Intracerebroventricular administration of <it>N</it>-acetylaspartylglutamate (NAAG) peptidase inhibitors is analgesic in inflammatory pain

1744-8069-4-31 1744-8069 Research Intracerebroventricular administration of <it>N</it>-acetylaspartylglutamate (NAAG) peptidase inhibitors is analgesic in inflammatory pain Yamamoto Tatsuo yamamotot@fc.kuh.kumamoto-u.ac.jp Kozikowski Alan kozikowa@uic.edu Zhou Jia Jia.Zhou@psychogenics.com Neale H Joseph nealej@georgetown.edu

Department of Anaesthesiology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba-shi, Chiba 260-8670, Japan

Drug Discovery Program, Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, Chicago, Illinois 60612, USA

PsychoGenics Inc., Tarrytown, NY 10591, USA

Department of Biology, Georgetown University, Washington, D.C., 20057, USA

Department of Anesthesiology at Kumamoto University, Kumamoto, Japan

Molecular Pain 1744-8069 2008 4 1 31 http://www.molecularpain.com/content/4/1/31 18673570 10.1186/1744-8069-4-31 07 5 2008 01 8 2008 01 8 2008 2008 Yamamoto et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background

The peptide neurotransmitter N-Acetylaspartylglutamate (NAAG) is the third most prevalent transmitter in the mammalian central nervous system. Local, intrathecal and systemic administration of inhibitors of enzymes that inactivate NAAG decrease responses to inflammatory pain in rat models. Consistent with NAAG's activation of group II metabotropic glutamate receptors, this analgesia is blocked by a group II antagonist.

Results

This research aimed at determining if analgesia obtained following systemic administration of NAAG peptidase inhibitors is due to NAAG activation of group II mGluRs in brain circuits that mediate perception of inflammatory pain. NAAG and NAAG peptidase inhibitors, ZJ43 and 2-PMPA, were microinjected into a lateral ventricle prior to injection of formalin in the rat footpad. Each treatment reduced the early and late phases of the formalin-induced inflammatory pain response in a dose-dependent manner. The group II mGluR antagonist reversed these analgesic effects consistent with the conclusion that analgesia was mediated by increasing NAAG levels and the peptide's activation of group II receptors.

Conclusion

These data contribute to proof of the concept that NAAG peptidase inhibition is a novel therapeutic approach to inflammatory pain and that these inhibitors achieve analgesia by elevating synaptic levels of NAAG within pain processing circuits in brain.

Introduction

The peptide N-acetylaspartylglutamate (NAAG) is by far the most prevalent 1 and widely distributed co-transmitter in the mammalian nervous system23. It is co-expressed in discrete subsets of neurons with most small amine transmitters, including glutamate and GABA. Consistent with other neuropeptides, NAAG is released under conditions of high neuronal activity and acts at presynaptic receptors 456. Synaptically released NAAG activates the group II metabotropic glutamate receptors [mGluR3 >> mGluR2; 678]. These receptors are expressed on astrocytes where they stimulate release of trophic factors and on presynaptic axons where they inhibit transmitter release 56910. Two enzymes that inactivate synaptically released NAAG, glutamate carboxypeptidase II and III, have been cloned and characterized 1112131415. Potent inhibitors (IC₅₀= 1–5 nM) of these enzymes are being tested in animal models of neurological conditions that are mediated by high levels of glutamate release 161718. While these NAAG peptidase inhibitors do not possess direct agonist activity at ionotropic or metabotropic glutamate receptors, they, like group II mGluR agonists, are effective in reducing perception of inflammatory, neuropathic pain and bone cancer pain in rat models 192021222324. Consistent with the conclusion that inhibitors of NAAG peptidases achieve analgesia by elevating the degree of NAAG activation of a group II mGluR, group II antagonists completely reverse these analgesic actions.

While group II mGluR agonists influence nociceptive responses of primary sensory afferents 192025262728, the widespread distribution of NAAG, NAAG peptidase activity 29 and group II mGluRs within pain pathways (reviewed in 3031) suggests that these receptors in the brain also might modulate pain perception following activation by NAAG. Group II mGluRs are upregulated in the central nervous system in response to inflammatory pain states 32333435. In the periaquaductal grey, a brain region that contributes to descending modulation of nociceptive transmission within the spinal cord 36, group II mGluR agonists act presynaptically to reduce GABAergic transmission 37. Speculation that this action contributes to analgesia derives from observations that opioid analgesia induced at the level of the periaquaductal grey also is mediated by reduction in GABAergic input to descending projections 3839. In this first test of the role of NAAG in regulation of pain perception via brain pain pathways, we administered NAAG and two NAAG peptidase inhibitors into the rat lateral ventricle prior to induction of inflammatory pain.

Methods

These experiments were executed in adherence with the guidelines of the Committee for Research and Ethical Issues of the International Association for the Study of Pain (1983). They were performed according to a protocol approved by the Institutional Animal Care Committee of Chiba University, Chiba, Japan. Male Sprague-Dawley rats (250 – 300 g, Japan SLC, Shizuoka, Japan) were prepared with ICV catheters and examined for the effect of the agents on the formalin test of inflammatory pain.

ICV cannulae

Implantation of the intracerebroventricular (ICV) injection cannula into the

right lateral ventricle was performed stereotaxically under halothane anesthesia. Stainless steel guide cannulae (24 gauge, 0.64 mm outer diameter, 15 mm long) were stereotaxically placed through a burr hole (0.5 mm caudal to coronal suture and 1 mm lateral to sagital suture; 3 mm deep to the dura) and affixed to the skull with stainless steel screws and cranioplastic cement. In our experience, drug injection via the canulae is optimal about 4 days after implantation as the canulae have not plugged with cells by that time, in contrast to 7 days after implantation. Thus, ICV cannula implantation was performed 4 days before the formalin test. All animals displayed normal feeding and drinking behaviors postoperatively. Rats showing neurological deficits were not studied.

Formalin test

To carry out the formalin test, 50 μl of 5% formalin was injected subcutaneously (SC) into the dorsal surface of the

right hind paw with a 25-gauge needle under brief halothane anesthesia. Within 1 min after the formalin injection, spontaneous flinching of the injected paw could be observed. Flinching is readily discriminated and is characterized as a rapid and brief withdrawal or flexion of the injected paw. This pain-related behavior was quantified by counting the number of flinches for 1 min periods at 1 – 2 and at 5 – 6 min, and then for 1 min periods at intervals during the period from 10 to 60 min after the injection. Two phases of spontaneous flinching behavior, an initial acute phase (phase 1: during the first 6 min after the formalin injection) and a prolonged tonic phase (phase 2: beginning about 10 min after the formalin injection), were observed. After the observation period, the animals were immediately killed with an overdose of barbiturate. Four to six rats were used for each treatment group reported here.

Behavioral analysis

The general behavior of each rat was carefully observed and tested. Motor functions were evaluated by the performance of two specific behavioral tasks, as follows. 1) The placing/stepping reflex: this response was evoked by drawing the dorsum of either hindpaw over the edge of a table top. In normal animals, this stimulus elicits an upward lifting of the paw onto the surface of the table, called stepping. Animals with any degree of hind limb flaccidity will demonstrate an altered or absent reflex. 2) The righting reflex: an animal placed horizontally with its back on the table will normally show an immediate coordinated twisting of the body around its longitudinal axis to regain its normal position on its feet. Animals displaying ataxic behavior will show a decreased ability to right themselves. To quantify the evaluation of motor functions, both tasks were scored on a scale of 0 to 2 in which 0 = absence of function and 2 = normal motor functions. Animals that were able to perform the motor tasks but did so more slowly than normal animals were assigned a score of 1. For example, the reflex withdrawal is typically immediate. Rats demonstrating a non-immediate reflex were scored 1. The normal righting reflex also is prompt and successful. Rats that either delayed the attempt or who were ultimately but not immediately successful were scored 1.

Drugs

(S)-2-[3-[(S)-1-carboxy-3-methylbutyl]ureido]pentanedioic acid (ZJ43) was synthesized following methods previously described 40. ZJ43 (molecular weight = 304.3) is an unsymmetrical urea, which was prepared by the addition reaction of an isocyanate, generated in situ from the tosylate salt of glutamic acid dibenzyl ester and triphosgene in the presence of Et₃N, with the second amino acid benzyl ester component. The subsequent debenzylation of the key intermediate by the catalytic hydrogenation affords ZJ43 in the final form. 2-(phosphonomethyl) pentanedioic acid (2-PMPA, molecular weight = 314) was purchased from Alexis Biochemicals, (San Diego, CA, USA). ZJ45 and 2-PMPA are NAAG peptidase inhibitors 161718. NAAG was purchased from Tocris (Bristol, UK). LY341495, a highly selective group II metabotropic glutamate receptor antagonist 41, was purchased from Tocris. The ICV administered drugs were delivered in a total volume of 3 μl.

Experimental protocol

ZJ43, 2-PMPA or NAAG were administered ICV 10 min before the formalin injection. To obtain control data, vehicle (saline) was injected ICV (n = 5). To verify that the analgesic effect of ICV administered drugs on the formalin test was mediated by the activation a group II mGluR, 1 mg/kg of LY341495 was administered intraperitoneally (i.p.) 10 min before the ICV injection of drugs. The effect of intraperitoneal (i.p.) administration of 1 mg/kg of LY341495 on the formalin test also was examined.

Statistical analyses

For the dose-response analysis, data from phase 1 (0 – 6 min) and phase 2 (10 – 60 min) observations were considered separately. The cumulative instances of formalin-evoked flinches during the phase 1 and phase 2 were calculated for each rat. To evaluate the dose-dependence, one-way analysis of variance (ANOVA) was used. Published data strongly suggest that underlying mechanisms that generate the phase 1 response is different from those that generate the phase 2 response. Thus, for the dose-response analysis, we considered phase 1 and phase 2 responses as independent samples. For multiple comparisons, Tukey's test was used. For comparison of the pain response in the peptidase inhibitor group versus the group that was treated with inhibitor and the group II antagonist, the unpaired t-test (two tailed) was used.

Whenever appropriate, results are expressed as mean ± SEM. Critical values that reached a p < 0.05 level of significance were considered significant.

Results

Behavioral analysis

ICV injection of 100 μg of ZJ43, caused restlessness in all animals and 20% of the rats receiving 100 μg of ZJ43 scored 1 (impairment of motor function) in the placing/stepping reflex and righting reflex. After ICV injection of 10 μg or less of NAAG, ZJ43 or 2-PMPA, all animals scored 2 (normal motor function) in the placing/stepping reflex and righting reflex tests. As a result, 10 μg was the highest dose of ZJ43 and 2-PMPA injected into the lateral ventricle in this study. After the i.p. administration of 1 mg/kg of LY341495, all animals scored 2 (normal motor function) in the placing/stepping reflex and righting reflex tests.

Responses in formalin model of inflammatory pain

ICV injection of ZJ43 (Figure 1a), 2-PMPA (Figure 1b) or NAAG (Figure 1c) decreased the sum of flinches induced by formalin injection into the footpad while the group II mGluR agonist LY341495 alone (Figure 1c) had no detectable effect. Pretreatment with the group II mGluR antagonist LY341495 (1 mg/kg, i.p.) blocked the analgesic effect of 10 μg of ZJ43 or 2-PMPA on both phases of the flinching behavior (Figures 1a and 1b; phase 1: p < 0.01; phase 2: p < 0.01, by t-test). LY341495 similarly antagonized the analgesic effect of 10 μg of NAAG on the phase 2, but not phase 1, flinching behavior (Figure 1c; phase 1: p > 0.2; phase 2: p < 0.005, by t-test).

Figure 1

Effects of intracerebroventricular (ICV) injection of 10 μg of ZJ43 (a), 2-PMPA (b) or NAAG (c) on the time course of the flinches observed after the formalin injection into the dorsal surface of the right rat hind-paw. Drugs were administered 10 min before the formalin injection. The group II mGluR antagonist LY341495 (1mg/kg, i.p.) was injected 10 min before ZJ43, 2-PMPA, NAAG or saline (d). The number of flinches/min is plotted vs time after the formalin injection. NAAG and the NAAG peptidase inhibitors ZJ43 and 2-PMPA significantly reduced both phases of the response to formalin injection (see text for statistical assessment). The effects of the peptidase inhibitors were blocked by the LY341495 as was the effect of NAAG on phase 2 of the response to formalin. Each line represents the group mean and S.E.M. of four to six rats.

The NAAG peptidase inhibitors reduced phase 1 and phase 2 flinching behaviors in a dose-dependent manner relative to saline treated rats (Figures 2a and 2b; phase 1: p < 0.01; phase 2: p < 0.01 by ANOVA). Similarly, ICV injection of NAAG itself decreased the sum of flinches, in both phases of the flinching behavior in a dose-dependent manner between 1 and 10 μg (Figures 2a and 2b; phase 1: p < 0.05; phase 2: p < 0.01 by ANOVA). The maximal effect of the peptide appeared to be less than that obtained by the peptidase inhibitors. Ten μg of NAAG provided a maximal effect inasmuch as 100 μg of NAAG gave no greater reduction (Figure 2a and 2b).

Figure 2

Dose-response curves for ICV injection of ZJ43, 2-PMPA and NAAG representing the cumulative instances of formalin evoked flinches during the phase 1 (a) and the phase 2 (b)

Dose-response curves for ICV injection of ZJ43, 2-PMPA and NAAG representing the cumulative instances of formalin evoked flinches during the phase 1 (a) and the phase 2 (b). ZJ43, 2-PMPA and NAAG reduced the number of phase 1 and the phase 2 flinching behaviors in a dose dependent manner. Each point represents the group mean and S.E.M. of responses by groups of 5–6 rats. * <0.05 and ** <0.005 versus rats given saline ICV prior to formalin-induced inflammation.

Discussion

We previously reported that systemic, intrathecal and local application of NAAG peptidase inhibitors reduced pain perception in rat models of inflammatory, neuropathic and bone cancer induced pain and that these effects are blocked by co-application of a group II mGluR 2021222324. These and other data support the conclusion that the analgesic effects of NAAG peptidase inhibition are mediated by increased activation of presynaptic group II mGluRs and a subsequent reduction in transmitter release in those neuronal circuits in which NAAG and the peptidase activity are expressed [reviewed in 16]. However, it was not clear from these data if systemic application of NAAG peptidase inhibitors achieved analgesia by activation of group II mGluRs in brain pain pathways. This research ultimately is aimed at testing the concept that NAAG peptidase inhibition represents a clinically significant and completely new strategy for the treatment of inflammatory and neuropathic pain. As such, defining the locus of action of these peptidase inhibitors relative to pain perception is an issue of central importance. The data presented here support the conclusion that while the spinal cord and sensory neurons represent loci at which NAAG peptidase inhibition may contribute to analgesia, there also is at least one locus in the brain where NAAG directly mediates analgesia and that this brain region is accessible via the lateral ventricles. Given the quantities of the ZJ43 and 2-PMPA that are required locally 20 and intrathecally 21 to obtain analgesia, it is possible that the primary target for systemically applied NAAG peptidase inhibitor-mediated analgesia is in the brain and/or brain stem.

Since the inhibitors were infused into the right ventricle and the inflammation was induced in the ipsilateral footpad, it seems likely that the inhibitors were not acting proximal to this ventricle but rather that the compounds were affecting the broader periventricular tissue of the brain including the contralateral descending pain modulating pathway in the periaquaductal grey. The periaquaductal grey is an important locus in the pathway for opioid mediated analgesia. Relevant to these NAAG peptidase inhibition data, the periaquaductal grey contains a relatively high concentration of both NAAG and NAAG peptidase activity 29. Further, group II mGluR agonist activation of presynaptic receptors on GABAergic neurons in the periaquaductal grey reduces GABA release 37. We previously reported that NAAG inhibits GABA release from cortical neurons via a presynaptic mechanism 5. A similar action of presynaptic opiate receptors on GABAergic neurons in the periaquaductal grey disinhibits a descending pathway that suppresses nociceptive transmission in the dorsal horn of the spinal cord 3639. While these data are consistent with speculation that intracerebroventricular administration of NAAG peptidase inhibitors might achieve analgesia via a mechanism that parallels that of the opiates in the PAG, a dose response study directly in the PAG is required to demonstrate this site of action. More importantly, these data together with our previous studies suggest that this peptide transmitter functions both spinally and centrally along pathways that mediate and modulate perception of inflammatory pain in a manner that is analogous to opiate peptides.

Given the wide distribution of group II mGluR receptors in the brain, it is surprising that direct infusion of NAAG did not result in a broad range of behavioral effects outside of the pain modulatory pathway and that its effects on the pain responses were not at least as profound as those of the peptidase inhibitors. This result is most likely due to the equally widespread distribution of extracellular NAAG peptidase activity in the brain. In the absence of peptidase inhibition, NAAG delivered by ICV is likely to be hydrolyzed rapidly as it diffuses through the extracellular space. Equally relevant to the development of new analgesic drugs, the systemic administration of the peptide would have the same drawbacks as synthetic group II agonists. That is, agonists activate receptors without a relationship to the normal level of information movement within circuits. In contrast, strategies that enhance the actions of endogenously released transmitters, such as NAAG peptidase inhibition, enhance the effects of the normal action of the circuit. This is, for example, the basis of the therapeutic efficacy of benzodiazepines, barbiturates and serotonin selective reuptake inhibitors.

ZJ43 is tricarboxylic acid with a urea core. Its hydrophilic structure contributes to its relatively low penetration across the CACO-2 cell culture model of tight junctions as found in the blood-brain barrier (Neale, et al., unpublished data). As a result, it is not surprising that despite its low nanomolar ^IC50 for NAAG peptidases, relatively high (50–100 mg/kg) systemic doses of ZJ43 are required to obtain significant analgesia in inflammatory and neuropathic pain models 21. This finding that some of the analgesic effects of ZJ43 are obtained by acting at pain circuits that are accessible via the lateral ventricle militates in favor of developing similarly potent NAAG peptidase inhibitors that cross the blood brain barrier more effectively. Toward this objective, we have synthesized a series of prodrug esters of ZJ43 that are themselves not NAAG peptidase inhibitors but that may enter the central nervous system more effectively due to their improved ClogP values. In preliminary studies, some of these prodrugs are more active in an inflammatory pain model than is ZJ43 (Yamamoto, unpublished). These results suggest that some esters of ZJ43 penetrate the blood-brain barrier effectively and are hydrolyzed efficiently by central nervous system esterases rather than by serum esterases. If NAAG peptidase inhibition continues to show promise as an analgesic pharmacotherapy, this prodrug development strategy is likely to be important in reducing the amount of drug required to achieve analgesia, thus reducing the probability of secondary effects and toxicity in other tissues.

Conclusion

NAAG and its receptors are present in circuits within the brain and brainstem that process pain perception. This study provides the first direct demonstration that NAAG peptidase inhibitors reduce inflammatory pain perception by acting on pain communication pathways within the brain. Currently, clinical therapy for pain is limited to opioids and non-steroidal analgesics. This discovery of the analgesic effects of NAAG and NAAG peptidase inhibitors in the brain represents an important step in understanding the cellular mechanism underlying this promising new approach to analgesia.

Abbreviations

N-acetylaspartylglutamate: NAAG; metabotropic glutamate receptor: mGluR.

Competing interests

Tatsuo Yamamoto and Joseph Neale have no competing interests. This work was completed during an interval when Acenta Discovery held the license from Georgetown University to the intellectual property related to ZJ43. Dr. Kozikowski was the major owner of Acenta Discovery, Inc. Dr. Zhou was an employee of Acenta Discovery. Acenta Discovery is now owned by PyschoGenics, Inc., and Dr. Zhou is now an employee of PsychoGenics Inc. As such, Dr. Kozikowski and Dr. Zhou could profit from this class of compounds should they ever become drugs. Neither had any direct involvement in collection or analysis of data presented herein.

Authors' contributions

TY executed the behavioral studies. JHN worked with AK and JZ on the characterization of ZJ43, generated the hypothesis, created the experimental design and wrote the manuscript. AK and JZ intellectual contribution was the designed ZJ43 within a series of related NAAG peptidase inhibitors. Additionally, JZ synthesized and chemically characterized ZJ43. All authors have reviewed and approved this final manuscript.

Acknowledgements

This research supported by a grant from the NIH (NS38080) to JHN, Grant-in-Aid for Scientific Research (B) of Japan (12470315) to TY and gifts to Georgetown University by Nancy and Daniel Paduano (JHN). Acenta Discovery, Inc. supported this research by providing ZJ43.

Distribution of <it>N</it>-acetyl-aspartic and <it>N</it>-acetyl-aspartyl-alutamic acids in nervous tissue Curatolo A D'Archangelo P Lino A Brancati A J Neurochem 1965 12 339 342 10.1111/j.1471-4159.1965.tb06771.x 14340686 The nagging question of the function of <it>N</it>-acetylaspartylglutamate Coyle JT Neurobiol Dis 1997 4 231 238 10.1006/nbdi.1997.0153 9361299 <it>N</it>-Acetylaspartylglutamate: the most abundant peptide neurotransmitter in the mammalian central nervous system Neale JH Bzdega T Wroblewska B J Neurochem 2000 75 443 452 10.1046/j.1471-4159.2000.0750443.x 10899918 NAAG peptidase inhibitor reduces acute neuronal degeneration and astrocyte damage following lateral fluid percussion TBI in rats Zhong C Zhao X Sarva J Kozikowski A Neale JH Lyeth BG J Neurotrauma 2005 22 266 276 10.1089/neu.2005.22.266 15716632 NAAG inhibits KCl-induced [(3)H]-GABA release via mGluR3, cAMP, PKA and L-type calcium conductance Zhao J Ramadan E Cappiello M Wroblewska B Bzdega T Neale JH Eur J Neurosci 2001 13 340 346 10.1046/j.1460-9568.2001.01396.x 11168538 GCP II (NAALADase) inhibition suppresses mossy fiber-CA3 synaptic neurotransmission by a presynaptic mechanism Sanabria ER Wozniak KM Slusher BS Keller A J Neurophysiol 2004 91 182 193 10.1152/jn.00465.2003 12917384 <it>N</it>-acetylaspartylglutamate selectively activates mGluR3 receptors in transfected cells Wroblewska B Wroblewski JT Pshenichkin S Surin A Sullivan SE Neale JH J Neurochem 1997 69 174 181 9202308 <it>N</it>-acetylaspartylglutamate activates cyclic AMP-coupled metabotropic glutamate receptors in cerebellar astrocytes Wroblewska B Santi MR Neale JH Glia 1998 24 172 179 10.1002/(SICI)1098-1136(199810)24:2<172::AID-GLIA2>3.0.CO;2-6 9728763 Neuroprotection by glial metabotropic glutamate receptors is mediated by transforming growth factor-beta Bruno V Battaglia G Casabona G Copani A Caciagli F Nicoletti F J Neurosci 1998 18 9594 9600 9822720 Pharmacological evidence for an involvement of group II and group III mGluRs in the presynaptic regulation of excitatory synaptic responses in the CA1 region of rat hippocampal slices Vignes M Clarke VR Davies CH Chambers A Jane DE Watkins JC Collingridge GL Neuropharmacology 1995 34 973 982 10.1016/0028-3908(95)00093-L 8532178 Molecular cloning of a complementary DNA encoding a prostate-specific membrane antigen Israeli RS Powell CT Fair WR Heston WD Cancer Res 1993 53 227 230 8417812 Prostate-specific membrane antigen is a hydrolase with substrate and pharmacologic characteristics of a neuropeptidase Carter RE Feldman AR Coyle JT Proc Natl Acad Sci USA 1996 93 749 753 40126 8570628 10.1073/pnas.93.2.749 Molecular cloning of a peptidase against <it>N</it>-acetylaspartylglutamate from a rat hippocampal cDNA library Bzdega T Turi T Wroblewska B She D Chung HS Kim H Neale JH J Neurochem 1997 69 2270 2277 9375657 Isolation and expression of a rat brain cDNA encoding glutamate carboxypeptidase II Luthi-Carter R Berger UV Barczak AK Enna M Coyle JT Proc Natl Acad Sci USA 1998 95 3215 3220 19722 9501243 10.1073/pnas.95.6.3215 The cloning and characterization of a second brain enzyme with NAAG peptidase activity Bzdega T Crowe SL Ramadan ER Sciarretta KH Olszewski RT Ojeifo OA Rafalski VA Wroblewska B Neale JH J Neurochem 2004 89 627 635 10.1111/j.1471-4159.2004.02361.x 15086519 The neurotransmitter <it>N</it>-acetylaspartylglutamate in models of pain, ALS, diabetic neuropathy, CNS injury and schizophrenia Neale JH Olszewski RT Gehl LM Wroblewska B Bzdega T Trends Pharmacol Sci 2005 26 477 484 16055199 NAAG peptidase inhibitors and their potential for diagnosis and therapy Zhou J Neale JH Pomper MG Kozikowski AP Nat Rev Drug Discov 2005 4 1015 1026 10.1038/nrd1903 16341066 Progress in the discovery and development of glutamate carboxypeptidase II inhibitors Tsukamoto T Wozniak KM Slusher BS Drug Discov Today 2007 12 767 776 10.1016/j.drudis.2007.07.010 17826690 Systemic pre-treatment with a group II mGlu agonist, LY37 reduces hyperalgesia <it>in vivo</it> Sharpe EF Kingston AE Lodge D Monn JA Headley PM Br J Pharmacol 9268 135 1255 1262 10.1038/sj.bjp.0704583 Local administration of <it>N</it>-acetylaspartylglutamate (NAAG) peptidase inhibitors is analgesic in peripheral pain in rats Yamamoto T Saito O Aoe T Bartolozzi A Sarva J Zhou J Kozikowski A Wroblewska B Bzdega T Neale JH Eur J Neurosci 2007 25 147 158 10.1111/j.1460-9568.2006.05272.x 17241276 Antinociceptive effects of <it>N</it>-acetylaspartylglutamate (NAAG) peptidase inhibitors ZJ-11, ZJ-17 and ZJ-43 in the rat formalin test and in the rat neuropathic pain model Yamamoto T Hirasawa S Wroblewska B Grajkowska E Zhou J Kozikowski A Wroblewski J Neale JH Eur J Neurosci 2004 20 483 494 10.1111/j.1460-9568.2004.03504.x 15233757 Inhibition of spinal <it>N</it>-acetylated-alpha-linked acidic dipeptidase produces an antinociceptive effect in the rat formalin test Yamamoto T Nozaki-Taguchi N Sakashita Y Inagaki T Neuroscience 2001 102 473 479 10.1016/S0306-4522(00)00502-9 11166133 Spinal <it>N</it>-acetyl-alpha-linked acidic dipeptidase (NAALADase) inhibition attenuates mechanical allodynia induced by paw carrageenan injection in the rat Yamamoto T Nozaki-Taguchi N Sakashita Y Brain Res 2001 909 138 144 10.1016/S0006-8993(01)02650-6 11478930 Ketamine and <it>N</it>-acetylaspartylglutamate peptidase inhibitor exert analgesia in bone cancer pain Saito O Aoe T Kozikowski A Sarva J Neale JH Yamamoto T Can J Anaesth 2006 53 891 898 16960267 Peripheral group II metabotropic glutamate receptors (mGluR2/3) regulate prostaglandin E2-mediated sensitization of capsaicin responses and thermal nociception Yang D Gereau RWt J Neurosci 2002 22 6388 6393 12151517 Peripheral group II metabotropic glutamate receptors mediate endogenous anti-allodynia in inflammation Yang D Gereau RWt Pain 2003 106 411 417 10.1016/j.pain.2003.08.011 14659524 Group II metabotropic glutamate receptors inhibit cAMP-dependent protein kinase-mediated enhancemednt of tetrodotoxin-resistant sodium currents in mouse dorsal root ganglion neurons Yang D Gereau RWt Neurosci Lett 2004 357 159 162 10.1016/j.neulet.2003.11.074 15003274 Role of peripheral group I and II metabotropic glutamate receptors in IL-1beta-induced mechanical allodynia in the orofacial area of conscious rats Ahn DK Kim KH Jung CY Choi HS Lim EJ Youn DH Bae YC Pain 2005 118 53 60 10.1016/j.pain.2005.07.017 16154694 The regional distribution of <it>N</it>-acetylaspartylglutamate (NAAG) and peptidase activity against NAAG in the rat nervous system Fuhrman S Palkovits M Cassidy M Neale JH J Neurochem 1994 62 275 281 8263527 Metabotropic glutamate receptors – important modulators of nociception and pain behavior Neugebauer V Pain 2002 98 1 8 10.1016/S0304-3959(02)00140-9 12098611 Metabotropic glutamate receptor involvement in models of acute and persistent pain: prospects for the development of novel analgesics Varney MA Gereau RWt Curr Drug Targets CNS Neurol Disord 2002 1 283 296 10.2174/1568007023339300 12769620 Up-regulation of metabotropic glutamate receptor 3 mRNA expression in the cerebral cortex of monoarthritic rats Neto FL Schadrack J Platzer S Zieglgansberger W Tolle TR Castro-Lopes JM J Neurosci Res 2001 63 356 367 10.1002/1097-4547(20010215)63:4<356::AID-JNR1030>3.0.CO;2-3 11170186 Up-regulation of metabotropic glutamate receptor subtypes 3 and 5 in spinal cord in a clinical model of persistent inflammation and hyperalgesia Dolan S Kelly JG Monteiro AM Nolan AM Pain 2003 106 501 512 10.1016/j.pain.2003.09.017 14659534 Differential expression of central metabotropic glutamate receptor (mGluR) subtypes in a clinical model of post-surgical pain Dolan S Kelly JG Monteiro AM Nolan AM Pain 2004 110 369 377 10.1016/j.pain.2004.04.018 15275788 Enhanced group II mGluR-mediated inhibition of pain-related synaptic plasticity in the amygdala Han JS Fu Y Bird GC Neugebauer V Mol Pain 2006 2 18 1471776 16681859 10.1186/1744-8069-2-18 Central nervous system mechanisms of pain modulation Fields HL Basbaum AI Textbook of Pain Textbook of Pain, Edingurgh; Churchill Livingston Wall PD, Melzack R 1999 309 322 Multiple metabotropic glutamate receptor subtypes modulate GABAergic neurotransmission in rat periaqueductal grey neurons <it>in vitro</it> Drew GM Vaughan CW Neuropharmacology 2004 46 927 934 10.1016/j.neuropharm.2004.01.015 15081789 Opioid inhibition of rat periaqueductal grey neurones with identified projections to rostral ventromedial medulla in vitro Osborne PB Vaughan CW Wilson HI Christie MJ J Physiol 1996 490 Pt 2 383 389 1158677 8821137 Presynaptic inhibitory action of opioids on synaptic transmission in the rat periaqueductal grey <it>in vitro</it> Vaughan CW Christie MJ J Physiol 1997 498 Pt 2 463 472 1159215 9032693 Synthesis of urea-based inhibitors as active site probes of glutamate carboxypeptidase II: efficacy as analgesic agents Kozikowski AP Zhang J Nan F Petukhov PA Grajkowska E Wroblewski JT Yamamoto T Bzdega T Wroblewska B Neale JH J Med Chem 2004 47 1729 1738 10.1021/jm0306226 15027864 LY341495 is a nanomolar potent and selective antagonist of group II metabotropic glutamate receptors Kingston AE Ornstein PL Wright RA Johnson BG Mayne NG Burnett JP Belagaje R Wu S Schoepp DD Neuropharmacology 1998 37 1 12 10.1016/S0028-3908(97)00191-3 9680254