Antinociceptive effects of N-acetylaspartylglutamate (NAAG) peptidase inhibitors ZJ-11, ZJ-17 and...

Antinociceptive effects of N-acetylaspartylglutamate(NAAG) peptidase inhibitors ZJ-11, ZJ-17 and ZJ-43 in therat formalin test and in the rat neuropathic pain model

Tatsuo Yamamoto,1 Serabi Hirasawa,1 Barbara Wroblewska,2 Ewa Grajkowska,4 Jia Zhou,3 Alan Kozikowski,3

Jarda Wroblewski4 and Joseph H. Neale21Department of Anaesthesiology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba-shi, Chiba 260–8670, Japan2Department of Biology, Georgetown University, Washington, DC, 20007, USA3Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, Chicago, Illinois 60612, USA4Department of Pharmacology, Georgetown University, Washington, DC, 20007, USA

Keywords: Fos, group II metabotropic glutamate receptors, inflammatory pain, LY-341495, mechanical allodynia, NAAG peptidase,NAAG, neuropathic pain

Abstract

The peptide neurotransmitter N-acetylaspartylglutamate (NAAG) acts as an agonist at group II metabotropic glutamate receptors(mGluRs). NAAG is inactivated by extracellular peptidase activity yielding glutamate and N-acetylaspartate. We recently developed aseries of potent NAAG peptidase inhibitors, including ZJ-11, ZJ-17 and ZJ-43. In the present study, we examined the effects ofintrathecally administered ZJ-11 and ZJ-17 and intravenously administered ZJ-11 and ZJ-43 in the rat formalin test (an inflammatorypain model) and in the rat partial sciatic nerve ligation model (a neuropathic pain model). Intrathecal injection of ZJ-11 or ZJ-17 orintravenous injection of ZJ-11 or ZJ-43 suppressed both phases of the agitation behaviour induced by paw formalin injection.Intrathecal and intravenous injection of ZJ-11 suppressed the expression of Fos-like immunoreactivity, induced by paw formalininjection, in laminae I–II in segments L4–L5 of the spinal cord, suggesting an action on sensory spinal transmission. Partial sciaticnerve ligation induced significant mechanical allodynia 7 days after the nerve injury. Intrathecal injection of ZJ-11 or ZJ-17 orintravenous administration of ZJ-11 or ZJ-43 attenuated the level of mechanical allodynia induced by this nerve ligation. These effectsof intrathecally or intravenously administered ZJ compounds in both the formalin test and the partial sciatic nerve ligation model werecompletely antagonized by pretreatment with LY-341495, a highly selective group II mGluR antagonist. Thus, elevation ofextracellular NAAG, induced by the inhibition of NAAG peptidase, activates group II mGluRs and produces an analgesic effect inneuropathic and inflammatory and pain models. In contrast, peptidase inhibition did not affect the threshold for withdrawal from anoxious mechanical stimulus or from an acute thermal stimulus in the hotplate test.

Introduction

N-acetylaspartylglutamate (NAAG) is the most prevalent and widely

distributed peptide transmitter in the mammalian nervous system

(reviewed by Neale et al., 2000). This peptide is present in highest

concentration in the spinal cord (Fuhrman et al., 1994), has been

identified in synaptic vesicles (Williamson &Neale, 1988; Renno et al.,

1997), and has been localized in spinal sensory neurons (Cangro et al.,

1987), in ascending and descending spinal tracts and in spinal

interneurons (Moffett et al., 1993, 1994). NAAG is released from

spinal cord cells by depolarizing stimuli (Zollinger et al., 1994). It

selectively activates the type 3 metabotropic glutamate receptor

(mGluR3), with 10-fold less activity at mGluR2 (Wroblewska et al.,

1997; Schweitzeret al., 2000).Group IImGluRs, primarilymGluR3, are

expressed by spinal and sensory neurons (Carlton et al., 2001; Ohishi

et al., 1993;Boxall et al., 1998;Berthele et al., 1999;Azkue et al., 2000).

Group II mGluR agonists markedly reducemonosynaptic excitation and

primary afferent synaptic transmission in the spinal cord (Ishida et al.,

1993; Gerber et al., 2000) and are antinociceptive in both inflammatory

and neurogenic thermal hyperalgesia (Dolan & Nolan, 2000;

Neugebauer, 2002; Sharpe et al., 2002). Additionally, activation of

presynaptic mGluR3 byNAAG suppresses synaptic transmission (Zhao

et al., 2001; Xi et al., 2002; Garrido Sanabria et al., 2004).

Following release from neurons, NAAG is inactivated by at least

two extracellular peptidases to produce glutamate and N-acetylaspar-

tate (Riveros & Orrego, 1984; Robinson et al., 1987; Serval et al.,

1990; Slusher et al., 1990; Bzdega et al., 1997; Luthi-Carter et al.,

1998; Bacich et al., 2002; Bzdega et al., 2004). Inhibition of NAAG

peptidase activity in the nervous system results in increased extracel-

lular levels of NAAG and decreased glutamate levels under conditions

of elevated neuronal activity (Slusher et al., 1999).

We recently found that intrathecal injection of 2-(phosphonomethyl)pen-

tanedioic acid (2-PMPA), an inhibitor of NAAG peptidase, failed to

reduce the threshold for perception of acute painful thermal stimulus but

did produce an analgesic effect in both phases of the formalin model and

in the carrageenan model of inflammatory pain (Yamamoto et al.,

2001a,b). In an elegant series of experiments, Carpenter et al. (2003)

Correspondence: Dr Tatsuo Yamamoto, as above.

E-mail: [email protected]

Received 12 February 2004, revised 10 May 2004, accepted 14 May 2004

European Journal of Neuroscience, Vol. 20, pp. 483–494, 2004 ª Federation of European Neuroscience Societies

doi:10.1111/j.1460-9568.2004.03504.x

assayed the effects of 2-PMPA in the isolated spinal cords of

anaesthetized rats and found that NAAG peptidase inhibition decreased

noxious evoked activity but not low threshold Ab fibre responses in

normal animals. These data suggested that NAAG peptidase plays an

important role in spinal nociceptive transmission during inflammation

and that decreasing the rate of inactivation of NAAG following synaptic

release and ⁄ or decreasing the extracellular glutamate level in the spinal

cord produces an analgesic effect during inflammation. However, the

precise mechanism through which inhibition of NAAG peptidase

produced an analgesic effect was not resolved.

In order to independently test the role of NAAG in nervous system

functions, we recently developed a series of novel urea-based NAAG

peptidase inhibitors (Nan et al., 2000; Kozikowski et al., 2001;

Kozikowski et al., 2004), including ZJ-11, ZJ-17 and ZJ-43 (Fig. 1).

In the present study, we tested the potential of these compounds to

interact with cloned mGluRs, their efficacy as inhibitors of glutamate

carboxypeptidase II (GCPII) and their efficacy in reducing pain

perception during inflammatory and neuropathic pain states. Specif-

ically, the effects of intrathecal administration of ZJ-11 and ZJ-17 and

intravenous administration of ZJ-11 and ZJ-43 were tested in the

formalin test, an inflammatory pain model, and the partial sciatic nerve

ligation model, a neuropathic pain model. The involvement of group II

mGluRs in peptidase-mediated analgesia also was tested using the

group II-selective antagonist LY341495.

Expression of the immediate–early protooncogene c-fos as assayed

by detection of Fos-like immunoreactivity (Fos-LI) has been widely

used to identify populations of neurons that are activated by noxious

stimuli (Hunt et al., 1987) and to concomitantly examine the ability of

drugs such as morphine to suppress activation of these pathways in

pain models (Hammond et al., 1998; Yamamoto et al., 2000). In the

present study, we tested the hypothesis that ZJ-11 would suppress

expression of Fos-LI in the spinal sensory pain pathway that is

activated by formalin injection in the rat footpad.

Materials and methods

The following investigations were performed according to a protocol

approved by the Institutional Animal Care Committee of Chiba

University, Chiba, Japan. Immediately following the completion of

formalin tests and partial sciatic nerve injury tests, the rats were killed

with an overdose of barbiturate.

Interactions with recominantly expressed metabotropicglutamate receptors

ZJ-11, ZJ-17 andZJ-34 (10 lm) were assayed for their ability to serve asagonists or antagonists on membranes from cell lines that expressed

group I (mGluR1a andmGluR5), group II (mGluR2 andmGluR3 [ZJ-43

only and at 100 lL) and group III (mGluR4 and mGluR6) metabotropic

glutamate receptors. Changes in the signal transduction responses

(increase in phosphoinositide hydrolysis or decrease in cyclic AMP)

were measured after the exposure to the ZJ compounds as has been

described previously (Wroblewska et al., 1997; Nan et al., 2000).

NAAG peptidase assays

Membranes from cell lines that express human and rat GCPII (Bzdega

et al., 1997; Nan et al., 2000) were assayed for their ability to

hydrolyse NAAG in vitro. Transfected cells were harvested in 50 mm

Tris–HCl buffer, pH 7.5, frozen at )80 �C, thawed and sonicated.

Membranes were sedimented in a refrigerated microcentrifuge for

15 min, washed once and resuspended in the same buffer. Protein

concentrations were determined by the bicinchoninic acid method

(Pierce, Rockford, IL, USA). NAAG peptidase activity was deter-

mined using 4 lm NAAG as a substrate. The GPCPII activity was

assayed in a two-step process. In the first step, aliquots of membranes

(4 lg in 100 lL) were incubated for 2 h at 37 �C in 50 mm Tris-HCl

(pH 7.4) in the presence of 4 lm NAAG and the given inhibitor, to

allow for the accumulation of glutamate produced in the GPCII

reaction. In the second step, the amount of accumulated glutamate was

measured using the Amplex Red Glutamic Acid Assay Kit (Molecular

Probes), and the fluorescence was read with a Dynex fluorescent plate

reader using excitation at 530 nm and emission at 590 nm. For all

inhibitors, dose–response curves were obtained and were used to

calculate IC50 values by nonlinear regression using the Sigma-Plot

software. In each series of experiments, a concentration curve of

NAAG was used to determine the Km. The final Ki values for each

Fig. 1. Chemical structures of ZJ-11, ZJ-17 and ZJ-43.

484 T. Yamamoto et al.

ª 2004 Federation of European Neuroscience Societies, European Journal of Neuroscience, 20, 483–494

inhibitor were calculated from the respective IC50 and Km values using

the Cheng and Prusoff equation.

Chemistry methods: preparation of ZJ-11, ZJ-17 and ZJ-43

ZJ-11, ZJ-17 and ZJ-43 were prepared following methods previously

described (Nan et al., 2000; Kozikowski et al., 2001; Kozikowski

et al., 2004; Olszewski et al., 2004). In general, these asymmetrical

ureas were prepared by the reaction of the isocyanate generated in situ

from the tosylate salt of glutamic acid dibenzyl ester and triphosgene–

Et3N with the second appropriately protected amino acid benzyl ester

component. The benzyl ester intermediates were then converted to the

free acids by catalytic hydrogenation to deliver the desired ureas.

Intrathecal catheters

Chronic intrathecal catheters were inserted, during halothane anaes-

thesia, by passing a PE-10 catheter through an incision in the atlanta–

occipital membrane to a position 8 cm caudal to the cisterna at the

level of lumbar enlargement (Yaksh & Rudy, 1976; Yamamoto et al.,

2001b).

Formalin test

The formalin test was excuted according to the protocol in Yamamoto

et al. (2001b). Briefly, rats were lightly anaesthetized with halothane

and the dorsal surface of the right hindpaw was injectd with 50 lL of

5% formalin, the animal was placed in a plexiglass box and the

spontaneous flinching of the paw as observed. For the intrathecal (IT)

dose–response study, ZJ-11 or ZJ-17 was administered intrathecally

10 min before the formalin injection. IT catheters were inserted 7 days

before the formalin injection. For the intravenous (IV) dose–response

study, ZJ-11 or ZJ-43 was administered intravenously 10 min before

the formalin injection. To verify that the analgesic effect of ZJ

compounds is mediated by the activation of mGluR3, 1 mg ⁄ kg of

LY-341495, a Group II mGluR antagonist, was administered intra-

peritoneally 20 min before either the intrathecal administration or

intravenous administration of ZJ compounds and formalin was

injected 10 min after the administration of ZJ compounds. To examine

the effect of intraperitoneal injection of 1 mg ⁄ kg of LY-341495 itself,

LY-341495 was administered intraperitoneally 20 min before either

the intrathecal administration or the intravenous administration of

saline and formalin was injected 10 min after the saline administra-

tion.

Partial sciatic nerve injury model

Anaesthesia was induced by inhalation of 5% halothane, maintained at

a concentration of 2–3%, as needed. After a local incision, the biceps

femoralis of right leg was bluntly dissected at mid-thigh to expose the

sciatic nerve. The right sciatic nerve was carefully mobilized with care

being taken to avoid undue stretching. After the mobilization of the

sciatic nerve, an 8–0 silicon-treated silk suture was inserted into the

right sciatic nerve just proximal to the sciatic trifurcation with a 3 ⁄ 8curved, reversed-cutting mini-needle, and tightly ligated so that the

dorsal 1 ⁄ 3–1 ⁄ 2 of the nerve thickness was trapped in the ligature

(Seltzer et al., 1990). The incision was closed layer to layer, with 3–0

silk suture, and the rats were allowed to recover from the anaesthetics.

All animals postoperatively displayed normal feeding and drinking

behaviour.

Mechanical thresholds were measured using von Frey filaments

with logarithmically incremental stiffness (0.41, 0.70, 1.20, 2.00,

3.63, 5.50, 8.50 and 15.1 g; Stoelting, Wood Dale, IL, USA) to

calculate the 50% probability thresholds for mechanical paw

withdrawal, as previously described (Chaplan et al., 1994). For the

measurement of mechanical threshold in the nerve injury model, rats

were placed in a plastic cage with a wire mesh bottom. Beginning

with the 2.00-g probe, filaments were applied to the plantar surface

of a right (nerve injured) hind paw for 6–8 s, in a stepwise

ascending or descending order following negative or positive

withdrawal responses, respectively, until six consecutive responses

were noted. Stimuli were presented at intervals of several seconds,

allowing for apparent resolution of any behavioural responses to

previous stimuli. Fifty per cent probability thresholds were calcu-

lated according to the method of Dixson (1980). In cases where

continuous positive or negative responses were observed to the

exhaustion of the stimulus set, values of 15.00 and 0.25 g,

respectively, were assigned.

A preliminary study performed revealed that the maximum level of

mechanical allodynia occurred 7 days after the nerve injury and lasted

for > 2 weeks after the nerve injury. Thus, we administered drugs

7 days after the partial sciatic nerve injury. Before and 7 days after the

nerve injury, the mechanical threshold of the right hind paw (nerve-

injured paw) was measured as control data. For the IT dose–response

study, ZJ-11 or ZJ-17 was administered intrathecally, and the right

hind paw was tested at 5, 15, 30, 45 and 60 min after the drug

administration. IT catheters were inserted 3 days before the nerve

injury. For the IV dose–response study, ZJ-11 or ZJ-43 was

administered intravenously and the right hind paw was tested at 5,

15, 30, 45 and 60 min after the drug administration. To verify that the

analgesic effect of ZJ compounds is mediated by the activation of

mGluR3, 1 mg ⁄ kg of LY-341495 was administered intraperitoneally

30 min before the administration of ZJ compounds. To examine the

effect of intraperitoneal injection of 1 mg ⁄ kg of LY-341495 itself,

LY-341495 was administered intraperitoneally 30 min before either

the intrathecal administration or the intravenous administration of

saline and the right hind paw was tested at 5, 15, 30, 45 and 60 min

after the saline administration.

For the measurement of the effect of NAAG peptidase inhibition

on the response to noxious mechanical stimulus (mechanical

threshold), rats were placed in a plastic cage with a wire mesh

bottom and allowed to habituate for a period of � 5 min. After

acclimation, pain-related behaviours were induced with a von Frey

monofilament (46.5 g) which was applied to the plantar surface of

the right and left hind paw every 2 s for 40 s (10 stimuli to each

hind paw). Before and 5, 15, 30, 45 and 60 min after the drug

administration (ZJ-11, ZJ-43 and saline for IV study and ZJ-11 and

saline for IT study), the number of paw withdrawal responses was

measured.

The hotplate test was carried out to assess the effect of NAAG

peptidase inhibitors on the thermal nociceptive threshold. Rats were

placed on 52.5 �C hotplate. The response latency to either a hindpaw

lick or jump was recorded. In the absence of a response, the animals

were removed from the hotplate at 60 s to avoid tissue injury, and a

60 s latency was assigned as the response.

Two baseline measurements were made before the drug adminis-

tration. For the IT study, ZJ-11 or saline was administered intrathecally

and the hotplate latency was measured at 5, 15, 30, 45 and 60 min

after the drug injection. For the IV study, ZJ-11, ZJ-43 or saline was

administered intravenously and the hotplate latency was measured at

5, 15, 30, 45 and 60 min after the drug injection.

NAAG peptidase inhibitor and analgesia 485


Behavioural analysis

The general behaviour of each rat was carefully observed and tested.

Motor functions were evaluated by the performance of two specific

behavioural tasks. (i) 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. (ii) 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 behaviour

will show a decreased ability to right themselves. To quantify the

evaluation of motor functions, both tasks were scored on a scale of

0–2 in which 0 ¼ absence of function and 2 ¼ 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.

Drugs

The intrathecally administered drugs were delivered in a total volume

of 10 lL followed by 10 lL of saline to flush the catheter. The

intravenously administered drugs were delivered in a total volume of

1 mL. For the intravenous administration, drugs were administered via

the tail vein. The agents used in this study were ZJ-11 (molecular

weight 322.3; Fig. 1), ZJ-17 (molecular weight 354.3; Fig. 1), and

ZJ-43 (molecular weight 304.3; Fig. 1). LY-341495, a very highly

selective group II metabotropic glutamate receptor antagonist (Kings-

ton et al., 1998).

Assay of Fos-like immunoreactivity

Fos-LI was assayed and quantified using methods previously

described (Yamamoto et al., 2000). For the IT study, a most-

effective dose (1000 lg) of ZJ-11 or saline was administered

intrathecally 10 min before the formalin injection, and the expression

of Fos-LI was examined 2 h after the formalin injection. For the IV

study, a most-effective dose (100 mg ⁄ kg) of ZJ-11 or saline was

administered intravenously 10 min before the formalin injection, and

the expression of Fos-LI was examined 2 h after the formalin

injection.

Statistical analyses

Formalin test

The time-response data are presented as the mean flinches (±SEM)

per min for the periods of 1–2 min and 5–6 min and then for

1-min periods at 5-min intervals up to 60 min. For the dose–

response analysis, data from phase 1 (0–6 min) and phase 2

(10–60 min) observations were considered separately. In each case,

the cumulative instances of formalin-evoked flinches during the

phase 1 and phase 2 were calculated for each rat. These individual

rat data were then used to construct phase 1 and phase 2 dose–

response curves. To compare the dose–response curves between

phase 1 and phase 2, the percentage of the phase 1 and phase 2

vehicle responses (% vehicle response) was calculated. To evaluate

the dose-dependence, one-way anova was used. For multiple

comparison, Dunnett’s test was used. To compare the dose–

response curve of phase 1 with that of phase 2 of each drug, two-

way anova was used. To evaluate the effect of LY-341495 on the

analgesic effect of ZJ compounds, the unpaired t-test (two-tailed)

was used.

Partial sciatic nerve ligation model

In the present study, we estimated the level of mechanical allodynia by

50% probability threshold for mechanical paw withdrawal of the right

(nerve injured) hind paw. To determine whether partial sciatic nerve

ligation induced significant mechanical allodynia, we compared the

preinjury mechanical threshold with predrug (7 days after nerve

injury) mechanical threshold with an unpaired t-test (two-tailed). To

analyse the effect of drugs on the mechanical threshold, the maximum

50% probability threshold was used. The maximum 50% probability

threshold was defined as the maximum value of the 50% probability

threshold during the entire time course. To analyse the dose-

dependency, one-way anova with Dunnett’s test was used. To

evaluate the effect of LY-341495 on the antiallodynic effect of ZJ

compounds, the unpaired t-test (two-tailed) was used.

Noxious mechanical stimulus model

To compare the base-line data (before the drug administration)

between groups, one-way analysis of variance (one way anova; IV

study) or t-test (IT study) was used. The time–response data are

presented as the mean number of paw withdrawal responses (± SEM)

induced by the 20 applications (10 stimuli to each hind paw) of a von

Frey filament. To analyse the effect of ZJ compounds on the

mechanical threshold, the minimum and maximum number of paw

withdrawal responses during the entire time course was used. To

analyse the drug effects, one-way anova (IV study) or t-test (IT study)

was used.

Thermal stimulus test

To compare the baseline hotplate latencies between groups, one-way

anova (IV study) or t-test (IT study) was used. To analyse the effects

of ZJ compounds on the hotplate test, the per cent maximum possible

effect (%MPE) was calculated, where %MPE ¼ ([postdrug maximum

response latency – predrug response latency] ⁄ [cut-off time (60 s) –

predrug response latency]) · 100. The postdrug maximum response

latency was defined as the single longest response latency during the

first 30 min after the intrathecal drug injection. To analyse the drug

effects, one-way anova (IV study) or t-test (IT study) was used.

Immunohistochemical study

For comparison of the number of Fos-LI neurons between 1000 lg of

ZJ-11 IT-treated group and saline IT-treated group or between

100 mg ⁄ kg of ZJ-11 IV-treated group and saline IV-treated group,

the unpaired t-test (two-tailed) was used.

Wherever appropriate, results are expressed as mean ± SEM.

Critical values that reached a P < 0.05 level of significance were

considered statistically significant.

Results

ZJ compounds

For inhibition of cloned human GCPII, ZJ-11, ZJ-17 and ZJ-43 had Ki

values of 1.4, 3.0 and 0.8 nm, respectively, under assay conditions in

which 2-PMPA had a Ki of 1.4 nm. For the recombinantly expressed

rat GCPII, the Ki values were 6, 1.5 and 3 nm.



ZJ-17, ZJ-11 and ZJ-43 failed to significantly interact at 10 lm as

agonists or antagonists with recombinantly expressed group I

metabotropic receptors (mGluR1 and 5), group II (mGluR2 and

mGluR3) or group III (mGluR4, 6 and 8) as shown in Table 1.

Behavioural analysis

After the IT injection of ZJ-11, ZJ-17 or saline and the IV injection of

ZJ-11, ZJ-43 or saline, all animals scored 2 (normal motor function) in

the placing ⁄ stepping reflex and righting reflex tests at doses applied in

this study. Similarly, systemic ZJ-43 had no significant effect on open

field locomotor behaviour in the same strain of rats (Olszewski et al.,

2004).

Formalin test

In the saline-treated rats (both IT and IV injection), a subcutaneous

injection of formalin resulted in a highly reliable biphasic display of

Fig. 2. Effects of intrathecal injection of 1000 lg of NAAG peptidase inhibitors ZJ-11, 1000 lg of ZJ-17 and saline, and effects of intravenous injection of100 mg ⁄ kg of ZJ-11, 100 mg ⁄ kg of ZJ-43 and saline on the time course of the flinches observed after the formalin injection into the dorsal surface of the ratright hindpaw. Drugs were administered 10 min before the formalin injection. The effects of pretreatment with 1 mg ⁄ kg of the group II mGluR antagonistLY341495 on the effect of the ZJ compounds were also plotted. Effects of 1 mg ⁄ kg of LY341495 + saline (intrathecal or intravenous) also are presented.LY341495 was injected intraperitoneally 20 min before either ZJ compound injection or saline injection. The number of flinches ⁄min is plotted vs. time after theformalin injection. Each point represents the mean response and SEM (n ¼ 5 or 6).

Table 1. Agonist and antagonist activity of ZJ compounds at recombinantly expressed mGluRs

Agonist activity(percentage of agonist activity)

Antagonist activity(percentage of inhibition)

ZJ-11 ZJ-17 ZJ-43 ZJ-11 ZJ-17 ZJ-43

mGluR1 0.4 ± 0.8 0.7 ± 0.3 )2.8 ± 2.2 1.8 ± 2.5 2.8 ± 6.1 3.3 ± 3.9mGluR5 4.2 ± 4.0 1.4 ± 1.5 )0.6 ± 0.3 4.0 ± 5.4 2.8 ± 7.3 5.1 ± 2.4mGluR2 4.0 ± 2.6 )1.8 ± 4.5 )3.0 ± 5.1 3.3 ± 2.4 )0.5 ± 2.0 4.0 ± 3.1mGluR3 NT NT 8.0 ± 4.2 NT NT 3.6 ± 2.0mGluR4 5.6 ± 5.7 )0.9 ± 2.9 3.2 ± 4.9 0.5 ± 4.5 )3.4 ± 2.1 5.8 ± 7.8mGluR6 3.9 ± 6.6 )4.8 ± 7.3 )0.1 ± 3.4 )4.2 ± 8.3 )4.8 ± 9.4 1.2 ± 4.8mGluR8 2.2 ± 5.9 4.9 ± 3.8 )2.5 ± 7.9 2.4 ± 4.7 2.0 ± 5.5 )2.5 ± 3.6

Activity assays were performed on membranes of cell lines expressing the individual mGluRs. All ZJ compounds were used at 10 lm concentrations (exceptmGluR3, at 100 lm). Agonist activity is expressed as a percentage of maximal activity induced by the agonist. Antagonist activity is expressed as the percentage ofinhibition obtained in the presence of the agonist. In inhibition experiments the following concentrations of agonists were used: 30 lm Glu for mGluR1, 10 lm Glufor mGluR5, 2 lm Glu for mGluR2, 100 lm for mGluR3, 1 lm AP4 for mGluR4, 0.5 lm AP4 for mGluR6 and 0.75 lm AP4 for mGluR8. Results are expressed asmeans ± SEM from 6–12 experiments. ZJ-11 and ZJ-17 were not tested (NT) against mGluR3.



flinching of the injected paw (Fig. 2), and this behaviour was similar to

that previously reported (Wheeler-Aceto et al., 1990; Yamamoto et al.,

2001b).

IT administration of either ZJ-11 or ZJ-17 decreased the cumulative

instances of formalin-evoked flinches of phase 1 and phase 2 (Fig. 2)

and this effect was dose-dependent (Fig. 3: ZJ-11, phase 1 P < 0.05,

phase 2 P < 0.001; ZJ-17, phase 1 P < 0.05, phase 2 P < 0.001; one-

way anova). While there was a significant effect of ZJ-11 and ZJ-17

on phase 2 at 100 lg and the phase 1 response was not significantly

affected by this dose of the peptidase inhibitor, the difference between

the dose–response curves of the phase 1 and phase 2 responses in

either IT ZJ-11 or ZJ-17 group did not reach statistical significance

(Fig. 3: P > 0.2, two-way anova). Pre-treatment with the group II

antagonist, LY-341495, blocked the effect of IT injection of 1000 lgof either ZJ-11 or ZJ-17 (Fig. 2: ZJ-11, phase 1 P < 0.01; phase 2

P < 0.001; ZJ-17, phase 1 P < 0.001, phase 2 P < 0.001, one-way

anova). Intraperitoneal injection of 1 mg ⁄ kg of LY-341495 + IT

saline injection had no effect on the cumulative instances of formalin-

evoked flinches of phase 1 and phase 2 as compared with the IT

saline-treated rats (Fig. 2: phase 1 P > 0.5, phase 2 P > 0.8, t-test).

IV administration of either ZJ-11 or ZJ-43 (Fig. 2) decreased the

cumulative instances of formalin-evoked flinches of phase 1 and

phase 2 in a dose-dependent manner (Fig. 3: ZJ-11, phase 1 P < 0.05,

phase 2 P < 0.005; ZJ-43, phase 1 P < 0.005, phase 2 P < 0.001;

one-way anova). There was no significant difference between the

dose–response curve of the phase 1 response and that of phase 2 in

either the IV ZJ-11 group or the IV ZJ-43 group (Fig. 3: P > 0.2, two-

way anova). Pre-treatment with LY-341495 significantly antagonized

the effect of IV administration of 100 mg ⁄ kg of ZJ-11 or ZJ-43

(Fig. 2: ZJ-11, phase 1 P < 0.05, phase 2 P < 0.005; ZJ-43, phase 1

P < 0.05, phase 2 P < 0.001; one-way anova). Intraperitoneal

injection of 1 mg ⁄ kg of LY-341495 + IV saline injection had no

effect on the cumulative instances of formalin-evoked flinches in

phase 1 or phase 2 compared with the IV saline-treated rats (Fig. 2:

phase 1 P > 0.5, phase 2 P > 0.8; t-test).

Partial sciatic nerve ligation model study

In the study of peptidase inhibitors administered by IT injection, the

level of 50% probability threshold of the injured paw 7 days after the

nerve injury was significantly less than the preinjury level (preinjury,

14.7 ± 1.2 g; 7 days after the nerve injury, 2.4 ± 1.4 g; n ¼ 53,

P < 0.001, t-test). This indicated that partial sciatic nerve injury

induced significant mechanical allodynia 7 days after the nerve

injury. There were no differences in the predrug (7 days after the

nerve injury, Fig. 4) level of 50% probability threshold between any

groups.

IT injection of either 100 or 1000 lg of ZJ-17 or IT injection of

1000 lg of ZJ-11 significantly increased the level of maximum 50%

probability threshold as compared with the saline-treated rats (Figs 4

and 5: ZJ-11, P < 0.001; ZJ-17, P < 0.001, one-way anova). Pre-

treatment with LY-341495 significantly antagonized the effect of IT

injection of either 1000 lg of ZJ-11 or 1000 lg of ZJ-17 (Fig. 4:

Fig. 3. Dose–response curves for intrathecal injection of ZJ-11 and ZJ-17 and intravenous administration of ZJ-11 and ZJ-43. The cumulative instances offormalin-evoked flinches in rats, expressed as a percentage of vehicle (saline)-evoked flinches during phase 1 and phase 2 of the formalin test are presented.Drugs were administered intrathecally 10 min before the formalin injection. Each point represents the mean and SEM of five or six rats. The abscissa showsthe log dose (lg in the intrathecal study and mg ⁄ kg in the intravenous study) and the ordinate shows the percentage of vehicle (saline) control flinches duringphase 1 and phase 2. *P < 0.05 as compared with responses at 10 lg of ZJ-11, ZJ-17 or ZJ-43.



ZJ-11, P < 0.001; ZJ-17, P < 0.001, one-way anova). Intraperitoneal

injection of 1 mg ⁄ kg of LY-341495 + IT saline injection had no effect

on the level of maximum 50% probability threshold as compared with

the IT saline-treated rats (Fig. 4: P > 0.7, t-test).

In the study of peptidase inhibitors administered by IV injection, the

level of 50% probability threshold of the injured paw 7 days after the

nerve injury again was significantly less than preinjury level of 50%

probability threshold (preinjury, 15.0 ± 0 g; 7 days after the nerve

injury, 1.7 ± 0.5 g; n ¼ 50, P < 0.001, t-test). There were no

differences in the predrug level of 50% probability threshold among

these groups.

IV injection of either 30 or 100 mg ⁄ kg of ZJ-11 or IV injection of

100 mg ⁄ kg of ZJ-43 significantly increased the level of maximum

50% probability threshold as compared with the saline-treated rats

(Figs 4 and 5: ZJ-11, P < 0.001; ZJ-43, P < 0.001; one-way anova).

Pre-treatment with LY-341495 completely blocked the effect of IV

injection of either 100 mg ⁄ kg of ZJ-11 or 100 mg ⁄ kg of ZJ-43 (Fig. 4:ZJ-11, P < 0.001; ZJ-43, P < 0.001; one-way anova). Intraperitoneal

injection of 1 mg ⁄ kg of LY-341495 + IV saline administration had no

effect on the level of maximum 50% probability threshold as compared

with the IV saline-treated rats (Fig. 4: P > 0.7, t-test).

Mechanical and thermal threshold tests

No difference was apparent between predrug number of paw

withdrawal responses to the mechanical stimuli in each group

following intrathecal administration (rats treated with 1000 lg of

ZJ-11, n ¼ 5, 13.2 ± 3.7; saline-treated rats, n ¼ 5, 13.4 ± 2.7;

P > 0.9, t-test). Intrathecal injection of 1000 lg of ZJ-11 had no

effect on the number of either the maximum or the minimum paw

withdrawal responses as compared with saline-treated rats (maximum

paw withdrawal responses: ZJ-11-treated rats, 15.6 ± 2.1; saline-

treated rats, 15.0 ± 2.0, P > 0.6; minimum paw withdrawal responses:

ZJ-11-treated rats, 13.0 ± 2.1; saline-treated rats, 12.0 ± 2.8; P > 0.5,

t-test).

Following intravenous injection, no difference was apparent

between predrug number of paw withdrawal responses (Fig. 6, time 0)

in each group. In the saline-treated group (n ¼ 5), the mean value was

15.4 ± 3.6 paw withdrawals. Intravenous injection of either

100 mg ⁄ kg of ZJ-11 or 100 mg ⁄ kg of ZJ-43 had no effect on the

number of either the maximum or the minimum paw withdrawal

responses as compared with saline-treated rats (maximum paw

withdrawal responses: ZJ-11-treated rats, 18.4 ± 1.5; ZJ-43-treated

rats, 18.8 ± 0.8; saline-treated rats, 17.0 ± 1.9; P > 0.1; minimum

paw withdrawal responses: ZJ-11-treated rats, 14.0 ± 3.2; ZJ-43-

treated rats, 15.6 ± 1.8; saline-treated rats, 13.4 ± 2.3; P > 0.3, one-

way anova).

In the hotplate assay following intrathecal injection of peptidase

inhibitor, no difference was apparent between the predrug % MPE in

each group (rats treated with 1000 lg of ZJ-11, n ¼ 5, 19.2 ± 4.7 s;

saline-treated rats, n ¼ 5, 14.7 ± 4.4 s; P > 0.1, t-test). Intrathecal

injection of 1000 lg of ZJ-11 had no effect on the %MPE as

compared with saline-treated rats (ZJ-11-treated rats, 13.4 ± 19.3;

saline-treated rats, 11.5 ± 15.2; P > 0.8, t-test).

Following intravenous injection of peptidase inhibitors in this

thermal stimulus assay (Fig. 7), no difference was apparent between

Fig. 4. Effects of intrathecal injection of 1000 lg of ZJ-11, 1000 lg of ZJ-17 and saline, and effects of intravenous injection of 100 mg ⁄ kg of ZJ-11, 100 mg ⁄ kgof ZJ-43 and saline on the time courses of the mechanical threshold in the partial sciatic nerve ligation model are presented. Drugs were administered 7 days after thenerve lesion. The effects of pretreatment with 1 mg ⁄ kg of LY341495 on the effect of either intrathecal or intravenous injection of ZJ compounds also are presented.LY341495 was injected intraperitoneally 30 min before the either ZJ compound injection or saline injection. Ordinate, 50% probability threshold of the nerve-injuredpaw (g); abscissa, time after drug administration (min). Each line represents the group mean and SEM of five to seven rats.



the predrug %MPE in each group (0 time). For the saline-treated group

(n ¼ 5), the mean time was 16.2 ± 4.4 s to paw withdrawal.

Intravenous injection of either 100 mg ⁄ kg of ZJ-11 or 100 mg ⁄ kgof ZJ-43 had no effect on the %MPE as compared with saline-treated

rats (ZJ-11-treated rats, )4.4 ± 11.8; ZJ-43-treated rats, )9.9 ± 25.5;

saline-treated rats, )9.5 ± 10.5; P > 0.8, one-way anova).

Formalin induction of Fos

Two hours after formalin injection in the footpad, there was a very

substantial increase in the number of neurons in spinal cord laminae I

and II that expressed detectable Fos-LI (Fig. 8). Intrathecal injection of

1000 lg of ZJ-11 or IV injection of 100 mg ⁄ kg of ZJ-11 significantly

depressed the number of these Fos-LI-positive neurons in laminae I–II

relative to IT or IV saline-treated rats (Fig. 8). ZJ-11 injection had no

significant effect on the numbers of the Fos-LI neurons in laminae III–

IV and lamina V as compared with the IT or IV saline-treated group

(Fig. 8).

Discussion

These data demonstrate that IT or IV injections of three potent

NAAG peptidase inhibitors substantially depressed the phase 1 and

phase 2 agitation behaviours in the rat formalin test. Similarly, IT and

IV injections of these inhibitors reduced the level of mechanical

allodynia that is induced by partial sciatic nerve injury. At the same

time, peptidase inhibition did not significantly affect acute mechan-

ical and thermal pain responses in control animals. These experi-

ments were based on the hypothesis that inhibition of NAAG

peptidase activity would lead to increased activation of group II

mGluR, particularly mGluR3, and result in reduced pain perception.

This hypothesis was tested directly by injection of the potent (IC50

15–20 nm) group II mGluR antagonist LY341495 prior to injection

of the ZJ compounds. LY341495 has been shown to be ineffective in

displacement of ligand binding at ionotropic glutamate receptors and

to be 1000-fold less effective as an antagonist at group I and III

mGluRs than group II mGluRs (Kingston et al., 1998). LY341495

completely reversed the effects of the peptidase inhibitors. These data

fully support the hypothesis that inhibition of NAAG peptidase

activity increases the activation of group II mGluRs and further that

this represents a novel approach to pain management that may be of

therapeutic value.

NAAG, Group II mGluRs and the spinal sensory axis

Group II mGluRs, NAAG and NAAG peptidase are prevalent in the

spinal sensory axis. NAAG is found in exceptionally high concentra-

tions in the spinal cord (Curatelo et al., 1965; Fuhrman et al., 1994)

and has been localized by immunocytochemistry in dorsal sensory

Fig. 5. Dose–response curves of the effect of intrathecal injection of ZJ-11and ZJ-17 (top) and intravenous administration of ZJ-11 and ZJ-43 (bottom)presenting the level of the maximum 50% probability threshold are presented.The maximum 50% probability threshold was defined as the maximum value ofthe 50% probability threshold for a paw withdrawal response during the entiretime course. Each point represents the mean and SEM (n ¼ 5–7 rats). Theabscissa shows the log dose. *P < 0.05 as compared with saline-treated rats.

Fig. 6. After acclimatisation, pain-related behaviour (paw withdrawal) wasinduced by a von Frey monofilament (46.5 g) that was applied to the plantarsurface of the right and left hind paw every 2 s for 40 s (10 stimuli to each hindpaw). Before (0) and 5, 15, 30, 45 and 60 min after the drug administration (ZJ-11, ZJ-43 and saline for IV study and ZJ-11 and saline for IT study), the numberof paw withdrawal responses was measured. No significant differences weredetected between the predrug number of paw withdrawal responses in eachgroup. Intravenous injection of 100 mg ⁄ kg of ZJ-11 or 100 mg ⁄ kg of ZJ-43had no significant effect on the number of either the maximum or the minimumpaw withdrawal responses as compared with saline-treated rats.



neurons, spinal interneurons, motoneurons and ascending and des-

cending spinal tract axons (Cangro et al., 1987; Moffett et al., 1993).

NAAG immunoreactivity is highest in rat spinal layers III–X and

lower in layers I–II with the exception of large-caliber dorsal root

fibers that pass through layers I–II and terminate in layers III–X

(Moffett et al., 1993, 1994). NAAG is released from spinal cord

neurons in a calcium-dependent manner following depolarization

(Zollinger et al., 1994). Substantial levels of NAAG peptidase activity

have been identified in spinal cord (Fuhrman et al., 1994; Bacich

et al., 2001) and dense NAAG peptidase immunoreactivity has been

reported in dorsal horn laminae I and II, while mild staining has been

detected in lamina X (Slusher et al., 1992). Group II mGluR,

predominantly mGluR3, message is expressed in spinal cord (Ohishi

et al., 1993; Jia et al., 1999). Carlton et al. (2001) also localized these

receptors in both the myelinated and unmyelinated central axons and

peripheral neurites of dorsal root ganglion cells. The highest density of

group II mGluRs is found in lamina IIi with lesser amounts of

immunoreactivity in laminae III and IV. Transection studies suggested

that most, but not all, of these receptors are localized on primary

afferent terminals (Carlton et al., 2001). These data provide a

substantial framework within which to interpret the influence of

NAAG peptidase inhibition on pain perception at the spinal sensory

level.

Site of analgesic action of NAAG peptidase inhibition

Neither the IT nor IV data permit a definitive conclusion as to the site

of action of the peptidase inhibitors with respect to analgesia.

However, the efficacy of IT and IV injection of ZJ-11 in reducing the

formalin-induced increase in Fos-LI in laminae I-II, but not in laminae

III-V, is highly supportive of the hypothesis that systemically applied

ZJ-11 suppresses the effect of nociceptive input from the primary

afferents. Intrathecal morphine administration has a similar effect in

the rat formalin test and on the reduction of inflammation-induced Fos

expression (Yamamoto et al., 2000). The observation that the

responses in both phases of the formalin test were decreased by

peptidase inhibition might suggest actions on primary afferents and

spinal cord neurons. Three reports are consistent with a role for

peripheral neurons in NAAG peptidase-induced analgesia. NAAG

peptidase inhibition by 2-PMPA attenuated afferent discharge in the

partial sciatic nerve ligation model (Chen et al., 2002). Injection of

group II agonists into the plantar surface of the mouse hindpaw did not

alter mechanical thresholds, but did moderate the mechanical thresh-

old increase induced by PGE2 or carrageenan (Yang & Gereau, 2003).

The conclusion that the sensory spinal synapse is an important site of

action of NAAG peptidase inhibitors in reducing pain perception is

further supported by the recent observation that 2-PMPA decreased

noxious evoked activity but not low threshold Ab fibre responses in

Fig. 8. Effect of intrathecal injection of 1000 lg of ZJ-11 and the effect ofintravenous injection of 100 mg ⁄ kg of ZJ-11 on the number of Fos-LI neuronsin different laminae of spinal segments L4 and L5 ipsilateral to the site offormalin injection. Drugs were administered 10 min before the formalininjection and the expression of Fos-LI was examined in tissue fixed 2 h afterthe formalin injection. Either intrathecal injection or intravenous injection ofZJ-11 decreased the number of Fos-LI neurons in laminae I–II, but not inlaminae III–V as compared with the saline-treated rats. Each bar represents thegroup mean and SEM of five rats.

Fig. 7. The hotplate test was carried out to assess the effect of NAAG pept-idase inhibitors on the thermal nociceptive threshold. Rats were placed on a52.5 �C hotplate. The response latency to either a hindpaw lick or jump wasrecorded. Two baseline measurements were made before the drug administra-tion. ZJ-11, ZJ-43 or saline was administered intravenously and the hotplatelatency was measured prior to (0) and at 5, 15, 30, 45 and 60 min after the druginjection. No significant differences were apparent between the predrug %MPEin each group. Intravenous injection of 100 mg ⁄ kg of ZJ-11 or 100 mg ⁄ kg ofZJ-43 had no significant effect on the %MPE as compared with saline-treatedrats.



normal animals (Carpenter et al., 2003). Strikingly, following

carrageenan-induced inflammation, 10-fold lower amounts of 2-PMPA

decreased the evoked activity. These reports together with the data

presented here suggest a significant role for sensory neurons in the

analgesia provide by NAAG peptidase inhibitors. The greater efficacy

of NAAG at mGluR3 and the low level of expression of mGluR2 in

the rat spinal cord (Ohishi et al., 1993) additionally emphasize the

potential role of mGluR3 in this process.

Group II mGluRs and pain perception

IT injection of a group II mGluR agonist significantly increased

mechanical withdrawal threshold in an inflammatory pain model in

adult female sheep (Dolan & Nolan, 2000). Similarly, IP injection of a

selective group II mGluR agonist, LY379268, reduced both

inflammatory and neurogenic thermal hyperalgesia in rats but did

not affect withdrawal latencies to mechanical or thermal stimulation

(Sharpe et al., 2002). Using group II- and group III-selective mGluR

agonists, Gerber et al. (2000) demonstrated that activation of receptors

in either group suppressed synaptic transmission via presynaptic

receptors on primary afferents in rat spinal cord slices. Similarly, a

group II agonist inhibited the C-fibre evoked response of dorsal horn

neurons in the rat carrageenan test (Stanfa & Dickenson, 1998). While

the mechanism by which group II mGluR activation contributes to

analgesia is unknown, these data suggest that inhibition of glutamate

release from primary afferent terminals may be involved. Consistent

with this, group II mGluR agonist inhibition of primary afferent

trasnsmission in spinal cord has been reported (Ishida et al., 1993).

Acting at presynaptic mGluR3 receptors, NAAG inhibits transmitter

release from cortical neurons in culture (Zhao et al., 2001) and

hippocampal neurons (Anwyl, 1999). More relevant to the actions of

the ZJ compounds, NAAG peptidase inhibition in hippocampal slices

reduces glutamate release via a presynaptic mechanism (Garrido

Sanabria et al., 2004).

The absence of an effect of LY341495 alone on pain perception

suggests that in these models the rate of NAAG release in the presence

of normal NAAG peptidase activity is not sufficient to provide

sustained activation of relevant group II mGluRs to overcome

consequences of the painful stimulus. As is the case for many peptide

cotransmitters, the rate of release of NAAG relative to the release of

small amine transmitters from the same cells is not known. It is clear,

however, that NAAG is rapidly hydrolysed by extracellular peptidase

activity in the nervous system (Robinson et al., 1987; Serval et al.,

1990; Slusher et al., 1990; Bzdega et al., 1997; Luthi-Carter et al.,

1998; Bacich et al., 2002; Bzdega et al., 2004). It may be, for

example, that the highest release of NAAG occurs outside the laminae

in which mGluR receptor activation is required to achieve analgesia.

In the absence of peptidase inhibition, hydrolysis would preclude the

extended diffusion required to reach these receptors. The data from

this study would suggest that inhibition of inactivation of NAAG

permits the extracellular peptide levels to rise to or to maintain an

elevated steady state for a longer period and to activate group II

mGluRs.

Specificity of ZJ actions

The three peptidase inhibitors used in this study were selected from a

group of more than 100 NAAG analogues that we have synthesized.

ZJ-11, -17 and -43 are among a group with considerable potency as

inhibitors of cloned human, rat and mouse NAAG peptidases.

Confidence in the conclusion that ZJ compounds are achieving

analgesia by elevating NAAG levels derives from several sources.

First, these actions are similar to those obtained by a structurally

different peptidase inhibitor, 2-PMPA. We have previously

demonstrated that this compound was antinociceptive in the formalin

pain model and in mechanical allodynia induced by carrageenan,

while also lacking an effect in the hotplate test (Yamamoto et al.,

2001a,b). Second, similar results are obtained from three different

potent peptidase inhibitors in our urea-based series of compounds. For

reasons of economy, we did not test all three compounds by both

routes of administration. These compounds do not significantly

interact with metabotropic glutamate receptors. ZJ-11 and ZJ-43

(100 lm) previously were found to be without significant activity in

competitive binding assays for a series of neurotransmitter receptors,

including NMDA receptors (Olszewski et al., 2004). Additionally,

ZJ-43 has been found to substantially reduce some of the schizophre-

nia-like behaviours induced by phencyclidine administration and this

action is similarly blocked by the group II antagonist LY341495

(Olszewski et al., 2004).

The data obtained using the group II mGluR antagonist, LY341495,

in these experiments militate against a direct role for the NMDA

receptor in the analgesic effects of this series of peptidase inhibitors.

The question of potential NMDA involvement arises because IT

injection of an NMDA receptor antagonist produced analgesia in the

formalin pain model (Yamamoto & Yaksh, 1992). However, ZJ-43

(100 lm) is without agonist or antagonist activity at NMDA receptor-

induced conductances or antagonist activity on glutamate-mediated

spontaneous end plate currents in cerebellar granule neurons

(Olszewski et al., 2004). Additionally, based on indirect data, some

have speculated that NAAG may act as a partial agonist at this

receptor without directly testing this concept (Puttfarcken et al., 1993;

Greene, 2001). We recently tested this hypothesis directly and found

no evidence of a partial agonist action of NAAG at NMDA receptors

or on NMDA-mediated spontaneous postsynaptic currents (Losi et al.,

2004).

Efficacy of ZJ compounds

The ZJ compounds are potent inhibitors of GCPIII. In preliminary

assays, ZJ-11, ZJ-17 and ZJ-43 also were found to inhibit the NAAG

peptidase activity of recombinantly expressed mouse GCPIII with Ki

values of � 25 nm. Nonetheless, relatively high concentrations of

these inhibitors were required for efficacy in pain assays following

both IT and IV administration. This is not surprising in that these

compounds are hydrophilic and highly charged, qualities that are

likely to limit diffusion into the spinal cord and passage via the blood–

brain barrier. In the intrathecal injection experiments, relatively high

amounts of these compounds were required to achieve effects in either

pain model. Indeed, we found that intrathecal injection of 100 lg of

2-PMPA attenuated the level of the agitation behaviour in the rat

formalin test (Yamamoto et al., 2001b) while10-fold higher amounts

of intrathecal ZJ-11 and ZJ-17 than 2-PMPA were required to obtain

maximal analgesia in this model. The difficulty with this mode of

administration as a means of assessing biological activity of a

compound relates to the requirement that the test compound be

sufficiently lipid soluble to penetrate the substantial layer of myeli-

nated tracks that surround the grey matter of the cord if it is to act

there. Given the amounts of ZJ-11 and ZJ-17 that were administered

intrathecally, it is not possible to determine whether the site of action

of the drug was in the spinal cord or whether a significant quantity of

peptidase inhibitor diffused within the intrathecal space to the brain or

sensory ganglia.



Taken together, these data suggest that NAAG peptidase activity

represents a significant target in the development of analgesic

compounds that function by enhancing normal processes of synaptic

transmission. Additionally important from a therapeutic perspective,

NAAG peptidase inhibition does not interfere directly with the ability

to detect and respond to acute mechanical and thermal stimuli under

normal conditions.

Acknowledgements

This study was supported by a Grant-in-Aid for Scientific Research (B) ofJapan 12470315 (T.Y.) and by NIH grants NS38080 and NS42672.

Abbreviations

2-PMPA, 2-(phosphonomethyl)pentanedioic acid; Fos-LI, Fos-like immunore-activity ⁄ immunoreactive; GCPII, glutamate carboxypeptidase II; IT, intrathe-cal; IV, intravenous; mGluR, metabotropic glutamate receptor; %MPE, per centmaximum possible effect; NAAG, N-acetylaspartylglutamate.

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Antinociceptive effects of N-acetylaspartylglutamate (NAAG) peptidase inhibitors ZJ-11, ZJ-17 and...

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