ARTICLE

Antinociceptive activity of a synthetic oxopyrrolidine-basedcompound, ASH21374, and determination of its possiblemechanismsZainul Amiruddin Zakaria, Mohammed Hijaz Sani, Mohammed Fazli Mohammat,Nurul Shulehaf Mansor, Zurina Shaameri, Teh Lay Kek, Mohd. Zaki Salleh, and Ahmad Sazali Hamzah

Abstract: This study was carried out to determine the antinociceptive activity of a novel synthetic oxopyrrolidine-basedcompound, (2R,3R,4S)-ethyl 4-hydroxy-1,2-dimethyl-5-oxopyrrolidine-3-carboxylate (ASH21374), and to elucidate the involvementof the opioid, vanilloid, glutamate, and nitric oxide – cyclic guanosine monophosphate (NO/cGMP) systems in modulating theobserved antinociception. ASH21374, in the doses of 2, 10, and 100 mg/kg body mass, was administered orally to mice 60 minsprior to exposure to various antinociceptive assays. From the results obtained, ASH21374 exhibited significant (P < 0.05)antinociceptive activity in the abdominal constriction, hot-plate, and formalin tests that was comparable with 100 mg/kgacetylsalicylic acid or 5 mg/kg morphine, respectively. ASH21374 also attenuated capsaicin- and glutamate-induced paw licking.Pre-treatment with 5 mg/kg naloxone significantly (P < 0.05) inhibited the activity in all assays, while pretreatment with 10 mg/kg�-funaltraxamine, 1 mg/kg naltrindole, or 1 mg/kg nor-binaltorphimine significantly (P < 0.05) reversed the activity in theabdominal constriction test. L-Arginine, NG-nitro-L-arginine methyl esters (L-NAME), methylene blue, and their combinations,failed to inhibit the ASH21374 antinociceptive activity. In conclusion, ASH21374 demonstrated antinociceptive activities on theperipheral and central nervous systems, mediated through the activation of opioid receptors, inhibition of the glutamatergicsystem, and attenuation of vanilloid-mediated nociceptive transmission. Further studies have been planned to determine thepharmacological potential of ASH21374.

Key words: ASH21374, oxopyrrolidine, antinociceptive, opioid, glutamatergic.

Résumé : L'étude présente a été réalisée afin d'évaluer l'activité anti-nociceptive d'un nouveau composé synthétique a based'oxopyrrolidine, le (2R, 3R, 4S)-éthyl 4-hydroxy-1,2-diméthyl-5-oxopyrrolidine-3-carboxylate, (ASH21374), et d'élucider l'implication dessystèmes des opioïdes, des vanilloïdes, du glutamate et de l'oxyde nitrique – guanosine monophosphate cyclique (NO/GMPc) dansla modulation de l'anti-nociception observée. Des doses de 2, 10 et 100 mg/kg de masse corporelle d'ASH21374 ont été adminis-trées par voie orale a des souris 60 minutes avant la réalisation de différentes analyses anti-nociceptives. Selon les résultatsobtenus, l'ASH21374 présentait une activité anti-nociceptive significative (P < 0,05) lors des tests de contraction abdominale, dela plaque chauffante et de la formaline, ce qui est comparable a l'effet de 100 mg/kg d'acide acétylsalicylique ou de 5 mg/kg demorphine, respectivement. L'ASH21374 atténuait aussi le léchage de la patte induit par la capsaïcine ou le glutamate. Unprétraitement avec 5 mg/kg de naloxone inhibait significativement (P < 0,05) l'activité lors de tous les tests alors qu'un prétrait-ement avec 10 mg/kg de �-funaltraxamine, 1 mg/kg de naltrindole ou 1 mg/kg de nor-binaltorphimine renversait significative-ment (P < 0,05) l'activité lors du test de contraction abdominale. La L-arginine, le NG-nitro-L-arginine méthyl ester (L-NAME), le bleude méthylène et leur combinaison ne parvenaient pas a inhiber l'activité anti-nociceptive du ASH21374. En conclusion,l'ASH21374 a démontré des activités anti-nociceptives a médiations périphérique et centrale au moyen de l'activation desrécepteurs des opioïdes, de l'inhibition du système glutamatergique, et de l'atténuation de la transmission nociceptive via lesrécepteurs des vanilloïdes. D'autres études ont été planifiées afin de d'évaluer de manière plus approfondie le potentiel phar-macologique du ASH21374. [Traduit par la Rédaction]

Mots-clés : ASH21374, oxopyrrolidine, anti-nociceptif, opioïde, glutamatergique.

IntroductionPain is essentially a sensation, and has been described as a multi-

dimensional sensory experience that is intrinsically unpleasant andassociated with suffering. Pain is a complex process, particularly inthe sense that it could be triggered through various transmitters and

receptor systems. Some of these well-known transmitter or receptorsystems include opioid, vanilloid, glutamate, and nitric oxide –cyclic guanosine monophosphate (NO/cGMP) (Russo 2001).

Opioid systems subsist all over the central (CNS) and peripheral(PNS) nervous systems, as well as in other tissues, and are engaged

Received 12 March 2013. Accepted 2 September 2013.

Z.A. Zakaria. Department of Biomedical Science, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia (UPM), 43400 Serdang, Selangor,Malaysia; Integrative Pharmacogenomics Institute, Universiti Teknologi MARA, Puncak Alam Campus, 42300 Bandar Puncak Alam, Selangor, Malaysia.M.H. Sani. Department of Biomedical Science, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia (UPM), 43400 Serdang, Selangor, Malaysia.M.F. Mohammat, N.S. Mansor, Z. Shaameri, and A.S. Hamzah. Institute of Science, Block C, Level 3, Universiti Teknologi MARA (UTM), 40450 ShahAlam, Selangor, Malaysia.T.L. Kek and M.Z. Salleh. Integrative Pharmacogenomics Institute, Universiti Teknologi MARA, Puncak Alam Campus, 42300 Bandar Puncak Alam,Selangor, Malaysia.Corresponding author: Zainul Amiruddin Zakaria (e-mail: dr_zaz@yahoo.com) and Ahmad Sazali Hamzah (e-mail: asazali@salam.uitm.edu.my).

1143

Can. J. Physiol. Pharmacol. 91: 1143–1153 (2013) dx.doi.org/10.1139/cjpp-2013-0099 Published at www.nrcresearchpress.com/cjpp on 16 September 2013.

Can

. J. P

hysi

ol. P

harm

acol

. Dow

nloa

ded

from

ww

w.n

rcre

sear

chpr

ess.

com

by

TE

MPL

E U

NIV

ER

SIT

Y o

n 11

/11/

14Fo

r pe

rson

al u

se o

nly.

in a various array of homeostatic functions and movement con-trol. However, the opioid system is mainly discussed in terms ofits role in the processing of noxious sensory input. Moreover, theopioid compounds exert their analgesic effects by binding to andactivating opioid receptors that transduce signals by binding toinhibitory G-coupled proteins, which comprise part of an endog-enous opioid system. This system normally works to modulatesensory input caused by noxious stimuli, wherein its response isactivated by endogenous peptide neurotransmitters. Exogenousopioids mimic and amplify the actions of these neurotransmit-ters. High densities of opioid receptors are located in all areas ofthe CNS known to be involved in integrating information aboutpain (Craig and Sorkin 2001; Trescot et al. 2008).

In addition, glutamate and NO are only a few of the many neu-rotransmitters proven to participate in central sensitization andchronic pain (Russo 2001). An accumulating body of electrophysi-ological, pharmacological, and behavioral evidence is emergingin support of the view that modulation of glutamate receptorsmay have therapeutic potential for several categories of persistentpain, including neuropathic pain resulting from injury and (or)disease of the central or peripheral nerves and inflammatory orjoint-related pain (Bleakman et al. 2006). Glutamate has been re-ported to be involved in descending pain modulation and facilita-tion of messages from the brainstem and midbrain centers to thespinal cord (Calejesan et al. 2000; Starowicz et al. 2007), whileother reports claim that glutamate takes part in the transmissionof nociceptive signals from the PNS to the dorsal horn of thespinal cord (Guimarães et al. 2010). It binds to various types ofglutamate receptors (e.g., N-methyl-D-aspartate (NMDA), �-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate, andmetabotropic glutamate receptors) located on the pre- and post-synaptic membranes, as well as at extra-synaptic sites. Glutamateconcentration in the synaptic cleft determines the extents of re-ceptor stimulation and excitatory synaptic transmission (Tao et al.2005).

On the other hand, NO is involved in numerous physiologicalprocesses in the PNS and CNS. However, its involvement in themodulation of pain processes is not simple, as it may exert pro- oranti-nociceptive effects depending on the circumstances. Most ofthe preclinical studies support a pronociceptive role of NO at thespinal level, whereas at the peripheral level, the information onthe effect of NO on pain and analgesia is conflicting (Hamza et al.2010).

Another receptor system that is important in the modulation ofpain processes involves vanilloid receptor 1, which is also knownas the transient receptor potential cation channel subfamilyV member 1 (TRPV1) receptor. Capsaicin, which is an active ingredi-ent in hot chili peppers, directly stimulates the TRPV1 receptors(Caterina et al. 1997). These receptors have been proven to takepart in the transmission and modulation of nociceptive activity,by selectively acting on unmyelinated C-fibers and thinly myelin-ated A primary sensory neurons located within the peripheral, aswell as the central, nociceptive pathways (Dray 1992; Szolcsanyi1993; Cui et al. 2006).

Although we are beginning to conceive of interventions thatcan terminate the development of pain mechanisms, the existinganalgesic treatments are still the choice for suppressing or con-trolling the symptoms (Woolf 2004). Of those analgesics, opioidanalgesics still remain the mainstay of medical therapy, and areused by millions of patients each year, especially for the treat-ment of acute and chronic pain (Kelly et al. 2008). However, thisclass of drugs possesses several side effects (e.g., constipation,nausea, urinary retention, respiratory depression, etc.), whichlimit their usage and indirectly cause the inadequate treatment ofpain (Lynch et al. 1998; Kalso et al. 2004). Moreover, the progresstowards opioid research has been disappointing and less signifi-cant despite immense advances made in our understanding of thepharmacology of the opioid systems and the extensive effort

spent in developing new and more selective molecules. This poorprogress is further hampered by the failure to develop a powerfulanalgesic drug that is free from the undesirable effects of mor-phine (Corbett et al. 2006). Thus, the quest to find new and effectiveanalgesic drugs with fewer or no side effects are still warranted andshould be continued.

Pyrrolidine-based compounds have been reported to exhibitvarious pharmacological activities (e.g., antinociceptive (Reilleyet al. 2010), anti-inflammatory (Chen et al. 2011), anticonvulsant(Kaminski et al. 2011), and anticancer (Bello et al. 2010)). Moreover,several oxopyrrolidine-based compounds, such as nefiracetam(Rashid and Ueda 2002), piracetam (Abdel Salam 2006), and leve-tiracetam (Micov et al. 2010), which are also classified under thenootropic drugs, have been reported to exert antinociceptive ac-tivity when assessed using various models of nociception, whichsuggests that this class of drugs is likely to alter nociception, andcould prove useful for treating certain types of pain (Navarro et al.2013). In light of the above, and since those nootropic agents sharethe same chemical backbone with the novel compound 2R,3R,4S)-ethyl4-hydroxy-1,2-dimethyl-5-oxopyrrolidine-3-carboxylate (ASH21374;Fig. 1 and 2), we believed that it could have biological propertiesthat include antinociceptive activity. Thus, we took this opportu-nity to study the antinociceptive activity of ASH21374 obtainedfrom previous work (Mohammat et al. 2009) using various in-vivoantinociceptive models in rats.

Materials and methods

Synthesis of (2R,3R,4S)-ethyl 4-hydroxy-1,2-dimethyl-5-oxopyrrolidine-3-carboxylate (referred to as ASH21374)

To a stirred solution of diethyl oxalate (15.0 g, 71.4 mmol/L) in100 mL ethanol, were added 40% methylamine in water (6.17 mL,71.4 mmol/L), and acetaldehyde (4.03 mL, 71.4 mmol/L). The reac-tion mixture was heated under reflux for one hour. After cooling,ice-water was added to the mixture, which was then acidified withHCl. The mixture was evaporated under reduced pressure at 60 °C,and the residue was extracted with dichloromethane, dried overanhydrous MgSO4, and evaporated at 40 °C. The crude productwas washed with diethyl ether to remove any trace of aldehyde, togive a yellow solid. Consequently, to a stirred solution of theyellow solid (5.0 g, 25.1 mmol/L) in CH2Cl2 (50 mL) was added aceticacid (1.44 mL, 25.1 mmol/L) then NaBH4 (1.05 g, 27.9 mmol) at 0 °C.The resulting mixture was stirred for an additional one hourat 0 °C and for 8 h at room temperature. Upon completion, thesolvent was removed in vacuo. The residue was partitioned be-tween EtOAc and washed with a saturated NaHCO3 solution. Theorganic phase was dried with anhydrous MgSO4 and concentratedin vacuo. The crude product was purified by washing with diethylether to yield ASH21374 as a white solid (3.04 g, 60%). The solid wasthen recrystallized from ethyl acetate to give colourless crystals.M.p. 94–96 °C. IR v cm−1: (3311 O–H), (1738 C=O), (1684 C=O);1H NMR (CDCl3, 300 MHz): � 1.25–1.30 (t, 3H, J = 7.2 Hz, CH3), 1.35–1.37, (d, 3H, J = 6.3 Hz, CH3), 2.63–2.69 (t, 1H, J = 8.4 Hz, CHCO2Et),2.80 (s, 3H, NCH3), 3.56–3.65 (qt, 1H, J = 6.3 Hz, MeCHNCH3), 4.17–4.25 (q, 2H, J = 7.2 Hz, OCH2); 4.55–4.58 (d, 1H, J = 8.4 Hz, CHCOH),4.62 (s br, 1H, OH); 13C (CDCl3, 75 MHz): 14.1 (CH3), 19.1 (CH3), 27.3(NCH3), 54.3 (MeCHNCH3), 54.3 (MeCHCO2Et), 61.5 (OCH2), 72.1(COH), 171.4 (C=O), 172.9 (C=O), MS–QTOF: m/z = calcd. for C9H15NO4 :201.2207 (MH)+; found 201.2235 (MH)+ (31).

Preparation of the drugsAcetic acid and dimethyl sulfoxide (DMSO) were purchased from

Fisher Scientific (UK). The acetylsalicylic acid (ASA; Bayer), morphinesulphate (MRP; Sigma), naloxone (NLX), �-funaltraxamine (�-FNA),naltrindole (NALT), and nor-binaltorphimine (nor-BNI), capsaicin,capsazepine, glutamate, L-arginine, NG-nitro-L-arginine methyl esters(L-NAME), methylene blue (MB; Sigma), and naloxone hydrochloride(NLX; Sigma) were used in this study. The ASA, MRP, ASH21374,

1144 Can. J. Physiol. Pharmacol. Vol. 91, 2013

Published by NRC Research Press

Can

. J. P

hysi

ol. P

harm

acol

. Dow

nloa

ded

from

ww

w.n

rcre

sear

chpr

ess.

com

by

TE

MPL

E U

NIV

ER

SIT

Y o

n 11

/11/

14Fo

r pe

rson

al u

se o

nly.

capsaicin, and capsazepine were prepared by dissolving them in1% DMSO. Glutamate, L-NAME, MB, L-arginine, and NLX were pre-pared by dissolving them in distilled water.

Experimental animalsMale BALB/c mice (25–30 g) and male Sprague–Dawley rats (200–

300 g) were obtained from the Animal Unit, Faculty of VeterinaryMedicine, UPM, Malaysia. During the acclimatization period (1 week),the animals were kept at the Animal House, Faculty of Medicineand Health Sciences, UPM, in rooms maintained at 22 ± 2 °C,70%–80% humidity, and a 12 h (light) – 12 h (dark) cycle and sup-plied with a standard commercial diet and water ad libitum. Theexperimental procedures were carried out in accordance withthe Animal Ethics Committee rules and regulations followed bythe University and the ethical guidelines for investigations of ex-perimental pain in conscious animals (Zimmermann 1983).

Antinociceptive assays

Abdominal constriction testThe 0.6% (v/v) acetic-acid-induced abdominal constriction test

was used as described by Zakaria et al. (2007) with slight modifi-cations, to assess the antinociceptive activity of ASH21374 on thePNS, as induced chemically. The mice were equally distributedamong 5 groups (n = 6 per group) and received 1% DMSO, andeither ASA (100 mg/kg body mass) or ASH21374 (2, 10, or 100 mg/kg)orally, 60 min prior to subjecting them to the abdominal constric-tion test. The abdominal constriction, which is created by an in-jection of acetic acid and consists of a contraction of theabdominal region together with a stretching of the hind limbs,was counted cumulatively over the period of 25 min, beginning5 min following the acetic acid administration. Antinociceptiveactivity was signified by a reduction in the mean number of ab-dominal constrictions in the test groups, as compared with thecontrol group. All of the test solutions were administered in thevolume of 10 mL/kg body mass. Reduction in the frequency ofwrithing is described as the percentage of antinociception, usingthe following formula:

(Control group mean � Test group mean)Control group mean

× 100

Hot-plate testThe 52 °C hot-plate test (Zakaria et al. 2007) was used to assess the

thermal-induced central antinociceptive activity of ASH21374. Themice were equally distributed among 5 groups (n = 6 mice pergroup) and received 1% DMSO, and either morphine (5 mg/kg) orASH21374 (10 or 100 mg/kg) orally, 60 min prior to administeringthe hot-plate test. Mice were placed onto the Plexiglas walls on theheated surface to constrain their locomotion on the plate, andlatency to a discomfort reaction (licking of the paws, shaking, orjumping) was recorded immediately before, and at 1, 2, 3, 4, and5 h following oral administration of DMSO, MRP, or ASH21374;they remained in their home cages between tests. A cut-off time of20 s was applied to indicate complete analgesia and to avoid tissueinjury. The prolongation of the latency time compared with thevalues of the control was used for statistical analyses. The testsolutions were administered in the volume of 10 mL/kg body mass.

Formalin testWe used the formalin test as described by Hunskaar and Hole

(1987) with slight modifications to assess the antinociceptive ef-fect of ASH21374 at different phases of nociception. The rats weredistributed among 6 groups and received 1% DMSO and either ASA(100 mg/kg), MRP (5 mg/kg), or ASH21374 (10 and 100 mg/kg),60 min prior to the subjection of the formalin test. All of the testsolutions were administered in the volume of 10 mL/kg body mass.Pain was induced by administration of 2.5 �L of 25% formalin inthe subplantar region of the right hind paw. The rats were indi-vidually placed in a Plexiglass observation chamber, and theamount of time the rat spent licking or biting the injected pawwas considered as an indicator of pain and was recorded for30 min following the formalin injection. The early phase of noci-ception was measured between 0–5 min while the late phase ofnociception was measured 15–30 min after formalin administra-tion.

Determination of the opioid receptors involvement in theASH21374 antinociceptive activity

The involvement of opioid receptors in mediating the ASH21374antinociceptive activity was also determined. Briefly, 9 groups ofmice (n = 6 mice per group) were pre-challenged subcutaneouslywith 5 mg/kg naloxone, 15 min prior to the administration of1% DMSO (oral), and either morphine (5 mg/kg morphine, subcu-taneous injection (s.c.)) or ASH21374 (100 mg/kg, orally). Sixty min-utes after the compound was administered, the mice weresubjected to the abdominal constriction, hot-plate, or formalintests (Zakaria et al. 2005).

Determining the involvement of opioid receptor subtypesin ASH21374 antinociceptive activity

Additional experiments were carried out to determine the opi-oid receptor subtypes that might take part in the modulation ofASH21374's antinociceptive activity. In this study, the specific �-,�-, and �-receptor antagonists, namely �-funaltraxamine (�-FNA),naltrindole (NALT), and nor-binaltorphimine (nor-BNI), respec-tively, were used. The determination of opioid receptor subtypeinvolvement was done using the hot-plate test as was describedearlier for naloxone. The doses of the selective opioid antagonistsand timing of administration were based on previous studies(Choi et al. 2003; Reeta et al. 2006). The �-FNA (10 mg/kg, i.p.), NALT(1 mg/kg, i.p.), and nor-BNI (1 mg/kg, i.p.) were administered 15, 30,and 90 min before oral administration of 100 mg/kg of ASH21374.The mice were then individually subjected to the heat stimulus60 min after administering the ASH21374.

Fig. 1. Molecular structure of (2R,3R,4S)-ethyl 4-hydroxy-1,2-dimethyl-5-oxopyrrolidine-3-carboxylate (ASH21374).

Fig. 2. Ortep diagram of (2R,3R,4S)-ethyl 4-hydroxy-1,2-dimethyl-5-oxopyrrolidine-3-carboxylate (ASH21374).

Zakaria et al. 1145

Published by NRC Research Press

Can

. J. P

hysi

ol. P

harm

acol

. Dow

nloa

ded

from

ww

w.n

rcre

sear

chpr

ess.

com

by

TE

MPL

E U

NIV

ER

SIT

Y o

n 11

/11/

14Fo

r pe

rson

al u

se o

nly.

Capsaicin-induced paw licking testThe procedure described by Goncalves et al. (2005), with slight

modification, was adopted to investigate the role of vanilloid re-ceptors in the modulation of ASH21374 antinociceptive action.Rats were orally pretreated with 1% dimethyl sulfoxide (DMSO, thevehicle), and either capsazepine (0.17 mmol/kg) or ASH21374 (10 or100 mg/kg) 60 min before capsaicin injection (1.6 �g/paw, 20 �L)into the intraplantar (i.pl.) region of the rat’s right hind paw. Imme-diately after administering the phlogistic agent the rats were indi-vidually placed in a transparent glass observation chamber andobserved individually for 5 min after the injection. The amount oftime the animals spent licking the injected paw was recorded with achronometer and was considered as an indication of nociception.

Capsaicin-induced thermal hyperalgesia testThe fact that the TRPV1 receptors are involved in thermal hy-

peralgesia has triggered this study to determine the potential ofASH21374 to affect the capsaicin-induced thermal hyperalgesia,using the plantar test. In this test, the hind paw withdrawal laten-cies (PWLs) to a noxious thermal stimulus were determined usingthe technique described by Hargreaves et al. (1988) and Pomoniset al. (2003) using a plantar test apparatus (model 37370; UgoBasile). Cut-off was set at 32 s, and the directed heat (infrared)intensity set to 50%. Twenty-four rats were distributed into 4 groups(n = 6 rats per group). Groups 1 and 2 were orally pretreated withthe vehicle (1% DMSO), whereas groups 3 and 4 were pretreatedwith ASH21374 (100 mg/kg, p.o.). Sixty minutes later, groups 2 and4 were injected with capsaicin (3 �g/�L, i.pl.) while groups 1 and 3were injected with distilled water (dH2O, i.pl.). PWLs were mea-sured 30 min after the capsaicin injection.

Capsaicin-induced hypothermiaThis study was carried out because TRPV1 receptors are involved in

thermal hyperalgesia, and capsaicin reduces body temperature. Theaim of this study was to determine the effect of ASH21374 oncapsaicin-induced decreases in body temperature, according to themethod described by Christoph et al. (2008). Capsaicin (3 mg/kg) orvehicle (1% DMSO) was administered (i.p.) followed by the adminis-tration of test solutions. All of the animals tested received capsaicinand vehicle in a cross-over design in a randomized order with a1-week washout period. Temperature was measured (rectal ther-mometer) twice before (baseline), and 7.5, 15, 30, and 60 min afteradministering capsaicin, using a digital thermometer (SK-1250MC,Sato Keiryoki Manufacturing Co., Ltd., Japan).

Glutamate-induced paw licking testTo study the way that the glutamatergic system modulates the

antinociceptive properties of ASH21374, the procedure describedby Beirith et al. (2002) was used, but with slight modifications.Rats were orally pretreated with 1% DMSO or ASH21374 (10 and100 mg/kg) 60 min prior to glutamate injection. A volume of glu-tamate (20 �L: 10 �mol/paw, in normal saline) was injected, i.pl.,in the right hind paw of the rats. Immediately after administeringthe phlogistic agent the rats were individually placed in a trans-parent glass chamber and observed individually from 0 to 15 minafter the glutamate injection. The amount of time the animalsspent licking or biting the injected paw was recorded with a chro-nometer, and was considered as an indication of nociception.

Involvement of the nitric oxide – cyclic guanosinemonophosphate pathway

To determine the role of the nitric oxide – cyclic guanosinemonophosphate (NO/cGMP) pathway in the modulation of ASH21374antinociceptive activity the method described by Abacioglu et al.(2000) was adopted with slight modifications. Mice (n = 6) were pre-treated intraperitoneally with 20 mg/kg L-arginine, L-NAME, MB,or their respective combinations (L-arginine with L-NAME orL-arginine with MB) followed 15 min later by oral pretreatment with

1% DMSO or 100 mg/kg ASH21374, respectively. Both L-NAME and MBwere also pre-challenged against 5 mg/kg morphine to indirectlyprove their effectiveness at the dose used to pre-treat for ASH21374.Sixty minutes after the administration of test solutions, the micewere injected (i.p.) with 0.6% acetic acid.

Acute oral toxicity studyThe acute toxicity test (mouse) followed the method described

previously by the Organization for Economic Cooperation andDevelopment (OECD) (1996). The mice were separated into controland test groups (n = 6 mice per goup). They were fasted overnightand then the ASH21374 (5000 mg/kg) was administered orally,while the control group only received 0.9% saline. After treatment,the mice were observed for 30 min and thereafter for 14 days toobserve any signs of toxicity, mortality, or behavioral changes.Food and water were supplied ad libitum.

Motor coordination evaluation

Rota-rod testTo investigate the possible effect of ASH21374 on motor coordina-

tion, the rota-rod test was employed as described by Kinnard andCarr (1957), with slight modifications. The test was performed usinga rod with a diameter of 3 cm, rotating at a constant speed of 14 rpm(Treadmill for mice 7600, Ugo Basile, Milano, Italy). Two days beforetesting, the mice underwent a selection process whereby they werepretrained, and only the animals able to remain on the rod for 60 sfor 2 consecutive trials (separated by 30 min pause between trials)were selected to be used in the experiments. On the day of the exper-iment, the mice were tested once again to obtain the predrug timespent on the rotarod, which had to be at least 60 s for all of the animals.Thirty minutes prior to the test, the selected mice were treated with0.9% saline, ASH21374 (2, 10, and 100 mg/kg, p.o.), or diazepam (4 mg/kg,i.p.). The number of animals per group unable to remain on the rod forat least 60 s was recorded at 30, 60, 90, and 120 min after the p.o. drugadministration (or the vehicle in the control group).

Statistical analysisThe results are the mean ± SEM. One-way ANOVA with Dun-

nett’s post-hoc test was used to analyze and compare the data,with P < 0.05 as the limit of significance.

Results

The antinociceptive profile of ASH21374

The abdominal constriction testFigure 3 shows the antinociceptive effect of ASH21374 assessed

using the abdominal constriction test in mice. The compoundexhibited significant (P < 0.05) antinociceptive activity in a dose-dependent manner. The percentage of antinociceptive activity re-corded for the dose of 2, 10, and 100 mg/kg was approximately17.1%, 50.6%, and 67.8%, respectively. ASA was found to give apercentage of antinociception of approximately 56.8%.

The hot-plate testThe antinociceptive effect of ASH21734 assessed using the hot-

plate test in mice is shown in Fig. 4. The compound also exhibitedsignificant (P < 0.05) antinociceptive activity in a dose-dependentmanner. Except for the 2 mg/kg dose, only the 10 and 100 mg/kgASH21374 exerted antinociceptive activity, with the activity fromthe 10 mg/kg dose seen 2 h after administration, lasting until theend of the experiment, while activity from the 100 mg/kg dose wasseen 0.5 h after administration, and also lasted until the end of theexperiment. MRP was found to produce significant (P < 0.05) anti-

1146 Can. J. Physiol. Pharmacol. Vol. 91, 2013

Published by NRC Research Press

Can

. J. P

hysi

ol. P

harm

acol

. Dow

nloa

ded

from

ww

w.n

rcre

sear

chpr

ess.

com

by

TE

MPL

E U

NIV

ER

SIT

Y o

n 11

/11/

14Fo

r pe

rson

al u

se o

nly.

nociceptive activity 0.5 h after administration, and the activitylasted until the end of the experiment. Overall, MRP was moreeffective than ASH21374.

The formalin testThe antinociceptive activity of ASH21374 assessed using the forma-

lin test in rats is shown in Figs. 5a and 5b. The compound was foundto produce significant (P < 0.05) dose-dependent antinociceptive ac-tivity in the early and late phases of formalin-induced nociception.Despite reduction in the latency of discomfort in the early phase, asshown for the 3 doses used, only 100 mg/kg ASH21374 exerted anysignificant effect. As for the late phase, only 10 and 100 mg/kgASH21374 exhibited significant reduction in the latency of discom-fort. MRP, an analgesic that acts on the CNS, was found to block bothphases of the test, while ASA, an example of an analgesic that acts onthe PNS, attenuated only the late phase of the test. Furthermore,MRP was the most effective antinociceptive in both phases whencompared with ASA or ASH21374 at all doses tested.

Possible mechanisms for the antinociceptive activity ofASH21374

The involvement of opioid systems and their receptor subtypesThe antinociceptive activity of 100 mg/kg ASH21374 was re-

duced after pre-treatment with 5 mg/kg naloxone, when assessedusing the abdominal constriction (Fig. 3), hot plate (Table 1), andformalin tests (Figs. 5a and 5b). Further studies revealed that thecompound’s antinociceptive activity was completely reversed bythe �- and �-opioid receptor antagonists NALT and nor-BNI, respec-tively. However, �-FNA only caused partial reversal of ASH21374'santinociceptive acivity (Table 1).

Effect on capsaicin-induced nociceptive transmissionsThe ability of ASH21374 to inhibit capsaicin-induced nocicpe-

tive transmission via inhibition of the TRVP1 receptor-induced isshown in Fig. 6a. The 10 and 100 mg/kg doses of ASH21374 signif-icantly (P < 0.05) attenuated the capsaicin-induced paw licking ina dose-dependent manner, with the percentage of analgesia re-corded ranging between 21% and 51%, respectively. Interestingly,the effectiveness of 100 mg/kg ASH21374 was comparable withthat of 0.17 mmol/kg capsazepine. In other studies, the attenua-tion of capsaicin-induced thermal hyperalgesia by ASH21374 wasalso determined. The 100 mg/kg dose of ASH21374 exerted signif-icant (P < 0.05) antinociceptive activity against thermally inducedhyperalgesia, as indicated by the increase in PWL value whencompared with the respective control vehicle. Moreover, the ad-dition of capsaicin alone significantly (P < 0.05) reduced PWL,which was significantly (P < 0.05) reversed following pretreatmentwith ASH21374 (Fig. 6b). ASH21374 (100 mg/kg) also significantly(P < 0.05) reversed the capsaicin-induced decrease in body temper-ature (Table 2), indicating that the compound has the potential toinhibit capsaicin-induced TRPV1 receptors.

The role of glutamatergic systemFigure 7 shows the antinociceptive profile of ASH21374 in the

glutamate-induced paw licking test. ASH21374 significantly (P < 0.05)attenuated the nociceptive effect assessed using the glutamate-induced paw licking test in a dose-dependent manner. The per-centage of analgesia recorded for the 10 and 100 mg/kg doses ofASH21374 ranged between 29% and 55%.

Involvement of the NO/cGMP pathwayFigure 8a shows the effects of L-NAME and L-arginine, alone or

combined, on the antinociceptive activity of 100 mg/kg ASH21374,as assessed using the abdominal constriction test. As determinedearlier, 100 mg/kg ASH21374 exerted significant (P < 0.05) antino-ciceptive activity, with approximately 68% analgesia when givenalone. L-NAME alone exhibited significant (P < 0.05) antinociceptiveactivity and maintained the antinociceptive activity of ASH21374,albeit with a slight reduction (approximately 57% analgesia). Onthe other hand, L-arginine alone did not significantly (P < 0.05)affect the nociception induced by the acetic acid, and when giventogether with the ASH21374 or L-NAME also failed to inhibitthe antinociceptive activity of these compounds. Moreover, pre-treatment with the combination of L-arginine and L-NAME alsofailed to change the antinociceptive level of ASH21374. On theother hand, L-NAME was found to significantly (P < 0.05) reversethe antinociceptive activity of morphine.

Figure 8b shows the effects of MB and L-arginine, alone andcombined, on the antinociceptive activity of 100 mg/kg ASH21374,as assessed using the abdominal constriction test. MB alone exhib-ited significant (P < 0.05) antinociceptive activity (approximately67% analgesia), and when given together with ASH21374 did notsignificantly (P < 0.05) change its antinociceptive activity (approx-imately 72% analgesia). Further, L-arginine did not affect theantinociceptive activity of MB, or a combination of MB andASH21374. On the other hand, MB was found to significantly(P < 0.05) inhibit the antinociceptive activity of morphine.

Fig. 3. Antinociceptive profile of (2R,3R,4S)-ethyl 4-hydroxy-1,2-dimethyl-5-oxopyrrolidine-3-carboxylate (ASH21374) assessed usingthe abdominal constriction test in mice. *, P < 0.05 when comparedwith the control group. Data are the mean ± SEM; n = 6 male BALB/cmice per group.

Fig. 4. Antinociceptive profile of (2R,3R,4S)-ethyl 4-hydroxy-1,2-dimethyl-5-oxopyrrolidine-3-carboxylate (ASH21374) assessed usingthe hot-plate test in mice. *, P < 0.05 when compared with thecontrol group at same the respective interval. Data are the mean ±SEM; n = 6 male BALB/c mice per group; BF, before treatment.

Zakaria et al. 1147

Published by NRC Research Press

Can

. J. P

hysi

ol. P

harm

acol

. Dow

nloa

ded

from

ww

w.n

rcre

sear

chpr

ess.

com

by

TE

MPL

E U

NIV

ER

SIT

Y o

n 11

/11/

14Fo

r pe

rson

al u

se o

nly.

Acute toxicity and motor performance assessmentOral administration of ASH21374, at all doses used, had no per-

ceptible effect on the behavioral and locomotor activity of mice.Furthermore, during the observation period, no mortality wasobserved at doses up to 1000 mg/kg. Oral administration ofASH21374 (2, 10, and 100 mg/kg) did not cause any significantvariation in the motor performance of the mice (data not shown).In contrast, diazepam (4 mg/kg, i.p.) significantly reduced the timethe animals spent on the rota-rod. Based on these findings, andbecause it does not affect motor performance, ASH21374 was con-sidered to be safe for oral use as an antinociceptive agent.

DiscussionIn this study, ASH21374, a novel oxopyrrolidine derivative, was

demonstrated to possess dose-dependent antinociceptive activ-ity when assessed using various animal models. The ability ofASH21374 to attenuate nociceptive responses in both the abdom-inal constriction and hot-plate tests suggested that the compoundexerts antinociceptive effects on both the PNS and CNS. In addi-tion, the ability of ASH21374 to attenuate both phases of theformalin test also suggested that the compound exerts antinoci-ceptive effects on both the PNS and CNS. Furthermore, the

Fig. 5. (a) Antinociceptive profile of (2R,3R,4S)-ethyl 4-hydroxy-1,2-dimethyl-5-oxopyrrolidine-3-carboxylate (ASH21374) at the early phase, asassessed using the formalin test in rats. (b) Antinociceptive profile of ASH21374 at the late phase, as assessed using the formalin test in rats.*, P < 0.05 when compared with the control group. Data are the mean ± SEM; n = 6 male Sprague–Dawley rats per group.

1148 Can. J. Physiol. Pharmacol. Vol. 91, 2013

Published by NRC Research Press

Can

. J. P

hysi

ol. P

harm

acol

. Dow

nloa

ded

from

ww

w.n

rcre

sear

chpr

ess.

com

by

TE

MPL

E U

NIV

ER

SIT

Y o

n 11

/11/

14Fo

r pe

rson

al u

se o

nly.

antinociceptive activity of ASH21374 was also demonstrated tobe mediated, partly, via the activation of opioid receptors.

We have developed an antinociceptive profile for ASH21374based on the compounds ability to attenuate different types ofantinociceptive models. The acetic-acid-induced nociception, asseen in the abdominal constriction test, involves the release ofcyclo-oxygenase (COX)-synthesized prostacyclin (Berkenkopf andWeichman 1988). Thus, the ability of ASH21374 to attenuate noci-ception in this assay suggests that the compound acted partly

via the inhibition of peripheral COX (Ballou et al. 2000). Mean-while, the nociceptive action induced by thermal stimuli, as seenwith the hot-plate test, was thought to involve activation of noci-ceptive mechanisms in the CNS (Pini et al. 1996). According to Piniet al. (1996), when compounds can prolong analgesic latency inthe hot-plate test, this indicates an ability to attenuate nociceptivemechanisms in the CNS. This test measures the complex responseto an acute, noninflammatory, nociceptive stimulus, and is influ-enced by drugs that act on the CNS (e.g., opioids), but not on thePNS (e.g., nonsteroidal anti-inflammatory (NSAID) (Pini et al.1996). Furthermore, the ability of ASH21374 to affect the nocicep-tive response induced by chemical and thermal stimuli also indi-cates its strong analgesic properties (Zakaria et al. 2007). On theother hand, the nociceptive effect induced by intraplantar injec-tions of formalin is characterized by the presence of the earlyand late phases. The early phase is associated with the non-inflammation-mediated nociception, while the late phase is at-tributed to inflammation-mediated nociception (Zakaria et al.2007). It is well known that analgesics that act on the CNS inhibitboth phases, while drugs acting on the PNS only inhibit the latephase of the test (Chen et al. 1995). Based on our findings, ASH21374inhibits both phases of the formalin test, which demonstrates thepotential to act on the CNS, and the capability to modulate anti-nociceptive activity in both the CNS and PNS. These findings fur-ther confirmed our observations with the abdominal constrictionand hot-plate tests. Furthermore, the results obtained with theformalin test suggest that ASH21374 could be used in the treat-ment of non-inflammation-mediated pain as well as inflammation-mediated pain (Sani et al. 2012).

In an attempt to establish the possible mechanisms of anti-nociception for ASH21374, we have studied the role of opioidsand glutamatergic receptor systems, NO/cGMP pathways, andvanilloid-receptor-induced nociception in modulating the com-pound’s pain-relieving activity. To study the role of opioid recep-tors, mice were pretreated with naloxone, a non-specific opioidantagonist, before administration of ASH21374, and then assessedusing the abdominal constriction, hot-plate, and formalin tests.Interestingly, naloxone inhibited ASH21374-induced antinocicep-tive activity in all models of nociception, suggesting the involve-ment of opioid receptors, in part, in modulating the compound’santinociceptive activity in the CNS and PNS (Abacioglu et al. 2000).Further attempts to determine the types of opioid receptors in-volved in the modulation of ASH21374 antinociception revealedthat the antinociceptive activity of ASH21374 was completely re-versed by the � and � receptor antagonists, but was only partiallyblocked by the � receptor antagonist. This suggests that theASH21374 acts as a nonselective opioid agonist.

To determine the involvement of the glutamatergic system inthe modulation of ASH21374's antinociceptive activity, the com-pound was used in the glutamate-induced paw licking test. Vari-ous studies have demonstrated the importance of glutamate andglutamatergic receptors, both the ionotropic and metabotropic,

Table 1. The effect of nonselective and selective opioid receptor antagonists on the antinociceptive effect of (2R,3R,4S)-ethyl 4-hydroxy-1,2-dimethyl-5-oxopyrrolidine-3-carboxylate (ASH21374) assessed using the hot-plate test on male BALB/c mice.

Latency time (s)

Treatment Dose (mg/kg) 0 min 60 min 90 min 120 min 150 min 180 min 210 min 240 min

dH2O+1% DMSO 6.12±0.26 6.55±0.25 6.62±0.19 6.07±0.38 5.03±0.54 5.73±0.42 5.42±0.45 5.51±0.37dH2O+ASH 100 5.98±0.25 9.43±0.66* 11.17±0.54* 10.00±0.62* 10.87±0.64* 9.55±0.42* 9.62±0.38* 9.31±0.24*�-FNA 10 6.30±0.34 6.03±0.55 6.72±0.45 6.97±0.43 6.62±0.21 6.57±0.28 6.52±0.29 6.04±0.43�-FNA + ASH 10 + 100 6.65±0.23 7.22±0.27# 9.32±0.67# 8.55±0.35# 9.35±0.50# 8.40±0.78 8.72±0.49 7.97±0.62nor-BNI 1 5.90±0.32 5.98±0.43 7.00±0.33 6.53±0.25 6.20±0.24 6.65±0.24 6.12±0.27 5.98±0.47nor-BNI + ASH 1 + 100 6.42±0.31 6.67±0.38 6.50±0.39 6.45±0.17 7.30±0.37 6.92±0.38 6.25±0.20 6.32±0.56NALT 1 6.20±0.23 6.93±0.16 7.03±0.49 6.45±0.15 6.57±0.24 6.48±0.29 6.33±0.30 6.21±0.11NALT + ASH 1 + 100 6.48±0.27 6.43±0.33 6.73±0.33 6.03±0.43 6.90±0.42 6.52±0.49 7.07±0.30 6.81±0.66

Note: DMSO, dimethylsufoxide; ASH, dH2O+ASH21374; �-FNA, �-funaltraxamine; nor-BNI, nor-binaltorphimine; NALT, naltrindole; *, P < 0.05 when compared withthe 1% DMSO treatment group. #, P < 0.05 when compared with the dH2O+ASH21374 treatment group.

Fig. 6. Antinociceptive profile of (2R,3R,4S)-ethyl 4-hydroxy-1,2-dimethyl-5-oxopyrrolidine-3-carboxylate (ASH21374) assessed(a) using the capsaicin-induced paw licking test and (b) capsaicin-induced thermal hyperalgesia in male Sprague–Dawley rats.*, P < 0.05 and **, P < 0.01 when compared with the control group;a, P < 0.05 when compared with the V/dH2O-treated group; b, P < 0.05when compared with the V/Caps-treated group; c, P < 0.05 whencompared with the ASH/Caps-treated group; Capz, capsezepine;V, vehicle (1% DMSO); dH2O, distilled water; ASH, ASH21374; Caps,capsaicin.

Zakaria et al. 1149

Published by NRC Research Press

Can

. J. P

hysi

ol. P

harm

acol

. Dow

nloa

ded

from

ww

w.n

rcre

sear

chpr

ess.

com

by

TE

MPL

E U

NIV

ER

SIT

Y o

n 11

/11/

14Fo

r pe

rson

al u

se o

nly.

in nociceptive neurotransmission at the peripheral, spinal, andsupraspinal levels (Aanonsen and Wilcox 1990; Mao et al. 1992;Fundytus 2001). The activation of glutamate receptors has alsobeen reported to contribute to the maintenance of peripheralnociceptive processes associated with inflammatory pain, but notphysiological pain (Neugebauer 2002). This finding is supportedby an earlier report from Bhave et al. (2001), who found that glu-tamate receptor antagonist inhibited the inflammatory, but notneurogenic, phase of the formalin test. Based on our findings,ASH21374 attenuates glutamate-induced nociception, indicatingits potential to act, in part, as a glutamate receptor antagonist.Therefore, the involvement of the glutamatergic system, in part,in the modulation of ASH21374 antinociception is proven.

To determine the ability of ASH21374 to inhibit nociceptivetransmission via vanilloid receptors, the compound was testedusing the capsaicin-induced paw licking and thermal hyperlagesiatest. Concurrently, antagonists of TRPV1 receptors exerted anantinociceptive activity when assessed using the inflammatoryas well as neuropathic pain models in rats (Huang et al. 2002;Khairatkar-Joshi and Szallasi 2009). Since ASH21374 exerted inhib-itory effects against the neurogenic pain induced by capsaicin, itis suggested that the compound inhibits nociceptive transmissiontriggered via the TRPV1 activation. TRPV1 receptors are also trig-gered by heat (Jhaveri et al. 2005) and, based on the ability ofASH21374 to inhibit thermal nociception as described earlier, theresults from this study suggest the potential of ASH21374 as anantagonist of TRPV1 receptors at in the PNS and CNS. Further,

ASH21374 was also found to prevent the decrease in rectal bodytemperature induced by the administration of capsaicin.

To study the role of the L-arginine–NO–cGMP pathway in mod-ulating the antinociceptive activity of ASH21374, the compoundwas pre-challenged with L-arginine (a NO donor), L-NAME (aninhibitor of NOS), and MB (an inhibitor of the cGMP pathway),followed by antinociceptive evaluation using the abdominal con-striction test. NO is a reactive biological molecule found insideand between cells, and mediates the conveyance of biochemicalsignals, resulting in a wide spectrum of effects on different bio-logical systems, including the PNS (Julius and Basbaum 2001) andCNS (Meller and Gebhart 1993). The consequences of NO produc-tion include the activation of soluble guanylate cyclase (sGC) andincrease in the level of cGMP within target cells (Julius andBasbaum 2001). NO modulates pain mechanisms in both the PNSand CNS (Duarte et al. 1990), depending on its concentration,where high levels of NO have been demonstrated to induce pain,and vice versa (Meller and Gebhart 1993). In addition, the role ofNO as a mediator or modulator in analgesic drug function has alsobeen documented (Talarek and Fidecka 2002). The discrepanciesin the effect of NO may be attributed to the different subsets ofprimary nociceptive neurons that predominantly innervate eachtissue (Patil et al. 2003). As a result of these discrepancies, theNO/cGMP pathway of the PNS has been particularly implicated invarious nociceptive states (Cunha et al. 2010). The link betweenNO and cGMP, and their pathway association with pain mecha-nisms have been established elsewhere (Vivancos et al. 2003;Cunha et al. 2010). MB is commonly used in research involvingpain perception to clarify the involvement of the cGMP pathwayin the effects of the NO system on pain. This is because MB acts asa less specific and potent guanyl cyclase (GC) inhibitor, directlyblocking NOS and decreasing the accumulation of cGMP, sinceGC is one of the main targets of NO (Vivancos et al. 2003). Inthis study, however, the respective administration of NO donor(L-arginine) or inhibitor (L-NAME), or their combination failed toattenuate the antinociceptive activity of ASH21374. Moreover, thecGMP inhibitor (MB) also failed to attenuate the antinociceptiveactivity of ASH21374. These findings, which are in line with findingsreported by Schmidt et al. (2010), indicate that the antinociceptiveactivity of ASH21374 did not involve activation of the NO/cGMP path-way. Interestingly, indomethacin-induced antinociception also didnot involve activation of the L-arginine–NO–cGMP pathway (Ortizet al. 2003). Moreover, our finding that morphine antinociceptiveactivity was inhibited by L-NAME and MB concurs with previousreports (Jain et al. 2003). Taking into account that the NO/cGMPpathway is important in the antinociceptive mechanisms of thePNS, it is worth mentioning the suggestions made by Duarte et al.(1990) and Meller and Gebhart (1993) that the effect of NO couldstill depend on the dosage and the rate and timing of its release.Moreover, even if NO was involved in the modulation of ASH21374'santinociceptive activity, as suggested by Duarte et al. (1990) andMeller and Gebhart (1993), its activity should not be linked to theactivation of cGMP pathway, as MB failed to affect the com-pound’s activity. This could suggest that the antinociceptive

Table 2. Effect of (2R,3R,4S)-ethyl 4-hydroxy-1,2-dimethyl-5-oxopyrrolidine-3-carboxylate (ASH21374) on the capsaicin-induced reduced body tem-perature assessed using the rectal body temperature of male Sprague–Dawley rats (n = 6 rats per group).

Time after administration

Rectal temperature (°C)

Treatment Dose (mg/kg) 0 min 7.5 min 15 min 30 min 60 min

Vehicle+dH2O 0 37.17±0.25 37.43±0.58 37.89±0.45 37.41±0.62 37.51±0.40Vehicle+3 mg/kg capsaicin 0 37.47±0.31 35.12±0.11* 33.27±0.38* 36.18±0.08* 37.84±0.23ASH21374+dH2O 100 36.92±0.87 37.23±0.41 37.61±0.84 37.27±0.89 37.77±0.36ASH21374 + 3 mg/kg capsaicin 100 37.44±0.57 36.89±0.65 37.08±0.09 37.32±0.23 37.39±0.16

Note: Values for the dose are in milligrams of treatment per kilogram of body mass; vehicle, 1% dimethyl sulfoxide; *, P < 0.05 when compared with thevehicle+dH2O treatment group.

Fig. 7. Antinociceptive profile of (2R,3R,4S)-ethyl 4-hydroxy-1,2-dimethyl-5-oxopyrrolidine-3-carboxylate (ASH21374) assessed usingthe glutamate-induced paw licking test in rats. *, P < 0.05 whencompared with the control group. Data are the mean ± SEM; n = 6male Sprague–Dawley rats per group; ASA, acetylsalicylic acid.

1150 Can. J. Physiol. Pharmacol. Vol. 91, 2013

Published by NRC Research Press

Can

. J. P

hysi

ol. P

harm

acol

. Dow

nloa

ded

from

ww

w.n

rcre

sear

chpr

ess.

com

by

TE

MPL

E U

NIV

ER

SIT

Y o

n 11

/11/

14Fo

r pe

rson

al u

se o

nly.

activity of ASH21374 involves activation of a NO-mediated cGMP-independent mechansism (Morioka et al. 2002). Based on the find-ings of this study, it is plausible to suggest that the antinociceptiveactivity of ASH21374 is due to the existence of different subsets ofnociceptive primary sensory neurons.

In conclusion, this study demonstrates, for the first time, theantinociceptive activities of ASH21374, mediated by the PNS andCNS, which were partly modulated via non-selective opioid andglutamatergic systems, and possibly involved the activation of anon-NO/cGMP pathway and attenuated transmission of TRPV1-mediated nociception. Further studies have been planned to de-termine the pharmacological potential of ASH21374.

Conflict of interestThe authors declare that there is no conflict of interest associ-

ated with this study.

AcknowledgementsThe authors thank the Faculty of Medicine and Health Sciences,

UPM, for providing the necessary facilities for this study, and thePharmacogenomics Centre UiTM (PROMISE) for the high resolu-tion MS analysis and Universiti Kebangsaan Malaysia (UKM) forsingle X-ray Crystallography Data. This work was supported by theFundamental Research Grant Scheme (FRGS; Project Code: 04-01-12-1128FR) awarded by the Ministry of Higher Education, Malaysia.

ReferencesAanonsen, L.M., and Wilcox, G.L. 1990. Excitatory amino acid receptors and

nociceptive neurotransmission in rat spinal cord. Pain, 41: 309–321. doi:10.1016/0304-3959(90)90008-2. PMID:1975091.

Abacioglu, N., Tunctan, B., Akbulut, E., and Cakici, I. 2000. Participation of thecomponents of L-arginine/nitric oxide/cGMP cascade by chemically-inducedabdominalconstriction in themouse. LifeSci.67: 1127–1137.doi:10.1016/S0024-3205(00)00711-6. PMID:10954047.

Abdel Salam, O.M.E. 2006. Vinpocetine and piracetam exert antinociceptiveeffect in visceral pain model in mice. Pharmacol. Rep. 58: 680–691. PMID:17085860.

Ballou, L.R., Botting, R.M., Goorha, S., Zhang, J., and Vane, J.R. 2000. Nociceptionin cyclooxygenase isozyme-deficient mice. Proc. Natl. Acad. Sci. U.S.A. 97:10272–10276. doi:10.1073/pnas.180319297. PMID:10954756.

Beirith, A., Santos, A.R., and Calixto, J.B. 2002. Mechanism underlying thenociception and paw edema caused by injection of glutamate into themouse paw. Brain Res. 924: 219–228. doi:10.1016/S0006-8993(01)03240-1.PMID:11750907.

Bello, C., Cea, M., Dal Bello, G., Garuti, A., Rocco, I., Cirmena, G., et al. 2010.Novel 2-((benzylamino)methyl) pyrrolidine-3,4-diol derivatives as alpha-mannosidase inhibitors and with antitumor activities against hematologicaland solid malignancies. Bioorg. Med. Chem. 18: 3320–3334. doi:10.1016/j.bmc.2010.03.009. PMID:20346684.

Berkenkopf, J.W., and Weichman, B.M. 1988. Production of prostacyclin in micefollowing intraperitoneal injection of acetic acid, phenylbenzoquinone andzymosan: its role in the writhing response. Prostaglandin, 36: 693–709. doi:10.1016/0090-6980(88)90014-7. PMID:2853424.

Bhave, G., Karim, F., Carlton, S.M., and Gereau, R.W. 2001. Peripheral group I

Fig. 8. Acetic-acid-induced abdominal constriction test. (a) Effects of L-arginine and L-NAME, alone or combined, on the antinociceptiveproperties of (2R,3R,4S)-ethyl 4-hydroxy-1,2-dimethyl-5-oxopyrrolidine-3-carboxylate (ASH21374). (b) Effects of L-arginine and methylene blue(MB), alone or combined, on the antinociceptive properties of ASH21374. *, P < 0.05 when compared with the control group. Data are themean ± SEM; n = 6 male BALB/c mice per group.

Zakaria et al. 1151

Published by NRC Research Press

Can

. J. P

hysi

ol. P

harm

acol

. Dow

nloa

ded

from

ww

w.n

rcre

sear

chpr

ess.

com

by

TE

MPL

E U

NIV

ER

SIT

Y o

n 11

/11/

14Fo

r pe

rson

al u

se o

nly.

metabotropic glutamate receptors modulate nociception in mice. Nat. Neu-rosci. 4: 417–423. doi:10.1038/86075. PMID:11276233.

Bleakman, D., Alt, A., and Nisenbaum, E.S. 2006. Glutamate receptors and pain.Semin. Cell Dev. Biol. 17: 592–604. doi:10.1016/j.semcdb.2006.10.008. PMID:17110139.

Calejesan, A.A., Kim, S.J., and Zhuo, M. 2000. Descending facilitatory modulationof a behavioral nociceptive response by stimulation in the adult rat anteriorcingulate cortex. Eur. J. Pain, 4: 83–96. doi:10.1053/eujp.1999.0158. PMID:10833558.

Caterina, M.J., Schumacher, M.A., Tominaga, M., Rosen, T.A., Levine, J.D., andJulius, D. 1997. The capsaicin receptor: a heat-activated ion channel in thepain pathway. Nature, 389: 816–824. doi:10.1038/39807. PMID:9349813.

Chen, T.F., Tsai, H.Y., and Wu, T.S. 1995. Anti-inflammatory and analgesic activ-ities from roots of Angelica pubescens. Planta Med. 61: 2–8. doi:10.1055/s-2006-957987. PMID:7700984.

Chen, Z., Wu, Y., Liu, Y., Yang, S., Chen, Y., and Lai, L. 2011. Discovery of dualtarget inhibitors against cyclooxygenases and leukotriene A4 hydrolyase. J.Med. Chem. 54: 3650–3660. doi:10.1021/jm200063s. PMID:21542630.

Christoph, T., Bahrenberg, G., De Vry, J., Englberger, W., Erdmann, V.A.,Frech, M., Kögel, B., Röhl, T., Schiene, K., Schröder, W., Seibler, J., andKurreck, J. 2008. Investigation of TRPV1 loss-of-function phenotypes in trans-genic shRNA expressing and knockout mice. Mol. Cell. Neurosci. 37: 579–589.doi:10.1016/j.mcn.2007.12.006. PMID:18249134.

Choi, S-S., Han, K-J., Lee, H-K., Han, E-J., and Suh, H-W. 2003. Possible antinoci-ceptive mechanisms of opioid receptor antagonists in the mouse formalintest.Pharmacol.Biochem.Behav.75: 447–457.doi:10.1016/S0091-3057(03)0144-8.

Corbett, A.D., Henderson, G., McKnight, A.T., and Paterson, S.J. 2006. 75 years ofopioid research: the exciting but vain quest for the Holy Grail. Br. J. Pharma-col. 147: S153–S162. doi:10.1038/sj.bjp.0706435. PMID:16402099.

Craig, A., and Sorkin, L. 2011. Pain and Analgesia. eLS. doi:10.1002/9780470015902.a0000275.pub3.

Cui, M., Honore, P., Zhong, C., Gauvin, D., Mikusa, J., Hernandez, G.,Chandran, P., Gomtsyan, A., Brown, B., Bayburt, E.K., Marsh, K., Bianchi, B.,McDonald, H., Niforatos, W., Neelands, T.R., Moreland, R.B., Decker, M.W.,Lee, C.H., Sullivan, J.P., and Faltynek, C.R. 2006. TRPV1 receptors in the CNSplay a key role in broad-spectrum analgesia of TRPV1 antagonists. J. Neurosci.26: 9385–9393. doi:10.1523/JNEUROSCI.1246-06.2006. PMID:16971522.

Cunha, T.M., Roman-Campos, D., Lotufo, C.M., Duarte, H.L., Souza, G.R.,Verri, W.A., Jr., et al. 2010. Morphine peripheral analgesia depends on acti-vation of the PI3K�/AKT/nNOS/NO/KATP signaling pathway. Proc. Natl. Acad.Sci. U.S.A. 107: 4442–4447. doi:10.1073/pnas.0914733107. PMID:20147620.

Dray, A. 1992. Mechanism of action of capsaicin-like molecules on sensoryneurones. Life Sci. 51: 1759–1765. doi:10.1016/0024-3205(92)90045-Q. PMID:1331641.

Duarte, I., Lorenzetti, B., and Ferreira, S. 1990. Peripheral analgesia and activa-tion of the nitric oxide–cyclic GMP pathway. Eur. J. Pharmacol. 186: 289–293.doi:10.1016/0014-2999(90)90446-D. PMID:1981187.

Fundytus, M.E. 2001. Glutamate receptors and nociception: implications for thedrug treatment of pain. CNS Drug, 15: 29–58. doi:10.2165/00023210-200115010-00004. PMID:11465012.

Goncalves, C.E., Araldi, D., Panatieri, R.B., Rocha, J.B., Zeni, G., andNogueira, C.W. 2005. Antinociceptive properties of acetylenic thiophene andfuran derivatives: evidence for the mechanism of action. Life Sci. 76: 2221–2234. doi:10.1016/j.lfs.2004.10.038. PMID:15733937.

Guimarães, A.G., Oliveira, G.F., Melo, M.S., Cavalcanti, S.C.H., Antoniolli, A.R.,Bonjardim, L.R., Silva, F.A., Santos, J.P.A., Rocha, R.F., Moreira, J.C.F.,Araújo, A.A.S., Gelain, D.P., and Quintans-Júnior, L.J. 2010. Bioassay-guidedevaluation of antioxidant and antinociceptive activities of carvacrol. BasicClin. Pharmacol. Toxicol. 107: 949–957. doi:10.1111/j.1742-7843.2010.00609.x.PMID:20849525.

Hamza, M., Wang, X.M., Wu, T., Brahim, J.S., Rowan, J.S., and Dionne, R.A. 2010.Nitric oxide is negatively correlated to pain during acute inflammation. Mol.Pain, 6: 55. doi:10.1186/1744-8069-6-55. PMID:20843331.

Hargreaves, K., Dubner, R., Brown, F., Flores, C., and Joris, J. 1988. A new andsensitive method for measuring thermal nociception in cutaneous hyperal-gesia. Pain, 32: 77–88. doi:10.1016/0304-3959(88)90026-7. PMID:3340425.

Huang, S.M., Bisogno, T., Trevisani, M., Al-Hayani, A., De Petrocellis, L., Fezza, F.,et al. 2002. An endogenous capsaicin-like substance with high potency atrecombinant and native vanilloid VR1 receptors. Proc. Natl. Acad. Sci. U.S.A.99: 8400–8405. doi:10.1073/pnas.122196999. PMID:12060783.

Hunskaar, S., and Hole, K. 1987. The formalin test in mice: Dissociation betweeninflammatory and non-inflammatory pain. Pain, 30: 103–104. doi:10.1016/0304-3959(87)90088-1. PMID:3614974.

Jain, N.K., Patil, C.S., Singh, A., and Kulkarni, S.K. 2003. Sildenafil, a phos-phodiesterase-5 inhibitor, enhances the antinociceptive effect of morphine.Pharmacology, 67: 150–156. doi:10.1159/000067802. PMID:12571411.

Jhaveri, M.D., Elmes, S.J., Kendall, D.A., and Chapman, V. 2005. Inhibition ofperipheral vanilloid TRPV1 receptors reduces noxious heat-evoked responsesof dorsal horn neurons in naïve, carrageenan-inflamed and neuropathicrats. Eur. J. Neurosci. 22: 361–370. doi:10.1111/j.1460-9568.2005.04227.x. PMID:16045489.

Julius, D., and Basbaum, A.I. 2001. Molecular mechanisms of nociception. Na-ture, 413: 203–210. doi:10.1038/35093019. PMID:11557989.

Kalso, E., Edwards, J.E., Moore, R.A., and McQuay, H.J. 2004. Opioid analgesics inchronic non-cancer pain: systematic review of efficacy and safety. Pain, 112:372–378. doi:10.1016/j.pain.2004.09.019. PMID:15561393.

Kaminski, K., Rzepka, S., and Obniska, J. 2011. Synthesis and anticonvulsantactivity of new 1-(2-oxo-2-(4-phenylpiperazin-1-yl)ethyl)pyrrolidine-2,5-diones.Bioorg. Med. Chem. Lett. 21: 5800–5803. doi:10.1016/j.bmcl.2011.07.118. PMID:21875804.

Kelly, J.P., Cook, S.F., Kaufman, D.W., Anderson, T., Rosenberg, L., andMitchell, A.A. 2008. Prevalence and characteristics of opioid analgesics use inthe US adult population. Pain, 138: 507–513. doi:10.1016/j.pain.2008.01.027.PMID:18342447.

Khairatkar-Joshi, N., and Szallasi, A. 2009. TRPV1 antagonists: the challenges fortherapeutic targeting. Trend Mol. Med. 15: 14–22. doi:10.1016/j.molmed.2008.11.004. PMID:19097938.

Kinnard, W.R., and Carr, C.J. 1957. A preliminary procedure for evaluation ofcentral nervous system depressants. J. Pharmacol. Exp. Ther. 121: 354–356.PMID:13481857.

Lynch, E.P., Lazor, M.A., and Gellis, J.E. 1998. The impact of postoperative pain onthe development of postoperative delirium. Anesth. Analg. 86: 781–785. doi:10.1097/00000539-199804000-00019. PMID:9539601.

Mao, J., Price, D.D., Hayes, R.L., Lu, J., and Mayer, D.J. 1992. Differential roles ofNMDA and non-NMDA receptor activation in induction and maintenance ofthermal hyperalgesia in rats with painful peripheral mononeuropathy. BrainRes. 598: 271–278. doi:10.1016/0006-8993(92)90193-D. PMID:1362520.

Meller, S.T., and Gebhart, G.F. 1993. Nitric oxide (NO) and nociceptive processingin the spinal cord. Pain, 52: 127–136. doi:10.1016/0304-3959(93)90124-8. PMID:8455960.

Micov, A., Tomic, M., Popovic, B., and Stepanovic-Petrovic, R. 2010. The antihy-peralgesic effect of levetiracetam in an inflammatory model of pain in rats:mechanism of action. Br. J. Pharmacol. 161: 384–392. doi:10.1111/j.1476-5381.2010.00877.x. PMID:20735422.

Mohammat, M.F., Shaameri, Z., and Hamzah, A.S. 2009. Synthesis of 2,3-dioxo-5-(subtituted)arylpyrroles and their 2-oxo-aryl-3-hydrazone pyrrolidine deriva-tives. Molecule, 14: 250–256. doi:10.3390/molecules14010250. PMID:19136912.

Morioka, N., Inoue, A., Hanada, T., Kumagai, K., Takeda, K., Ikoma, K., et al. 2002.Nitric oxide synergistically potentiates interleukin-1�-induced increase ofcyclooxygenase-2 mRNA levels, resulting in the facilitation of substance Prelease from primary afferent neurons: involvement of cGMP-independentmechanisms. Neuropharmacol. 43: 868–876. doi:10.1016/S0028-3908(02)00143-0.

Navarro, S.A., Serafim, K.G., Mizokami, S.S., Hohmann, M.S., Casagrande, R., andVerri, W.A., Jr., 2013. Analgesic activity of piracetam: effect on cytokine pro-duction and oxidative stress. Pharmacol. Biochem. Behav. 105: 183–192. doi:10.1016/j.pbb.2013.02.018. PMID:23474372.

Neugebauer, V. 2002. Metabotropic glutamate receptors: important modulatorsof nociception and pain behavior. Pain, 98: 1–8. doi:10.1016/S0304-3959(02)00140-9. PMID:12098611.

OECD (Organization for Economic Cooperation and Development). 1996. OECDguidelines for testing of chemicals 423: acute oral toxicity-acute toxic classmethod, 1st adoption. Organization for Economic Cooperation and Develop-ment, Paris, France.

Ortiz, M.I., Granados-Soto, V., and Castaneda-Hernadez, G. 2003. The NO–cGMP–K+ channel pathway participates in the antinociceptive effect of di-clofenac, but not of indomethacin. Pharmacol. Biochem. Behav. 76: 187–195.doi:10.1016/S0091-3057(03)00214-4. PMID:13679232.

Patil, C.S., Jain, N.K., Singh, A., and Kulkarni, S.K. 2003. Modulatory Effect ofcyclooxygenase inhibitors on sildenafil-induced antinociception. Pharmacol-ogy, 69: 183–189. doi:10.1159/000073662. PMID:14624058.

Pini, L.A., Vitale, G., Ottani, A., and Sandrini, M. 1996. Naloxone-reversible anti-nociception by paracetamol in the rat. J. Pharmacol. Exp. Ther. 280: 934–940.PMID:9023309.

Pomonis, J.D., Harrison, J.E, Mark, L., Bristol, D.R., Valenzano, K.J., andKatharine, W. 2003. N-(4-Tertiarybutylphenyl)-4-(3-cholorphyridin-2-yl)tetrahydropyrazine-1(2H)-carbox-amide (BCTC), a novel, orally effective vanil-loid receptor 1 antagonist with analgesic properties: II. In vivo characteriza-tion in rat models of inflammatory and neuropathic pain. J. Pharmacol. Exp.Ther. 306: 387–393. doi:10.1124/jpet.102.046268. PMID:12721336.

Rashid, M.H., and Ueda, H. 2002. Nonopioid and neuropathy-specific analgesicaction of the nootropic drug nefiracetam in mice. J. Pharmacol. Exp. Ther.303: 226–231. doi:10.1124/jpet.102.037952. PMID:12235255.

Reeta, Kh., Mediratta, P.K., Rathi, N., Jain, H., Chugh, C., and Sharma, K.K. 2006.Role of �- and �-opioid receptors in the antinociceptive effect of oxytocin informalin-induced pain response in mice. Regulatory Peptides, 135: 85–90.doi:10.1016/j.regpep.2006.04.004.

Reilley, K.J., Giulianotti, M., Dooley, C.T., Nefzi, A., McLaughlin, J.P., andHoughten, R.A. 2010. Identification of two novel, potent, low-liability an-tinociceptive compounds from the direct in vivo screening of a largemixture-based combinatorial library. AAPS J. 12: 318–329. doi:10.1208/s12248-010-9191-3. PMID:20422341.

Russo, C.M. 2001. Pain: Control. Encyclopedia of Life Sciences. Macmillan Pub-lisher Ltd., Nature Publishing Group. pp. 1–5.

Sani, M.H., Zakaria, Z.A., Balan, T., The, L.K., and Salleh, M.Z. 2012. Antinocice-

1152 Can. J. Physiol. Pharmacol. Vol. 91, 2013

Published by NRC Research Press

Can

. J. P

hysi

ol. P

harm

acol

. Dow

nloa

ded

from

ww

w.n

rcre

sear

chpr

ess.

com

by

TE

MPL

E U

NIV

ER

SIT

Y o

n 11

/11/

14Fo

r pe

rson

al u

se o

nly.

ptive activity of methanol extract of Muntingia calabura leaves and the mech-anisms of action involved. Evid. Based Complement. Alternat. Med. Article ID890361. doi:10.1155/2012/890361. PMID:22611437.

Schmidt, A., Böhmer, A., Schallenberger, C., Antunes, C., Tavares, R.,Wofchuk, S., Elisabetsky, E., and Souza, D. 2010. Mechanisms involved in theantinociception induced by systemic administration of guanosine in mice.Br. J. Pharmacol. 159: 1247–1263. doi:10.1111/j.1476-5381.2009.00597.x. PMID:20132210.

Starowicz, K., Maione, S., Cristino, L., Palazzo, E., Marabese, I., Rossi, F.,de Novellis, V., and Di Marzo, V. 2007. Tonic endovanilloid facilitation ofglutamate release in brainstem descending antinociceptive pathways. J. Neu-rosci. 27: 13739–13749. doi:10.1523/JNEUROSCI.3258-07.2007. PMID:18077685.

Szolcsanyi, J. 1993. Actions of capsaicin on sensory neurones. In Capsaicin in thestudy of pain. Edited by J.N. Wood. Academic Press, London, UK. pp. 1–26.

Talarek, S., and Fidecka, S. 2002. Role of nitric oxide in benzodiazepines-inducedantinociception in mice. Pol. J. Pharmacol. 54: 27–34. PMID:12020041.

Tao, Y.-X., Gu, J., and Stephens, R.L. 2005. Role of spinal cord glutamate trans-porter during normal sensory transmission and pathological pain states.Mol. Pain, 1: 30. doi:10.1186/1744-8069-1-30. PMID:16242033.

Trescot, A.M., Helm, S., Hansen, H., Benyamin, R., Glaser, S.E., Adlaka, R.,Patel, S., and Manchikanti, L. 2008. Opioids in the management of chronic

non-cancer pain: an update of American Society of the Interventional PainPhysicians' (ASIPP) Guidelines. Pain Physician, 11: S5–S62. PMID:18443640.

Vivancos, G.G., Parada, C.A., and Ferreira, S.H. 2003. Opposite nociceptive effectsof the L-arginine/NO/cGMP pathway stimulation in dermal and subcutaneoustissues. Br. J. Pharmacol. 138: 1351–1357. doi:10.1038/sj.bjp.0705181. PMID:12711636.

Woolf, C.J. 2004. Pain: Moving from symptom control toward mechanism-specific pharmacologic management. Ann. Intern. Med. 140: 441–451. PMID:15023710.

Zakaria, Z.A., Safarul, M., Valsala, R., Sulaiman, M.R., Fatimah, C.A.,Somchit, M.N., and Mat Jais, A.M. 2005. Influence of temperature on theopioid-mediated antinociceptive activity of Corchorus olitorius L. in mice.Naunyn-Schmiedebergs Arch. Pharmacol. 372: 55–62. doi:10.1007/s00210-005-1089-8. PMID:16133487.

Zakaria, Z.A., Mohd. Nor Hazalin, N.A., Mohd. Zaid, S.N.H., Abd. Ghani, M.,Hassan, M.H., Hanan Kumar, G., et al. 2007. Antinociceptive, anti-inflammatory and antipyretic effects of Muntingia calabura aqueous extract inanimal models. J. Nat. Med. 61: 443–448. doi:10.1007/s11418-007-0167-2.

Zimmermann, M. 1983. Ethical guidelines for investigation of experimental painin conscious animals. Pain, 16: 109–110. doi:10.1016/0304-3959(83)90201-4.PMID:6877845.

Zakaria et al. 1153

Published by NRC Research Press

Can

. J. P

hysi

ol. P

harm

acol

. Dow

nloa

ded

from

ww

w.n

rcre

sear

chpr

ess.

com

by

TE

MPL

E U

NIV

ER

SIT

Y o

n 11

/11/

14Fo

r pe

rson

al u

se o

nly.