and after 2 h in 70% of rats a weakening of the audiogenic seizures to the level of motor excitation only was observed. After 3 hrs after administration of blockers its anticonvulsive action weakened significantly (p < 0.01). Dicationic blockers that block both NMDA and AMPA/kainate receptors, IEM-1925 (in doses of 0.001-20.0 mg/kg) and IEM-1754 (0.025-50.0 mg/kg), did not affect audiogenic clonic-tonic convulsive reactions. The involvement of activation of NMDA and calcium permeable AMPA/kainate receptors in the pathogenesis of audiogenic seizures is discussed.

Topiramate in the treatment of migraine: a kainate (glutamate) receptor antagonist within the trigeminothalamic pathway.

BACKGROUND: The development of new agents for the preventive treatment of migraine is the greatest unmet need in the therapeutics of primary headaches. Topiramate, an anticonvulsant drug, is an effective anti-migraine preventive whose mechanism of action is not fully elucidated. Since glutamate plays a major role in migraine pathophysiology, the potential action of topiramate through glutamatergic mechanisms is of considerable interest. METHODS: Recordings of neurons in the trigeminocervical complex (TCC) and the ventroposteromedial thalamic nucleus (VPM) of anesthetized rats were made using electrophysiological techniques. The effects of intravenous or microiontophorezed topiramate on trigeminovascular activation of second- and third-order neurons in the trigeminothalamic pathway were characterized. The potential interactions of topiramate with the ionotropic glutamate receptors were studied using microiontophoresis. RESULTS: Both intravenous and microiontophorized topiramate significantly inhibited trigeminovascular activity in the TCC and VPM. In both nuclei microiontophoretic application of topiramate significantly attenuated kainate receptor-evoked firing but had no effect on N-methyl-d-aspartic acid or α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptor activation. CONCLUSION: The data demonstrate for the first time that topiramate modulates trigeminovascular transmission within the trigeminothalamic pathway with the kainate receptor being a potential target. Understanding the mechanism of action of topiramate may help in the design of new medications for migraine prevention, with the data pointing to glutamate-kainate receptors as a fruitful target to pursue.

Antinociceptive effects of MSVIII-19, a functional antagonist of the GluK1 kainate receptor.

The ionotropic glutamate receptor subunit, GluK1 (GluR5), is expressed in many regions of the nervous system related to sensory transmission. Recently, a selective ligand for the GluK1 receptor, MSVIII-19 (8,9-dideoxy-neodysiherbaine), was synthesized as a derivative of dysiherbaine, a toxin isolated from the marine sponge Lendenfeldia chondrodes. MSVIII-19 potently desensitizes GluK1 receptors without channel activation, rendering it useful as a functional antagonist. Given the high selectivity for GluK1 and the proposed role for this glutamate receptor in nociception, we sought to test the analgesic potential of MSVIII-19 in a series of models of inflammatory, neuropathic, and visceral pain in mice. MSVIII-19 delivered intrathecally dose-dependently reduced formalin-induced spontaneous behaviors and reduced thermal hypersensitivity 3 hours after formalin injection and 24 hours after complete Freund's adjuvant-induced inflammation, but had no effect on mechanical sensitivity in the same models. Intrathecal MSVIII-19 significantly reduced both thermal hyperalgesia and mechanical hypersensitivity in the chronic constriction injury model of neuropathic pain, but had no effect in the acetic acid model of visceral pain. Peripheral administration of MSVIII-19 had no analgesic efficacy in any of these models. Finally, intrathecal MSVIII-19 did not alter responses in Tail-flick tests or performance on the accelerating RotaRod. These data suggest that spinal administration of MSVIII-19 reverses hypersensitivity in several models of pain in mice, supporting the clinical potential of GluK1 antagonists for the management of pain.

BMAA selectively injures motor neurons via AMPA/kainate receptor activation.

The toxin beta-methylamino-l-alanine (BMAA) has been proposed to contribute to amyotrophic lateral sclerosis-Parkinsonism Dementia Complex of Guam (ALS/PDC) based on its ability to induce a similar disease phenotype in primates and its presence in cycad seeds, which constituted a dietary item in afflicted populations. Concerns about the apparent low potency of this toxin in relation to estimated levels of human ingestion led to a slowing of BMAA research. However, recent reports identifying potential new routes of exposure compel a re-examination of the BMAA/cycad hypothesis. BMAA was found to induce selective motor neuron (MN) loss in dissociated mixed spinal cord cultures at concentrations ( approximately 30 muM) significantly lower than those previously found to induce widespread neuronal degeneration. The glutamate receptor antagonist NBQX prevented BMAA-induced death, implicating excitotoxic activation of AMPA/kainate receptors. Using microfluorimetric techniques, we further found that BMAA induced preferential [Ca(2+)](i) rises and selective reactive oxygen species (ROS) generation in MNs with minimal effect on other spinal neurons. Cycad seed extracts also triggered preferential AMPA/kainate-receptor-dependent MN injury, consistent with the idea that BMAA is a crucial toxic component in this plant. Present findings support the hypothesis that BMAA may contribute to the selective MN loss in ALS/PDC.

Structure-activity relationship studies on N3-substituted willardiine derivatives acting as AMPA or kainate receptor antagonists.

N3-substitution of the uracil ring of willardiine with a variety of carboxyalkyl or carboxybenzyl substituents produces AMPA and kainate receptor antagonists. In an attempt to improve the potency and selectivity of these AMPA and kainate receptor antagonists a series of analogues with different terminal acidic groups and interacidic group spacers was synthesized and pharmacologically characterized. (S)-1-(2-Amino-2-carboxyethyl)-3-(2-carboxythiophene-3-ylmethyl)pyrimidine-2,4-dione (43, UBP304) demonstrated high potency and selectivity toward native GLU(K5)-containing kainate receptors (K(D) 0.105 +/- 0.007 microM vs kainate on native GLU(K5); K(D) 71.4 +/- 8.3 microM vs (S)-5-fluorowillardiine on native AMPA receptors). On recombinant human GLU(K5), GLU(K5)/GLU(K6), and GLU(K5)/GLU(K2), K(B) values of 0.12 +/- 0.03, 0.12 +/- 0.01, and 0.18 +/- 0.02 microM, respectively, were obtained for 43. However, 43 displayed no activity on homomeric GLU(K6) or GLU(K7) kainate receptors or homomeric GLU(A1-4) AMPA receptors (IC(50) values > 100 microM). Thus, 43 is a potent and selective GLU(K5) receptor antagonist.

Dual roles in feeding for AMPA/kainate receptors: receptor activation or inactivation within distinct hypothalamic regions elicits feeding behavior.

We have previously shown that hypothalamic injections of glutamate, or agonists of its ionotropic receptors (iGluRs), elicit intense feeding responses in satiated rats [Brain Res. 613 (1993) 88, Brain Res. 630 (1993) 41]. While attempting to clarify the role of the AMPA and kainate (KA) receptor subtypes in glutamatergic feeding systems, we discovered that lateral hypothalamic (LH) injection of high doses of the competitive AMPA/KA receptor antagonist, NBQX (10 and 30 nmol), elicited a pronounced feeding response. We questioned whether this effect was due to inactivation of AMPA or possibly KA receptors. To determine whether other AMPA/KA antagonists can also elicit feeding, we tested whether injection of CNQX, another AMPA/KA receptor antagonist, also stimulates eating and whether these feeding stimulatory effects were due to antagonists' actions in the LH or in other hypothalamic sites. Here we report that NBQX and CNQX elicit feeding in a dose dependent manner and are most effective when injected into the perifornical hypothalamus (PFH), or into the paraventricular nucleus (PVN) and, to a lesser extent, into the LH of satiated rats. In contrast, AMPA was most effective in stimulating feeding when injected into the LH, confirming previous reports. These data suggest that either activation or inactivation of AMPA/KA receptors in distinct but overlapping hypothalamic sites may be sufficient to induce feeding behavior, indicating a broadened role for glutamate in hypothalamic feeding mechanisms.

Kainate receptor (GluR5)-mediated disinhibition of responses in rat ventrobasal thalamus allows a novel sensory processing mechanism.

Kainate receptors have been studied extensively in vitro, but how they might function physiologically remains unclear. We studied kainate receptor modulation of synaptic responses in the rat ventrobasal thalamus using the novel antagonist LY382884 and the agonist ATPA (selective for GluR5-containing kainate receptors) as tools. No evidence could be found for a direct contribution of kainate receptors to responses of thalamic relay cells to lemniscal (sensory) input in thalamic slices studied with the aid of intracellular and field potential recordings, using selective AMPA and NMDA receptor antagonists and LY382884. However, the GluR5 agonist ATPA reduced the IPSPs originating from the thalamic reticular nucleus. Extracellular single-neurone recordings in anaesthetised rats showed that excitatory responses evoked by physiological vibrissa afferent stimulation were reduced by LY382884 applied iontophoretically at the recording site. This action of the antagonist was occluded when GABA receptors were blocked, indicating that the reduction in excitatory sensory responses by LY382884 is due to an action on GABAergic inhibition arising from the thalamic reticular nucleus. Further experiments showed that these actions depended on whether inhibition was evoked during activation of the excitatory receptive field rather than when inhibition was evoked from a surround vibrissa. We suggest that GluR5 is located presynaptically on inhibitory GABAergic terminals of thalamic reticular nucleus neurones, and that it is normally activated by glutamate spillover from synapses between excitatory afferents and relay neurones during physiological stimulation. We propose that this GluR5-activated disinhibition has an important novel role in extracting sensory information from background noise.

Antagonists of GLU(K5)-containing kainate receptors prevent pilocarpine-induced limbic seizures.

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.

Glutamate release inhibiting properties of the novel mGlu(5) receptor antagonist 2-methyl-6-(phenylethynyl)-pyridine (MPEP): complementary in vitro and in vivo evidence.

We have previously demonstrated that neuronal release of the excitatory amino acid glutamate is facilitated by the selective activation of presynaptic Group I metabotropic autoreceptors. Here we report the release inhibiting actions of the novel mGlu(5) receptor-selective antagonist, 2-methyl-6-(phenylethynyl)-pyridine (MPEP), both in vitro and in vivo. These data provide compelling evidence for the presence of functional positive modulatory mGlu(5) subtype autoreceptors in the mammalian central nervous system.

[The search for selective blockers of NMDA and AMPA/kainate receptors in a series of bis-ammonium compounds with adamantyl radicals]. / Poisk izbiratel'nykh blokatorov NMDA i AMPA/kainatnykh retseptorov v riadu bis-ammonievykh soedinenii s adamantil'nymi radikalami.

Two groups of substances capable of selectively blocking the NMDA and AMPA/kainate receptors in experiments on intact animals were found in a series of bis-ammonium compounds with adamantyl radicals. The selective NMDA receptor blockers (IEM-1754, IEM-1755, IEM-1752), as well as the reference agents MK-801 and memantine, produced anticonvulsant, anti-ischemic, and antihypoxant effects and prevented the loss of experimental animals from toxic doses of NMDA. The selective AMPA/kainate receptor blockers (IEM-1553, IEM-1751, IEM-1592, and DNQX)) also produced the anticonvulsant, anti-ischemic, and antihypoxant effects, but did not prevent from the loss of animals caused by the toxic doses of NMDA. The maximum activity was observed for IEM-1754, the activity of which exceeded that of MK-801 (by a factor of 5-10) and memantine (by a factor of 300-800) in all the test objects.

Synthesis and anticonvulsant activity of novel and potent 2,3-benzodiazepine AMPA/kainate receptor antagonists.

We have previously shown that 1-aryl-3,5-dihydro-7, 8-methylenedioxy-4H-2,3-benzodiazepin-4-ones (3) possess marked anticonvulsant properties and antagonize seizures induced by 2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA) in analogy to the structurally related 1-(4-aminophenyl)-4-methyl-7, 8-methylenedioxy-5H-2,3-benzodiazepine (1, GYKI 52466), a well-known noncompetitive AMPA/kainate receptor antagonist. We now report the synthesis of 3-(N-alkylcarbamoyl)-1-aryl-3,5-dihydro-7, 8-methylenedioxy-4H-2,3-benzodiazepin-4-ones (4a-h) and 1-aryl-3, 5-dihydro-7,8-methylenedioxy-4H-2,3-benzodiazepine-4-thiones (5a-c). The activity of all compounds, intraperitoneally (ip) injected, was evaluated against audiogenic seizures in DBA/2 mice and against seizures induced by maximal electroshock (MES) and pentylenetetrazole (PTZ) in Swiss mice. Some of the new compounds 4 and 5 showed remarkable anticonvulsant activity, and their toxicity, as evidenced by the rotarod test, is lower than that of 1. The time course of anticonvulsant activity of derivatives 4b and 5b,c was studied and compared to that of 1 and 3b,c. Compounds 4a,b and 5a-c antagonize seizures induced by AMPA and kainate (KA) and their anticonvulsant activity is reversed by pretreatment with aniracetam. Using the patch-clamp technique, the capability of derivatives 3c, 4b, and 5c to antagonize KA-evoked currents in primary cultures of granule neurons was tested and compared with that of the parent compounds 1 and 1-(4-aminophenyl)-3, 4-dihydro-4-methyl-3-methylcarbamoyl-7,8-methylenedioxy-5H-2, 3-benzodiazepine (2, GYKI 53655).

NS-257, a novel competitive AMPA receptor antagonist, interacts with kainate and NMDA receptors.

In this study, we examined the effects of a novel, water-soluble, putative competitive AMPA receptor antagonist, 1,2,3,6,7, 8-hexahydro-3-(hydroxyimino)-N,N,7-trimethyl-2-oxobenzo[2,1- b:3, 4-c']dipyrrole-5-sulfonamide (NS-257) on AMPA, kainate and NMDA receptors using the two-electrode voltage-clamp technique in Xenopus oocytes. All glutamate receptor subtypes were inhibited by NS-257 in a voltage-independent way. When kainate was applied to oocytes injected with total mouse brain mRNA, mainly AMPA receptors were activated. The antagonistic effects of NS-257 on these kainate-induced currents were concentration-dependent and competitive. In the same way, NS-257 blocked kainate-induced currents recorded from oocytes expressing homomeric GluR-1 receptors. In our experiments higher concentrations (>1 microM) of NS-257 also produced inhibitory effects on kainate and to a lesser extent on NMDA receptor function as indicated by recordings from GluR-6 or NR-1b/2A cRNA injected oocytes. While NMDA receptor function was inhibited in a competitive fashion, kainate responses recorded from homomeric GluR-6 receptors were blocked in a mixed competitive-noncompetitive manner. This mixed antagonistic action of NS-257 might have been caused by preincubating oocytes with concanavalin A, which blocks desensitization of kainate receptors. Although NS-257 appeared to be a less potent AMPA receptor antagonist then other known antagonists like NBQX, its main advantage over all other reported compounds so far is its higher aqueous solubility which still represents the major weakness of the other AMPA receptor antagonists, especially for clinical use.