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NEUROPHARMACOLOGY

Antiallodynic and Antihyperalgesic Effects of Selective Competitive GLU_K5 (GluR5) Ionotropic Glutamate Receptor Antagonists in the Capsaicin and Carrageenan Models in Rats

Lilly Research Laboratories, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, Indiana (C.K.J., A.A., A.M.O., D.B., R.M.A.S., S.I., P.L.O., H.E.S.); and Centro de Investigación Lilly, Madrid, Spain (E.D.)

Received April 2, 2006; accepted June 29, 2006.

	Abstract

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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 -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-ethylbutyl 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.

Glutamate is a major excitatory neurotransmitter in primary sensory afferent pathways (e.g., Fundytus, 2001). For example, noxious stimulation, such as the administration of formalin into the hindpaw, increases the release of glutamate and aspartate from dorsal horn neurons (Skilling et al., 1988). The persistent release of glutamate in pain pathways can lead to the development of central sensitization, characterized by altered responsiveness of dorsal horn and thalamic neurons, expansion of receptive fields, and plasticity of neuronal connections (e.g., Coderre, 1993; Urban et al., 1994). Furthermore, repetitive C-fiber stimulation produces a "wind-up" of dorsal horn neuron activity that is mimicked by the application of L-glutamate (Zieglgänsberger and Herz, 1971) and NMDA (King et al., 1988). Blockade of the activation of postsynaptic ionotropic receptors has been shown to produce antinociception and decrease central sensitization (e.g., Coderre and van Empel, 1994).

Evidence has begun to accumulate indicating that GLU_K5 glutamate receptors play an important role in nociception and central sensitization (for review, see Ruscheweyh and Sandküler, 2002). Kainate receptors are present on small diameter C-fibers, and GLU_K5 receptors have been identified on dorsal root ganglion cells as well as in the spinal cord on spinothalamic tract neurons (e.g., Agrawal and Evans, 1986; Tölle et al., 1993; Furuyama et al., 1993). Nonselective AMPA/kainate receptor antagonists, including NBQX, 6-cyano-7-nitroquinoxaline-2,3-dione, and NS1209, have been shown to produce antinociception in a variety of animal models of acute and persistent pain (e.g., Jackson et al., 1995; Pogatzki et al., 2003; Blackburn-Munro et al., 2004). However, Simmons et al. (1998) demonstrated that the relatively selective GLU_K5 antagonist LY382884 as well as the nonselective AMPA/kainate receptor antagonists NBQX and LY293558 but not the nonselective AMPA receptor antagonist LY300164, produced antinociception in the formalin test in rats. LY382884 also attenuated the responses of spinothalamic tract neurons to mechanical and thermal stimuli in normal and neuropathic monkeys (Palecek et al., 2004). Recently, Ko et al. (2005) reported that in GLU_K5-deficient mice, responses to capsaicin and inflammatory pain were substantially reduced. In addition, the mixed AMPA/GLU_K5 receptor antagonist LY293558 has been demonstrated to produce analgesia in the capsaicin model in humans (Sang et al., 1998) and is efficacious in acute migraine (Sang et al., 2004) and in postdental surgery pain (Gilron et al., 2000). Taken together, these data suggest that GLU_K5 receptors play an important role in persistent, but not acute, pain. However, most of the studies to date have been conducted using compounds that lack a high degree of selectivity for GLU_K5 versus AMPA or GLU_K6 receptors, therefore limiting the strength of this conclusion.

We recently described a new series of decahydroisoquinoline GLU_K5-selective competitive antagonists (Dominguez et al., 2005) with compounds that have greater potency and selectivity than previous compounds in this pharmacologic class. The improved selectivity of these compounds allows for a more definitive examination of the role of GLU_K5 signaling in persistent pain states. The purpose of the present experiments was to evaluate the efficacy of these newer antagonists in models of C-fiber activation and inflammatory persistent pain. Concentration-response curves were determined for representative compounds (see Fig. 1) for antagonizing glutamate-induced calcium influx in HEK293 cells stably expressing GLU_K5, GLU_K6, or GLU_A2 homomeric receptors. The selectivity of representative antagonists for blocking kainate-induced currents at the native GLU_K5 receptors in DRG cells relative to AMPA- or NMDA-induced currents in hippocampal pyramidal cells was determined electrophysiologically. Dose-response curves were also determined after s.c. administration in the capsaicin and carrageenan models in rats. Because the amino acid antagonists exhibited low efficacy after systemic administration, dose-response curves also were determined for selected compounds after direct intracisternal administration. To improve oral bioavailability, ester prodrugs of the parent amino acids were prepared (Dominguez et al., 2005; see also Fig. 1) and dose-response curves determined after oral administration.

View larger version (19K): [in this window] [in a new window]   Fig. 1. Structures of competitive GluK5 ionotropic glutamate receptor antagonists.

	Materials and Methods

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Cell Culture. All cell and tissue culture reagents were from Invitrogen (Grand Island, New York). Recombinant human glutamate receptors [GLU_A2 (flip), GLU_K5(Q), or GLU_K6(Q)] were stably expressed in HEK293 cells. Cells were grown as monolayers under 5% CO₂ at 37°C. Medium used for GLU_K5- and GLU_K6-expressing cells was minimum essential medium (catalog no. 11095-080; Invitrogen), with 10% fetal bovine serum and 250 µg/ml geneticin added. Medium for GLU_A2 cells was Dulbecco's modified Eagle's medium (catalog no. 11965-092; Invitrogen), with 5% fetal bovine serum, 250 µg/ml hygromycin, 1000 units/ml penicillin G sodium, and 1 mg/ml streptomycin sulfate added.

Measurement of Calcium Influx Using Fluo-3. Cells were seeded into poly-D-lysine-coated 96-well plates (Becton Dickinson Labware, Bedford, MA) 1 or 2 days before experiments at a density of 60,000 cells/well (1 day) or 30,000 cells/well (2 days). Cells were washed three times with 100 µl of assay buffer composed of Hanks' balanced salt solution without phenol red (Invitrogen) with 20 mM HEPES and 3.7 mM CaCl₂ added (final [CaCl₂] = 5 mM). Plates were then incubated for 2 to 3 h in the dark at room temperature in 40 µl of assay buffer with 8 µM Fluo3-AM dye (Molecular Probes Inc., Eugene, OR). After dye incubation, cells were rinsed once and incubated with 100 µl of assay buffer containing 250 µg/ml concanavalin A (Sigma, St. Louis, MO) for 0.5 h to prevent desensitization of kainate receptors. Finally, 50 µl of concanavalin A-containing assay buffer was added to wells and fluorescence measured using a fluorometric imaging plate reader (Molecular Devices, Sunnyvale, CA). A first addition of 50 µl of concanavalin A-containing assay buffer was followed by a second addition of 100 µl of concanavalin A-containing buffer 3 min later. Test compounds were added in the absence of agonist during the first addition and in the presence of glutamate during the second addition. Glutamate concentration was 100 µM when testing compounds at GLU_K5 or GLU_K6 receptors and 200 µM when testing compounds at GLU_A2 (approximate EC₈₀ concentrations). Concanavalin A was omitted from experiments using GLU_A2 receptors.

Electrophysiological Recording Conditions. Whole-cell voltage clamp recordings (V_h = -70 mV) were made from single cells using the tight-seal whole-cell configuration of the patch-clamp technique (Hamill et al., 1981). Glass fragments of coverslips with adherent cells were placed in a perfusion chamber and rinsed with buffer of the composition: 140 mM NaCl, 5 mM CaCl₂, 5 mM KCl, 1 mM MgCl₂, and 10 mM HEPES, and 10 mM glucose, pH 7.4, with NaOH (osmolality, 315 mosm/kg). Pipette solutions contained 140 mM CsCl, 1 mM MgCl₂, 14 mM diTris creatine phosphate, 50 U/ml creatine phosphokinase, 14 mM MgATP, 10 mM HEPES, and 15 mM BAPTA, pH 7.2, with CsOH (osmolality, 295 mosm/kg). Experiments were performed at room temperature (20-22°C) and recorded on an Axopatch 200A amplifier using pClamp 8.0 Software (Axon Instruments Inc., Union City, CA). Pipette resistance was typically 1.5 to 2.5 M. Drug application was via a multibarreled perfusion array. IC₅₀ values for compounds were evaluated using 30 µM kainate, 30 µM AMPA, or 10 µM NMDA. Data are expressed as mean ± S.E.M. (n = 3-7).

Kainate currents were measured in acutely prepared isolated DRG from P4 to P7 rat neonates as described previously (Bortolotto et al., 1999) in the presence of 250 µg/ml concanavalin A to prevent agonist-induced desensitization. AMPA and NMDA currents were measured in cultured hippocampal pyramidal neurons prepared from E17 rat embryos as described previously (Bleakman et al., 1999; Bortolotto et al., 1999). NMDA currents were activated by application of NMDA in the absence of magnesium in the perfusion buffer with added glycine (10 µM).

Subjects. Male Sprague-Dawley rats (Harlan Sprague-Dawley, Indianapolis, IN) weighing 250 to 290 g for the capsaicin experiments, 200 to 225 g for the Rotorod test, and 70 to 90 g for the carrageenan experiments were used. Rats were housed in groups of up to six per cage in a large colony room on a 12-h light/dark cycle (lights on 6:00 A.M.), with food and water provided ad libitum. Each animal was used only once. Test sessions were conducted between 8:00 A.M. and 6:00 P.M. All treatment or dose groups consisted of six rats. All experiments were conducted in accordance with the National Institutes of Health regulations of animal care covered in Principles of Laboratory Animal Care, National Institutes of Health publication 85-23, and were approved by the Institutional Animal Care and Use Committee.

Capsaicin-Induced Mechanical Allodynia. Groups of six rats were injected s.c. with vehicle or a dose of drug 15 min before capsaicin (30 µg in 25 µl) was injected into the plantar surface of the right hindpaw. Ten minutes after the injection of capsaicin, mechanical hyperalgesia was evaluated with a calibrated series of von Frey filaments using the up-and-down method of Chaplan et al. (1994). In brief, rats were placed in clear plastic cages (17.5 x 15 x 15 cm) fitted with wire mesh flooring and allowed to acclimate for approximately 5 min; the withdrawal thresholds were determined by the up- and-down method (Chaplan et al., 1994) by applying each filament of a graded series of filaments to the midplantar surface of each hindpaw in a perpendicular fashion at 5, 10, and 15 mm from the primary injection site and depressed slowly (4-5 s) until bending occurred, and the maximum force of the fiber was exerted. Any paw withdrawal response in the 5, 10, or 15 mm region outside the primary site of injection was scored as a response to the filament.

For intracisternal injections, animals were lightly anesthetized with isoflurane, and the back of the rat's head was shaved. The head of the animal was placed perpendicular to the body axis as a 25-gauge needle, attached to a 25-µl syringe, was inserted to a depth of 5 mm from the surface of the skin into the cisterna magna. A 10-µl injection was delivered over approximately 10 s, and the needle was held in place for an additional 10 s before being withdrawn. Intracisternal injections were administered approximately 15 min before the intraplantar injection of capsaicin, and animals were tested 15 min after capsaicin administration.

Carrageenan-Induced Thermal Hyperalgesia. Groups of six rats were injected s.c. with -carrageenan (100 µl of a 1.5% solution) into the plantar surface of the right hindpaw at time 0 followed 90 min later by a p.o. or i.p. injection of vehicle or a dose of drug. Withdrawal responses to mechanical and thermal stimuli were determined after approximately an additional 30 and 40 min, respectively. Withdrawal latencies to a nociceptive thermal stimulus were assessed using a modification of the methods of Hargreaves et al. (1988). Each rat was placed in a Plexiglas cubicle with a glass floor through which an infrared photobeam was projected onto the plantar surface of the hindpaws, and the latency to withdrawal from the thermal stimulus was determined. The intensity of the infrared photobeam from the plantar reflex device (Plantar Test; Ugo Basile, Comerio, Italy) was adjusted to produce a mean response latency in untreated rats of approximately 12 to 15 s and terminated automatically after 27 s in the absence of a response. The response latency was determined using a timer linked to the photodiode motion sensors in the plantar reflex device. Response latency was defined as the time from the onset of exposure to the infrared photobeam to the cessation of the photobeam when the photodiode motion sensors detected the withdrawal response of the paw of the rat. Response to the thermal stimulus was reported as the difference in withdrawal latency between the treated and untreated paws in seconds and was calculated using the following formula: withdrawal latency of the carrageenan-treated paw - withdrawal latency of the untreated paw.

Rotorod Test. Twenty-four hours before compound testing, rats were given three training trials to maintain posture on an accelerating rod (Omnitech Electronics Inc., Columbus, OH), 17 rpm for 5 s and maintaining that speed for 40 s (Simmons et al., 1998). The following day, Rotorod testing was conducted at time points corresponding to the pain testing. Animals that did not fall off the Rotorod were given a maximum score of 40 s. Compounds were evaluated over several doses, varying from a dose that was without effect on the Rotorod and increasing in 2- or 3-fold steps until a dose that produced a statistically significant motor impairment, or 100 mg/kg, was reached.

Drugs. LY293558, LY377770, LY382884, and compounds 1, 2a, 2b, 3a, 3b, 4a, 4b, 5, and 6 (Lilly Research Laboratories; see Fig. 1) were dissolved in distilled water or 5% solutol. Morphine sulfate (Sigma/RBI, Natick, MA) and -carrageenan (Sigma) were dissolved in double-deionized water. Doses refer to the form of the drug listed. All drugs were administered s.c. or p.o. by gavage in a volume of 1.0 ml/kg or intracisternally in a volume of 10 µl. Capsaicin (Sigma) was prepared as a 3 mg/2.5 ml solution, dissolved in olive oil (Sigma), and sonicated for 25 min in a 45°C water bath.

Statistical Analysis. Data were expressed as means ± S.E.M. Affinities of test compounds in transfected cell lines were determined from concentration-response curves for antagonism of glutamate-evoked Ca²⁺ influx. The curves were analyzed using GraphPad Prism 3.02 software (GraphPad Software Inc., San Diego, CA), with slope factor not fixed and top and bottom fixed at 100 and 0% inhibition, respectively. The dissociation constant (K_b) was calculated from the IC₅₀ value for inhibiting glutamate-induced Ca²⁺ influx according to the Cheng-Prusoff equation (Cheng and Prusoff, 1973): K_b = IC₅₀/(1 + [Glu]/EC_{50 Glu}), where [Glu] is the concentration of glutamate (100 or 200 µM), and EC_{50 Glu} is the EC₅₀ value of glutamate for evoking calcium influx in the given cell line, determined from glutamate concentration-response curves run in the same plates as the antagonist concentration-response curves. In vivo, treatment groups were compared with appropriate control groups using one-way analysis of variance and Dunnett's t test. Statistical analyses were performed using JMP statistical software (SAS Institute Inc., Cary, NC). ED₅₀ values and 95% confidence limits were determined using GraphPad Prism. A probability of p 0.05 was taken as the level of statistical significance.

	Results

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Ion Flux in HEK293 Cells. To determine the selectivity of this series of amino acid antagonists for GLU_K5 versus GLU_K6 or AMPA receptors, the ability of compounds to inhibit glutamate-evoked calcium influx was measured in HEK293 cells stably expressing cloned GLU_K5, GLU_K6, or GLU_A2. Table 1 shows calculated K_b values for the antagonists at each cell line.

In the GLU_K5-expressing cell line, the rank order of potency was compound 6 (K_b = 3 nM) 4a > 5 3a 2a > LY377770 4b (prodrug of 4a) LY293558 LY382884 2b (prodrug of 2a) 3b (prodrug of 3a; K_b = 3 µM). Compound 1 (prodrug of LY382884) had no effect at GLU_K5 receptors at concentrations up to 100 µM.

In GLU_A2-expressing cells, the antagonists displayed potency in the order of LY293558 (K_b = 0.4 µM) 3a 2a 4a >> LY377770 2b. LY382884, 1, 3b, 4b, 5, and 6 each produced no significant inhibition in GLU_A2-expressing cells at concentrations up to 100 µM. None of the compounds tested had any significant effect in GLU_K6-expressing cells at concentrations up to 100 µM.

Electrophysiological Recordings. To confirm antagonist potency and selectivity for GLU_K5 versus AMPA receptors in native tissues, as well as to determine the selectivity for GLU_K5 versus NMDA receptors, selected compounds were examined for their ability to antagonize kainate-evoked inward currents in rat DRG neurons and AMPA- or NMDA-evoked currents in cultured rat hippocampal neurons (Fig. 2). Compound 2a antagonized kainate-evoked currents in rat DRG with IC₅₀ = 0.15 ± 0.06 µM and AMPA- and NMDA-evoked currents in hippocampal neurons with IC₅₀ values of 2.1 ± 0.8 and 90 ± 41 µM, respectively. Compound 3a blocked kainate-, AMPA-, and NMDA-evoked currents with IC₅₀ values of 0.18 ± 0.11, 1.4 ± 0.9, and 1.9 ± 1.6 µM, respectively.

View larger version (24K): [in this window] [in a new window]   Fig. 2. Dose-related antagonism of kainate-induced currents in rat DRG neurons and of AMPA- and NMDA-induced currents in rat hippocampal neurons in vitro by 2a and 3a. Each point represents the mean of three to seven cells. Vertical lines represent ± S.E.M. and are absent when less than the size of the point. Abscissa, concentration of drug in micromolar; ordinate, percent inhibition.

LY293558, LY377770, and LY382884 s.c. in Capsaicin Test. LY293558 is a mixed AMPA/KA receptor antagonist (Table 1), whereas LY377770 and LY382884 are relatively selective GLU_K5 receptor antagonists. All three compounds produced dose-related reduction in mechanical allodynia in the capsaicin test (Fig. 3). LY293558 was somewhat more potent than the other two compounds (Table 2), and doses of 5.6 and 10 mg/kg s.c. produced effects that were significantly different from vehicle. LY377770 and LY382884 were approximately equipotent to each other (Table 2) with a dose of 10 mg/kg s.c. or higher, producing statistically significant effects.

View larger version (21K): [in this window] [in a new window]   Fig. 3. Dose-related reversal of capsaicin-induced mechanical allodynia by competitive GluK5 ionotropic glutamate receptor antagonists after s.c. administration in rats. Each point represents the mean of six rats. Vertical lines represent ± S.E.M. and are absent when less than the size of the point. Points above V represent the effects of vehicle. Abscissa, dose of drug in mg/kg; ordinate, withdrawal threshold in grams to a mechanical stimulus. *, p < 0.05 versus vehicle, Dunnett's t test.

LY382884 s.c. and Compound 1 p.o. in Capsaicin Test. We have reported previously that amino acid compounds such as LY382884 have poor oral bioavailability. Therefore, ester prodrugs were synthesized to increase oral bioavailability (Dominguez et al., 2005). The isobutyl ester of LY382884, compound 1, had virtually no measurable affinity for GLU_K5 receptors (Table 1) but produced a dose-related antiallodynic effect in the capsaicin test, with doses of 10 and 30 mg/kg producing statistically significant effects (Fig. 4). However, the effects of the prodrug administered p.o. were relatively modest in magnitude when compared with the complete reversal of capsaicin-induced allodynia produced by the parent compound LY382884 administered s.c.

View larger version (21K): [in this window] [in a new window]   Fig. 4. Dose-related reversal of capsaicin-induced mechanical allodynia by LY382884 s.c. and the ester prodrug 1 after oral administration in rats. Each point represents the mean of six rats. Vertical lines represent ± S.E.M. and are absent when less than the size of the point. Points above V represent the effects of vehicle. Points above 10 M represent the effects of 10 mg/kg morphine s.c. Abscissa, dose of drug in mg/kg; ordinate, withdrawal threshold in grams to a mechanical stimulus. *, p < 0.05 versus vehicle, Dunnett's t test.

2b 4b

View larger version (25K): [in this window] [in a new window]   Fig. 5. Dose-related reversal of capsaicin-induced mechanical allodynia by the ester prodrugs of competitive GluK5 antagonists after oral administration in rats. Each point represents the mean of six rats. Vertical lines represent ± S.E.M. and are absent when less than the size of the point. Points above V represent the effects of vehicle. Points above 10 M represent the effects of 10 mg/kg morphine s.c. Abscissa, dose of drug in mg/kg; ordinate, withdrawal threshold in grams to a mechanical stimulus. *, p < 0.05 versus vehicle, Dunnett's t test.

Comparison of Amino Acid Decahydroisoquinolines Administered s.c. and Intracisternally. In addition to being relatively inefficacious after oral administration, a number of amino acid GLU_K5 antagonists, with a range of affinities for the GLU_K5 receptor, were also ineffective in the capsaicin test after s.c. administration (Fig. 6, left). LY382884 produced a dose-related reversal of allodynia over the dose range of 1.0 to 30 mg/kg s.c., and compound 5 was effective at a dose of 100 mg/kg s.c., whereas compound 6 had little or no efficacy over the dose range of 3.0 to 30 mg/kg s.c., even though the latter compound has a higher affinity than LY382884 for GLU_K5 receptors (Table 1). The lack of efficacy of these amino acid compounds raised the question of whether the efficacy was due to activity at receptors other than GLU_K5 receptors or due to a lack of blood-brain barrier penetration of the amino acids.

View larger version (21K): [in this window] [in a new window]   Fig. 6. Dose-related reversal of capsaicin-induced mechanical allodynia by competitive GluK5 antagonists after s.c. or intracisternal (IC) administration in rats. Each point represents the mean of six rats. Vertical lines represent ± S.E.M. and are absent when less than the size of the point. Points above V represent the effects of vehicle. Data for LY382884 administered s.c. are reproduced from Fig. 3. Abscissa, dose of drug in mg/kg s.c. or nanomoles per rat IC; ordinate, withdrawal threshold in grams to a mechanical stimulus. *, p < 0.05 versus vehicle, Dunnett's t test.

If the lack of efficacy after s.c. administration was due to poor brain penetration, then the amino acid GLU_K5 receptor antagonists would be expected to be efficacious after central administration, whereas if the efficacy of LY382884 and compound 5 was due to activity via other mechanisms, then compound 6 would not be expected to be efficacious after central administration, and/or the rank order of potencies in vivo might be expected to differ from the rank order of potencies determined in vitro. We therefore evaluated the efficacy in the capsaicin test of these amino acid antagonists, with a range of affinities for the GLU_K5 receptor, after intracisternal administration. The GLU_K5 antagonists evaluated all produced dose-related antiallodynic effects after intracisternal administration (Fig. 6, right). All three of the amino acids completely reversed the capsaicin-induced mechanical allodynia (Fig. 6, right) with a rank order of potencies of compound 6 > 5 > LY382884. Thus, the rank order of potencies in vivo after central administration was the same as the rank order of potencies at GLU_K5 receptors in vitro.

Carrageenan-Induced Thermal Hyperalgesia. The efficacy of the prodrug esters was also evaluated after oral administration on carrageenan-induced thermal hyperalgesia. The prodrug esters examined all produced dose-related antihyperalgesic effects (Fig. 7), except compound 2b produced a virtually complete reversal of thermal hyperalgesia; it is possible compound 2b would have produced greater efficacy had higher doses been tested. The rank order of potencies in the carrageenan test was compound 4b > 3b 2b (Fig. 7; Table 2). The rank order of potencies of the prodrugs in reversing thermal hyperalgesia was similar to that for the binding affinities of the parent compounds to cloned GLU_K5 receptors in vitro and for reversing capsaicin-induced mechanical allodynia (see Fig. 5).

View larger version (21K): [in this window] [in a new window]   Fig. 7. Dose-related reversal of carrageenan-induced thermal hyperalgesia by ester prodrugs of competitive GluK5 antagonists after oral administration in rats. Each point represents the mean of six rats. Vertical lines represent ± S.E.M. and are absent when less than the size of the point. Points above V represent the effects of vehicle. Abscissa, dose of drug in mg/kg s.c. Ordinate, withdrawal latency difference in seconds to a thermal stimulus. *, p < 0.05 versus vehicle, Dunnett's t test.

	Discussion

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The present studies evaluated the antimechanical allodynic and antithermal hyperalgesic effects of a series of amino acid decahydroisoquinoline GLU_K5 receptor competitive antagonists in the capsaicin and carrageenan tests in rats. Although these antagonists were efficacious when administered intracisternally, the majority of the amino acid GLU_K5 receptor antagonists were ineffective after either oral or s.c. administration, suggesting that they had very low oral bioavailability, poor blood-brain barrier penetration, or both. We have previously reported that these amino acid decahydroisoquinoline compounds have very low oral bioavailability (Dominguez et al., 2005). We therefore evaluated ester prodrugs that are well absorbed after oral administration and deliver the parent compound in plasma (see also Dominguez et al., 2005). The diethyl, isobutyl, or 2-ethylbutyl ester prodrugs tested herein were efficacious in reversing mechanical allodynia in the capsaicin assay after oral administration, which, together with our previous report (Dominguez et al., 2005), demonstrate that ester prodrugs of amino acid decahydroisoquinoline GLU_K5 receptor antagonists can reverse allodynia after oral administration.

GLU_K5 receptors are located in multiple sites on pain pathways (for review, see Ruscheweyh and Sandküler, 2002). GLU_K5 receptors are well documented to occur in dorsal root ganglion cells and small diameter C-fibers (Agrawal and Evans, 1986; Huettner, 1990; Furuyama et al., 1993; Tölle et al., 1993). GLU_K5 receptors are also located on spinothalamic tract neurons and contribute to high-threshold primary afferent fiber stimulation (Li et al., 1999; Palecek et al., 2004). In addition, GLU_K5 receptors are abundant in the adult rat in the thalamus and brain stem structures important in pain modulation (Bettler et al., 1990). Moreover, GLU_K5 receptors have also been reported to be located in rat skin and peripheral sensory and sensorimotor nerves (e.g., Carlton et al., 1995; Coggeshall and Carlton, 1998).

The GLU_K5 amino acid antagonists used in the current study were examined for their potency at GLU_K5 receptors and their selectivity for GLU_K5 versus GLU_K6 or GLU_A2 receptors, using calcium influx measurements at recombinant human glutamate receptors in vitro. Potency at GLU_K5 receptors ranged from K_b = 3 nM for compound 6 to K_b = 0.02 µM for compound 5. Ester prodrugs exhibited approximately 40- to 100-fold lower potency than the parent amino acid compounds (or, in the case of compound 1, showed no measurable potency at GLU_K5 receptors).

All of the amino acid parent compounds tested were found to be completely selective for GLU_K5 receptors versus GLU_K6 receptors in that none displayed any measurable affinity (up to 100 µM) for GLU_K6 receptors. Selectivity for GLU_K5 versus GLU_A2 receptors varied. LY293558 was found to be approximately equipotent at GLU_K5 and GLU_A2 receptors, in agreement with previous reports (Simmons et al., 1998). The remaining compounds all displayed 10-fold or greater selectivity for GLU_K5 versus GLU_A2 receptors. Several of the compounds that were efficacious when administered intracisternally in the capsaicin model (compounds 5 and 6) showed no measurable affinity (up to 100 µM) at GLU_A2 receptors, strengthening the interpretation that the efficacy observed was due to antagonism of GLU_K5 receptors rather than AMPA receptors.

The selectivity profile of selected antagonists was also confirmed electrophysiologically in primary neuronal cultures. Compounds 2a and 3a displayed approximately 8- and >100-fold selectivity, respectively, for blocking kainate-versus AMPA-evoked currents in native rat tissues and were in general agreement with the results obtained using recombinantly expressed human channels. In addition, the compounds tested displayed selectivity for GLU_K5 versus NMDA receptors ranging from approximately 11-fold (compound 3a) to 500-fold or greater (compound 2a).

Although ester prodrugs of selected amino acid decahydroisoquinoline GLU_K5 receptor antagonists were efficacious in reversing allodynia and hyperalgesia, not all parent amino acid decahydroisoquinolines were efficacious even after s.c. administration, suggesting that these compounds might vary in their ability to penetrate the blood-brain barrier. We therefore selected a series of compounds that varied over a wide range in their affinity for the GLU_K5 receptor and administered them by the intracisternal route to circumvent the blood-brain barrier. After intracisternal administration, the compounds reversed allodynia in the capsaicin test, and the rank order of potencies of the antagonists in the capsaicin test was the same as the rank order of potencies for GLU_K5 receptors determined in vitro. Thus, the present correlative data provide additional strong support for the interpretation that the reversal of allodynia and hyperalgesia produced by these amino acid decahydroisoquinolines is mediated by antagonism of GLU_K5 receptors. Moreover, the present results indicate that the efficacy of GLU_K5 antagonists is mediated, at least in part, at supraspinal levels. Previous studies have demonstrated the importance of kainate receptors on dorsal root ganglion cells, as well as on intrinsic spinal cord dorsal horn neurons (e.g., Palecek et al., 2004) and in skin (Carlton et al., 1995; Coggeshall and Carlton, 1998) in nociception. Thus, the present studies are the first to provide evidence for a supraspinal site of action of kainate receptors in modulating nociceptive signaling.

The present studies replicate and extend previous findings that GLU_K5 receptor antagonists are efficacious in reversing mechanical allodynia and/or thermal hyperalgesia induced by direct stimulation of C-fiber afferents by capsaicin or by carrageenan-induced inflammation. Turner et al. (2003) reported that the desensitizing kainate receptor agonist SYM 2081 reduced the frequency of hindlimb withdrawal to a normally non-noxious mechanical stimulus and increased the latency to a thermal stimulus. Moreover, SYM 2081 was efficacious after i.t. administration in the capsaicin test (Turner et al., 2003). SYM 2081 also reversed ongoing carrageenan-induced mechanical allodynia and partially reduced ongoing heat hyperalgesia (Turner et al., 2003). Likewise, Guo et al. (2002) found that the i.t. administration of LY382884, as well as NBQX and NS-102, attenuated thermal hyperalgesia induced by complete Freund's adjuvant. In addition, in the present study, the prodrugs were efficacious at doses 4- to 20-fold lower than the minimal dose that caused motor impairment on the Rotorod, replicating and extending similar findings in previous studies (Simmons et al., 1998; Blackburn-Munro et al., 2004), indicating that the effects of these antagonists are not simply due to motor impairment. Thus, the preponderance of evidence indicates that selective antagonists of GLU_K5 receptors, or desensitization by selective GLU_K5 agonists, can effectively reduce allodynia or hyperalgesia produced by direct stimulation of C-fibers or inflammation at doses that do not produce motor impairment.

A growing body of evidence also indicates that selective GLU_K5 receptor antagonists, and possibly desensitizing agonists, are also efficacious in other persistent or neuropathic pain states. As mentioned previously, Simmons et al. (1998) demonstrated that the relatively selective GLU_K5 antagonist LY382884 as well as the mixed AMPA/kainate receptor antagonists NBQX and LY293558, but not the nonselective AMPA antagonist LY300164, produced antinociception in the formalin test in rats. Furthermore, Procter et al. (1998) demonstrated that LY382884 and LY294486 (the racemate of LY377770), but not the AMPA receptor-selective antagonist GYKI 53655, reduced nociceptive responses recorded electrophysiologically from hemisected spinal cords from neonatal rats in vitro as well as from dorsal horn neurons in adult rats in vivo and in the hot-plate test in conscious mice. Moreover, Ta et al. (2000) found that SYM-2081 reduced mechanical allodynia and thermal hyperalgesia in a freeze nerve injury model of neuropathic pain. In addition, LY382884 attenuated the responses of spinothalamic tract neurons to mechanical and thermal stimuli in normal and neuropathic monkeys (Palecek et al., 2004). Recently, Ko et al. (2005) reported that in GLU_K5-deficient mice, responses to capsaicin and inflammatory pain were substantially reduced. Taken together, the data indicate that GLU_K5 receptors play an important role in a variety of persistent pain states and that GLU_K5 receptor antagonists may thus be efficacious in the treatment of persistent pain states.

In summary, the present studies compared a series of amino acid selective GLU_K5 antagonists and their ester prodrugs in reversing mechanical allodynia and thermal hyperalgesia induced by capsaicin and carrageenan. The amino acid parent compounds, in general, exhibited little efficacy after oral or s.c. administration but were efficacious after intracisternal administration. In contrast, the ester prodrugs generally were efficacious in both the capsaicin and carrageenan tests after oral administration. The present findings replicate and extend previous observations that selective GLU_K5 receptor antagonists produce antinociceptive effects in persistent pain models including the capsaicin and carrageenan tests, in addition to the formalin test and neuropathic pain models. Furthermore, taken together with previous reports, the site of action of GLU_K5 antagonists in producing antinociception appears to be both supraspinal and spinal. The present findings thus provide further evidence that GLU_K5 receptors are probably involved in mechanisms of central sensitization such as observed in persistent pain states and suggest they may have therapeutic utility in the clinical treatment of persistent pain states.

	Footnotes

 
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.106.105601.

ABBREVIATIONS: NMDA, N-methyl-D-aspartate; AMPA, -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid hydrobromide; NS1209, (R,S)-8-methyl-5-(4-(N,N-dimethylsulfamoyl)phenyl)-6,7,8,9,-tetrahydro-1H-pyrrolo[3,2-h]-isoquinoline-2,3-dione-3-O-(4-hydroxybutyrate-2-yl)oxime; LY382884, 3S,4aR, 6S,8aR-6-(4-carboxyphenyl)methyl-1,2,3,4,4a,5,6,7,8,8a-deca-hydroisoquinoline-3-carboxylic acid; LY293558, (3S,4aR, 6R,8aR)-6-[2-(1(2)H-tetrazole-5-yl)ethyl]decahydroisoquinoline-3-carboxylic acid; NBQX, 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline; LY300164, (R)-7-acetyl-5-(4-aminophenyl)-8,9-dihydro-8-methyl-7H-1,3-dioxolo[4,5-H][2,3]benzodiazepine; LY377770, (3SR,4aR,6R,8aR)-6[2-(1(2)H-tetrazole-5yl)ethyl]decahydroisoquinoline-3-carboxylic acid; HEK, human embryonic kidney; DRG, dorsal root ganglion; MED, minimal effective dose; SYM 2081, 2S,4R-4-methylglutamate; NS-102, 6,7,8,9-tetrahydro-5-nitro-1H-benz[g]indole-2,3-dione 3-oxime.

¹ Current affiliation: Department of Pharmacology, Vanderbilt University Medical Center, Nashville, TN.

Address correspondence to: Dr. Harlan E. Shannon, Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, IN 46285. E-mail: H.Shannon{at}Lilly.Com

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