Effects of NAAG peptidase inhibitor 2-PMPA in model chronic pain e relation to brain concentration Jens Nagel a , Irina Belozertseva b , Sergio Greco a , Vladimir Kashkin b , Andrey Malyshkin b , Aigars Jirgensons c , Elena Shekunova b , Bernd Eilbacher a , Anton Bespalov b,1 , Wojciech Danysz a, * a Preclinical R & D, Merz Pharmaceuticals GmbH, Eckenheimer Landstrasse 100, 60318 Frankfurt am Main, Germany b Institute of Pharmacology, Pavlov Medical University, 6/8 Lev Tolstoy Street, St. Petersburg 197089, Russia c Institute of Organic Chemistry, 21 Aizkraukles str., 1006 Riga, Latvia Received 10 April 2006; received in revised form 27 June 2006; accepted 13 July 2006 Abstract N-acetylated-alpha-linked-acidic peptidase (NAAG peptidase) converts N-acetyl-aspartyl-glutamate (NAAG, mGluR3 agonist) into N-acetyl- aspartate and glutamate. The NAAG peptidase inhibitor 2-PMPA (2-(phosphonomethyl)pentanedioic acid) had neuroprotective activity in an animal model of stroke and anti-allodynic activity in CCI model despite its uncertain ability to penetrate the bloodebrain barrier. The NAAG concentration in brain ECF under basal conditions and its alteration in relation to the brain ECF concentration of 2-PMPA is unclear. We therefore assessed those brain concentrations after i.p. administration of 2-PMPA, using in vivo microdialysis combined with LC/MS/MS analysis. Administration of 2-PMPA (50 mg/kg) produced a mean peak concentration of 2-PMPA of 29.66  8.1 mM. This concentration is about 100,000 fold more than is needed for inhibition of NAAG peptidase, and indicates very good penetration to the brain. Application of 2-PMPA was followed by a linear increase of NAAG-concentration reaching a maximum of 2.89  0.42 mM at the end of microdialysis. However, during the time the anti-allodynic effects of 2-PMPAwere observed, the NAAG concentration in the ECF did not reach levels which are likely to have an impact on any known target. It appears therefore that the observed behavioural effects of 2-PMPA may not be mediated by NAAG nor, in turn, by mGluR3 receptors. Ó 2006 Elsevier Ltd. All rights reserved. Keywords: Anxiety; NAAG; NAAG peptidase; 2-PMPA; Brain microdialysis; Brain concentration; HPLCMSMS; Pain; CCl; Carboxypeptidase II (GCPII) 1. Introduction Glutamate receptors and glutamatergic system in general are attractive targets for new drugs, due to their widespread involvement in pathomechanisms or symptomatological ex- pression of many diseases (Danysz et al., 1995; Kew and Kemp, 2005). However, such a widespread distribution and large array of functions means that certain physiological processes might be indiscriminately affected by glutamatergic drugs, resulting in undesirable side-effects. Indeed, only a few drugs in clinical use target the glutamatergic system, such as memantine, tampanel, riluzole, and acamprosate (Parsons et al., 1998). More discrete and selective interference may result in an improved therapeutic profile, and it has been sug- gested that inhibition of metallopeptidase N-acetylated-alpha- linked-acidic dipeptidase (Blakely et al., 1988) (also known as NAAG peptidase, glutamate carboxypeptidase II, GCP II, NAALADase) could fulfil these expectations (Tsai and Coyle, 1995; Neale et al., 2000). The existence of another NAAG peptidase (called glutamate carboxypeptidase III) was shown recently (Bzdega et al., 2004). The differences between GC * Corresponding author. Tel.: þ49 69 150 3564; fax: þ49 69 596 2150. E-mail address: wojciech.danysz@merz.de (W. Danysz). 1 Present address: Neuroscience Discovery, Abbott GmbH & Co. KG, P.O. Box 210805, 67008 Ludwigshafen, Germany. 0028-3908/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2006.07.018 Neuropharmacology 51 (2006) 1163e1171 www.elsevier.com/locate/neuropharm