SB225002

The effects of the selective and non-peptide CXCR2 receptor antagonist SB225002 on acute and long-lasting models of nociception in mice
Marianne N. Manjavachi a, Nara L.M. Quintão a, Maria Martha Campos b, Isa K. Deschamps a,
Rosendo A. Yunes c, Ricardo J. Nunes c, Paulo C. Leal c, João B. Calixto a,*
a Departament of Pharmacology, Campus Universitário, Universidade Federal de Santa Catarina, 88049-900 Florianópolis, SC, Brazil
b School of Dentistry (M.M.C.), Pontifícia Universidade Católica do Rio Grande do Sul, Porto Alegre, RS, Brazil
c Departament of Chemistry, Universidade Federal de Santa Catarina, Florianópolis, SC, Brazil

a r t i c l e i n f o

Article history:
Received 11 September 2008
Received in revised form 7 January 2009 Accepted 27 January 2009
Available online 4 March 2009

Keywords: Nociception SB225002 CXCR2
Competitive antagonists Mice
a b s t r a c t

This study evaluated the antinociceptive effects of the selective and non-peptide CXCR2 antagonist SB225002 in mouse models of pain. As assessed in different tests of spontaneous nociception, intraperi- toneal (i.p.) administration of SB225002 caused consistent and dose-related reduction of acetic acid- induced abdominal constrictions, whereas it did not signiﬁcantly affect the nociception evoked by forma- lin, capsaicin, glutamate or phorbol ester acetate (PMA). Systemic treatment with SB225002 strikingly reduced the spontaneous nociception induced by 8-bromo-cAMP (8-Br-cAMP), or mechanical hypernoci- ception induced by prostaglandin E2 (PGE2), epinephrine, or the keratinocyte-derived chemokine (KC). In the carrageenan model, SB225002 markedly reduced mechanical hypernociception when administered by i.p., intrathecal (i.t.) or intracerebroventricular (i.c.v.) routes, or even when co-administered with car- rageenan into the mouse paw, indicating peripheral and central sites of action for SB225002. In addition,
i.p. treatment with SB225002 signiﬁcantly attenuated the increase in MPO activity or the elevation of IL- 1b, TNFa or KC levels following carrageenan injection. In the persistent models of pain evoked by com- plete Freund’s adjuvant (CFA) or by the partial ligation of the sciatic nerve (PLSN), the repeated admin- istration of SB225002 displayed prominent and long-lasting antinociceptive effects. Notably, SB225002 did not evoke unspeciﬁc central effects, as evaluated in the open-ﬁeld and rota-rod tests, or even in the latency responses for thermal stimuli. Our data conﬁrm the previous notion on the critical role
exerted by chemokines in pain, indicating that selective CXCR2 antagonists, such as SB225002, might well represent interesting and innovative alternatives for the management of both acute and chronic pain.
© 2009 European Federation of International Association for the Study of Pain Chapters. Published by
Elsevier Ltd. All rights reserved.

⦁ Introduction

Inﬂammation is the most common cause of clinical pain result- ing from tissue injury. It is well known that several biological mediators, such as neurotrophic factors, neuropeptides, prosta- noids and kinins, are all able to conduct and amplify the nocicep- tive responses (Coutaux et al., 2005). In addition, it is broadly accepted that cytokines, produced by either immune or central nervous system cells, might directly sensitize the peripheral noci- ceptors (Obreja et al., 2002). Chemokines are a group of small cyto- kines (8–10 kDa) which regulate the recruitment and migration of leucocytes from the blood stream to the inﬂamed tissue by inter- acting with G protein-coupled receptors (Baggiolini, 2001). They are classiﬁed in different families according to the number and localization of amino terminal cysteine residues. For instance, in

* Corresponding author. Tel.: +55 48 331 9491; fax: +55 48 232 9139.
E-mail addresses: [email protected], [email protected] (J.B. Calixto).
the CXC chemokine family, the cysteine residues are separated by any amino acid residue (Charo and Ransohoff, 2006). Of high interest, the last decade of research revealed that chemokines are probably implicated in several pathophysiological events, includ- ing the processing of both acute and chronic pain (Charo and Ransohoff, 2006; White et al., 2007).
Some members of the CXC chemokine family display a gluta- mate–leucine–arginine (ELR+) domain in the amino terminal por- tion of the molecule and are known especially for their ability to attract neutrophils to the sites of inﬂammation. The best known components of this subfamily are represented by CXCL8 (interleu-
kin-8, IL-8), CXCL1 (keratinocyte-derived chemokine, KC; GROa),
CXCL2 (GROb) and CXCL5 (ENA-78) (Bizzarri et al., 2006; Charo and Ransohoff, 2006), which display their effects by interaction with CXCR1 and CXCR2 receptors. Relevantly, compelling evidence demonstrates that CXCL8 (IL-8) induces marked nociceptive alterations in several animal models (Endo et al., 1994; Ahn et al., 2005; Verri et al., 2006).

The compound SB225002 (N-(2-hydroxy-4-nitrophenyl)-N0-(2- bromophenyl) urea) was identiﬁed in 1998 as the ﬁrst non-pep- tide, selective and competitive CXCR2 receptor antagonist, and it was later reported that this compound is able to inhibit IL-8 bind- ing to the CXCR2 receptor in a nanomolar range (White et al., 1998). Further studies conﬁrmed the selectivity of SB225002 for the CXCR2 receptor and demonstrated striking effects for this antagonist in certain models of neutrophil migration (Rosenkilde and Schwartz, 2004; Bizzarri et al. 2006). As neutrophils are impli- cated in a series of pathological alterations, a potential therapeutic application for CXCR2 antagonists, including SB225002, is forth- rightly expected (Reutershan et al., 2006; Chapman et al., 2007). In fact, a recent publication (Barsante et al., 2008) revealed that DF 2162, a non-competitive allosteric inhibitor of CXCR1 and CXCR2, signiﬁcantly ameliorated adjuvant-induced arthritis in rats. The present study was designed to further evaluate whether the selective blocking of CXCR2 receptors might be useful in the con- trol of painful alterations by assessing the effects of treatment with SB225002 in different models of acute and long-lasting nociception in mice. Taken in concert with the literature data, the present work brings new evidence on the therapeutic potential of CXCR2 antag- onists for the treatment of painful states.

⦁ Material and methods

⦁ Animals

Male Swiss mice (20–28 g) obtained from the Department of Pharmacology, Universidade Federal de Santa Catarina (UFSC, Flor- ianópolis, Brazil), were used in this study. Animals were housed under conditions of optimum light, temperature and humidity (12 h light-dark cycle, 22 ± 2 °C, 60–80% humidity), with food and water provided ad libitum. All procedures used in the present study followed the ‘‘Principles of Laboratory Animal Care” from NIH pub- lication Nos. 85-23 and were approved by the Animal Ethics Com- mittee of the Universidade Federal de Santa Catarina. (protocol numbers 262/CEUA and 23080.035334/2003-16/UFSC). The num- ber of animals and the intensity of noxious stimuli used were the minimum necessary to demonstrate the consistent effects.

⦁ Acute nociceptive behavior

⦁ Acetic acid-induced abdominal constrictions
Abdominal constrictions induced by the intraperitoneal (i.p.) injection of acetic acid (0.6%) were accomplished according to the procedures described previously (Vaz et al., 1996). Animals were treated with different doses of SB225002 (0.1–1 mg/kg, i.p.) or vehicle (10 ml/kg, 1% Tween 80 in 0.9% NaCl solution), 30 min prior to the injection of acetic acid. The animals were observed individually and the number of abdominal constrictions was cumulatively counted over a period of 20 min after acetic acid injection, and considered as indicative of nociception. Dipyrone (60 mg/kg, i.p., 30 min) was used as a positive control drug.

⦁ Formalin-induced nociception

The procedure used was similar to that described previously (Mendes et al., 2000). Animals were treated with SB225002 (1 or 3 mg/kg, i.p.) or with vehicle (10 ml/kg, 1% Tween 80 in 0.9% NaCl solution), 30 min before formalin injection. Subsequently, mice re- ceived a 20-ll intraplantar (i.pl.) injection of 2.5% formalin solution into the right hindpaw. The animals were placed immediately in a glass cylinder (20 cm in diameter), and the time spent licking the injected paw over a period of 30 min was considered as indicative
of nociception.
⦁ Overt nociception models

The effects of SB225002 were further evaluated in other mouse models of spontaneous nociception. For that purpose, animals were pre-treated with SB225002 (1 or 3 mg/kg, i.p.) or vehicle (10 ml/kg, 1% Tween 80 in 0.9% NaCl solution), 30 min before the algogen injection. Mice received a 20-ll i.pl. injection of one of the following agents: capsaicin (5.2 nmol/paw; Sakurada et al., 1992), glutamate (30 lmol/paw; Beirith et al., 2002), phorbol myr- istate acetate (PMA) (30 ng/paw; Taniguchi et al., 1997), or 8-Br- cAMP (10 nmol/paw; Otuki et al., 2005), into the right hindpaw. The animals were observed individually in transparent glass cylin- ders (20 cm in diameter) during a period of 5 min after capsaicin, 15 min after glutamate, 45 min after PMA, or 10 min after 8-Br- cAMP. The amount of time spent licking the injected paw was reg- istered with a chronometer and considered as indicative of nociception.

⦁ Mechanical hypernociception induced by KC, PGE2 or epinephrine

To evaluate mechanical hypernociception, mice were treated with SB225002 (1 mg/kg, i.p.) or vehicle (10 ml/kg, 1% Tween 80 in 0.9% NaCl solution), 30 min before the i.pl. injection of KC (10 ng/paw, Cunha et al., 2005), prostaglandin E2 (PGE2) (0.1 nmol/paw, Kassuya et al., 2007) or epinephrine (100 ng/paw, Khasar et al., 2005). The mechanical hypernociception was mea- sured with Von Frey ﬁlaments (VFH), as described below, at differ- ent time-points after the i.pl. injection of the algogenic mediators.

⦁ Mechanical hypernociception induced by carrageenan

For the induction of inﬂammatory pain, mice received an i.pl. injection of 50 ll of carrageenan (300 lg/paw) under the surface of the right hindpaw (Quintão et al., 2005). To assess the systemic effect of drug treatment, mice received SB225002 (0.1–3 mg/kg, i.p.) or vehicle (10 ml/kg, 1% Tween 80 in 0.9% NaCl solution), 30 min before carrageenan injection. In order to evaluate the pos- sible site of action of SB225002 (central or peripheral), a separate group of animals received an i.pl. injection of SB225002 (35– 106 lg/paw), co-administered with carrageenan (300 lg/paw). Additional groups of animals received an intrathecal (i.t.) or intra- cerebroventricular (i.c.v.) injection of 5 ll of SB225002 (3.5–35 lg/ site), 10 min before the application of carrageenan. The i.t injec- tions were performed according to the method described by Hyl- den and Wilcox (1980) with some modiﬁcations. For the i.t. injections, the animals were conscious to avoid possible anesthetic interference. The needle connected to a microsyringe by a polyeth- ylene tube was introduced through the skin, and a volume of 5 ll of vehicle solution (control) or SB225002 was injected into the L5– L6 vertebral space. For i.c.v. injections, the animals were lightly anesthetized with isoﬂurane and 5 ll of SB225002 solution was in- jected directly into the lateral ventricle (coordinates from bregma: 1 mm lateral; 1 mm rostral; 3 mm vertical) as described previously by Laursen and Belknap (1986). The mechanical hypernociception of all groups was assessed by means of VFH, for up to 24 h after carrageenan administration, as described below.

⦁ Mechanical hypernociception induced by complete Freund adjuvant (CFA)

To produce a persistent inﬂammatory response, mice received a 20-ll i.pl. injection of CFA (1 mg/ml heat-killed and dried Mycobac- terium tuberculosis; each ml of vehicle contained 0.85 ml parafﬁn oil plus 0.15 ml mannide monooleate) into the right hindpaw (Quintão et al., 2005). To observe the effects of repeated treatment, SB225002 (1 mg/kg) was administered orally twice a day

×
(12 12 h) for a period of 7 days. The evaluation of the mechanical hypernociception was assessed every day using VFH, 6 h after the ﬁrst daily administration. Control animals received the vehicle (10 ml/kg, 1% Tween 80 in 0.9% NaCl solution), under the same schedule of treatment adopted for SB225002.

⦁ Surgical procedures of partial ligation of sciatic nerve (PLSN)

×
To evaluate neuropathic pain-like behaviour, the procedure used was similar to that described by Quintão et al. (2005). Mice were anesthetized with 7% chloral hydrate (8 ml/kg; i.p.). PLSN was performed by tying 1/3–1/2 of the dorsal portion of the sciatic nerve with an 8.0 silk suture (Ethicon, Edinburgh). In sham-oper- ated control group, the sciatic nerve was exposed without ligation. Following a period of recovery (4 days after the surgical proce- dures), animals submitted to PLSN were treated systemically with SB225002 (1 mg/kg, i.p.) or vehicle (10 ml/kg, Tween 80 plus 0.9% NaCl solution), twice a day (12 12 h) for a period of 5 days after the surgery, and then evaluated with VFH for up to 6 h after the treatment.

⦁ Hindpaw withdrawal response induced by von Frey hairs

× ×
For the evaluation of mechanical allodynia, mice were placed individually in clear Plexiglas boxes (9 7 11 cm) on elevated wire mesh platforms to allow access to the ventral surface of the right hindpaw. The withdrawal response frequency was measured following 10 applications (duration of 1 s each) of von Frey hairs (VFH, Stoelting, Chicago, IL, USA). Stimuli were delivered from be- low, to the plantar surface of the right hindpaw. The animals were acclimatized for 30 min before behavioural testing and the mechanical hypernociception was evaluated at several time- points. The VFH of 0.6 g produces a mean withdrawal frequency of about 15%, which is considered to be an adequate value for the measurement of mechanical hypernociception (Quintão et al., 2005). Therefore, 0.6 g VFH was used throughout this study. In or- der to determine the basal mechanical thresholds, all the groups were evaluated before the test or surgical procedures.

⦁ Biochemical assays

⦁ Myeloperoxidase (MPO) activity
Neutrophil recruitment to the mouse paw was assessed indi- rectly by means of tissue myeloperoxidase (MPO) activity, accord- ing to the method described beforehand (Cunha et al., 2005). For this purpose, animals were treated with SB225002 (0.1–3 mg/kg,
i.p.) and 30 min after, they received a 50-ll i.pl. injection of carra- geenan (300 lg/paw) into the right paw. Saline-injected paws
were used as control. Animals were sacriﬁced 6 h after the applica- tion of carrageenan. The subcutaneous tissue of the paws was re- moved, homogenized at 5% (w/v) in EDTA/NaCl buffer (pH 4.7) and centrifuged at 10,000 rpm for 15 min at 4 °C. The pellet was resuspended in 0.5% hexadecyltrimethyl ammonium bromide buf- fer (pH 5.4), and the samples were frozen and thawed three times in liquid nitrogen. Upon thawing, the samples were recentrifuged
(10,000 rpm, 15 min, 4 °C), and 25 ll of the supernatant was used
for the MPO assay. The enzymatic reaction was assessed with
1.6 mM tetramethylbenzidine, 80 mM NaPO4, and 0.3 mM hydro- gen peroxide. The absorbance was measured at 650 nm, and the re- sults are expressed as OD per milligram of tissue.

⦁ Determination of IL-1b, TNFa or KC levels in the mouse paw

Tissue levels of the proinﬂammatory cytokines IL-1b, TNFa or KC were measured according to the protocol described by Cunha et al. (2005). The animals were treated with SB225002 (0.1–
3 mg/kg, i.p.), and after 30 min they received a 50-ll i.pl. injection of carrageenan (300 lg/paw) into the right paw. The mice were
sacriﬁced 6 h after the injection of carrageenan. Saline-injected paws were used as control. Tissues were placed in PBS (PBS; pH 7.4; NaCl 137 mM, KCl 2.7 mM, Na2HPO4 8.1 mM, KH2PO4
—
1.5 mM) containing NaCl 0.4 M, PMSF 0.1 M, EDTA 10 mM, 0.05% of Tween 20, 0.5% of BSA and 2 mg/ml of aprotinin, homogenized, centrifuged at 3000g for 10 min and stored at 70 °C until further analysis. Cytokine levels were evaluated using an ELISA kit accord- ing to the manufacturer’s recommendations (R&D Systems).

⦁ Measurement of non-speciﬁc effects

To exclude possible non-speciﬁc effects of SB225002 on motor coordination, locomotor activity, or the response latencies, mice were tested on the rota-rod, open-ﬁeld and hot-plate paradigms, respectively (Quintão et al., 2005). Different groups of animals were pre-treated with SB225002 (1 and 3 mg/kg i.p.) or vehicle (10 ml/kg, i.p.) and they were submitted to all tests.

⦁ Drugs and reagents

N-(2-hydroxy-4-nitrophenyl)-N9-(2-bromophenyl) Urea (SB22 5002) was synthesized as described before (White et al., 1998). SB225002 was identiﬁed by comparing the 1H NMR data with those published beforehand (Yield 70%, m.p. = 193–194 °C. 1H NMR (400 MHz) (ppm) (DMSO-d6) 11.05 (br. s, 1H), 9.48 (s, 1H),
9.12 (s, 1H), 8.36 (d, 1H), 7.93 (d, 1H), 7.75 (dd, 1H), 7.69 (s, 1H),
7.63 (d, 1H), 7.36 (t, 1H), 7.03 (t, 1H); IR (cm—1) 3364, 3212,
1692, 1588, 1536, 1507, 1430, 1306, 1267, 1212, 1086, 746, 649.
LC–MS-ESI (m/z) 350 (M-H)—. SB225002 was dissolved in 1% Tween 80 in 0.9% NaCl solution. Formalin and acetic acid were from Merck (Darmstadt, Germany); capsaicin was from Calbiochem (San Diego, CA); glutamate, 8-Br-cAMP, PGE2, carrageenan, PMA, epinephrine, dipyrone, Tween 80, EDTA, aprotinin, phosphate-buffered saline
and CFA were purchased from Sigma Chemical Co. (St. Louis, Mis- souri, USA). Mouse KC, KC, IL-1beta and TNF-a DuoSet kits were obtained from R&D Systems (Minneapolis, MN, USA).

⦁ Statistical analysis

The results are presented as the mean ± S.E.M. of 5–7 animals, except for the ID50 values (i.e. the dose of SB225002 that reduced the nociceptive responses by 50% relative to control values), which are presented as the means accompanied by their respective 95% conﬁdence limits. The ID50 values were determined by the use of the least squares method. The percentages of inhibition are re- ported as the mean ± S.E.M. of inhibitions obtained for each indi- vidual experiment. Statistical comparison of the data was performed by two-way analysis of variance (ANOVA) followed by Bonferroni’s post-test or one-way ANOVA followed by Newman– Keuls’ test. P-values less than 0.05 (P < 0.05 or less) were consid- ered signiﬁcant.

⦁ Results

The results obtained with SB225002 in different models of spontaneous nociception are presented in Table 1. SB225002 (0.1–1 mg/kg, i.p.) caused a signiﬁcant and dose-related reduction of acetic acid-evoked abdominal constrictions. The percentage of inhibition observed at the dose of 1 mg/kg was 94 ± 3%, with an estimated mean ID50 value (accompanied by the 95% conﬁdence limit) of 0.3 (0.13–0.63) mg/kg. However, SB225002 (1–3 mg/kg, i.p.) was not able to signiﬁcantly affect either the neurogenic (0– 5 min) or the inﬂammatory (15–30 min) phases of the formalin

Table 1
Effect of SB225002 in several mouse models of nociception.

Model Control Treatment (mg/kg, i.p., 30 min prior)
SB 225002
0.3
1
3 Dipyronea 60
Writhing testb 48 ± 5 15 ± 6* 3± 2* – 12 ± 4 s*
Formalin 1st phase 76 ± 3 s – 90 ± 8 s 76 ± 5 s –
Formalin 2nd phase 291 ± 30 s – 234 ± 27 s 240 ± 18 s –
Capsaicin 44 ± 3 s – 43 ± 3 s – –
Glutamate 170 ± 13 s – 118 ± 20 s 151 ± 16 s 50 ± 10 s*
PMA 114 ± 8 s – 134 ± 8 s 116 ± 5 s –
8-Br-cAMP 34 ± 3 s – 28 ± 2 s 24 ± 2 s* –
All the data represents the mean ± S.E.M. of 5 to 7 animals. Signiﬁcanlty differ from control values *p < 0.05.
a Dipyrone was used as a positive control drug.
b This data is presented as the number of writhes.

test. The same pretreatment with SB225002 (1–3 mg/kg, i.p.), dosed 30 min before the induction of nociception by capsaicin, glu- tamate or PMA, also did not signiﬁcantly alter the spontaneous nociception evoked by these algogenic agents (Table 1). Con- versely, the systemic administration of SB225002 (3 mg/kg, i.p.) produced a marked decrease of the nociception induced by 8-Br- cAMP (32 ± 7%, Table 1).
As illustrated in Fig. 1, the i.pl. injection of PGE2 (0.1 nmol/paw), epinephrine (100 ng/paw) or KC (10 ng/paw) into the mouse hind- paw produced prominent mechanical hypernociceptive effects that lasted for up to 24 h, as indicated by a marked increase from base- line values in response to 0.6 g VFH stimulation. The CXCR2 recep-

tor antagonist SB225002 (1 mg/kg, i.p., 30 min) was found to be effective in signiﬁcantly preventing the mechanical hypernocicep- tion caused by either PGE2 (Fig. 1A) or epinephrine (Fig. 1B). The percentages of reduction were 41 ± 2% and 55 ± 1%, respectively. The treatment with SB225002 (1 mg/kg, i.p., 30 min prior) also pro- duced a signiﬁcant reduction of the mechanical hypernocicepion evoked by KC, with an inhibition of 49 ± 3% (Fig. 1C).
The i.pl. injection of carrageenan (300 lg/paw) induced a
marked mechanical hypernociception, with a duration of 6 h, as shown by a large increase from baseline values in response to
0.6 g VFH stimulation (Fig. 2). The systemic administration of SB225002 (0.1 to 3 mg/kg, i.p.) produced a signiﬁcant and dose-

Fig. 1. Response frequency of the right hindpaw assessed in control group and in mice treated systemically with SB225002 (1 mg/kg, 30 min before) at different intervals following i.pl. injection of (A) PGE2 (0.1 nmol/paw), (B) epinephrine (100 ng/paw), or (C) KC (10 ng/paw). Signiﬁcantly different from control values *p < 0.05, **p < 0.01. Basal threshold (B), (two-way ANOVA followed by Bonfferroni’s post-hoc test).

dependent reduction of the mechanical hypernociceptive response induced by carrageenan, with a maximal inhibition of 52 ± 3% at the dose of 1 mg/kg, and an estimated mean ID50 value of 1.19 (0.58–2.42) mg/kg (Fig. 2A and B).
In an attempt to verify the peripheral or central sites of action of SB225002, this compound was dosed by different routes of admin- istration and tested in the carrageenan model of pain. Fig. 2C dem- onstrates that i.pl. co-injection of SB225002 (35–106 lg/paw) markedly reduced the mechanical hypernociception induced by carrageenan (300 lg/paw). The observed inhibition obtained at
the dose of 106 lg/paw was 46 ± 1%. Moreover, the administration
of SB225002, by either i.t. or i.c.v. pathways (3.5–35 lg/site), was
capable of reducing the mechanical hypernociception induced by carrageenan, with inhibitions of 55 ± 2% and 41 ± 1%, both at the doses of 10.6 lg/site (Fig. 2D and E).
We next investigated whether the systemic treatment with SB225002 (1 mg/kg), administered for 7 days, twice a day by i.p. route, was able to prevent the persistent mechanical hypernoci- ception following CFA application (20 ll/paw). The repeated administration of SB225002 signiﬁcantly reduced the mechanical hypernociception induced by CFA injection for up to 5 days after
the treatment with an inhibition of 76 ± 9%, (Fig. 3A). Interestingly, the prolonged treatment with SB225002 (1 mg/kg), dosed twice a day by i.p. route, was also found to be markedly effective in revert- ing the mechanical allodynia induced by PLSN, for up to 4 days after the treatment. The percentages of reduction were 60 ± 2%, 2 h after the ﬁrst dose of SB225002, and 35 ± 3% on the 3rd day of repeated treatment (Fig. 3B).
The results in Fig. 4A indicate that the increase of MPO activity induced by the i.pl. injection of carrageenan (300 lg/paw) was sig- niﬁcantly inhibited by the treatment with SB225002 (0.3 and 1 mg/kg, i.p., 30 min prior), when compared to the control group (39 ± 2% and 46 ± 3%, respectively). The i.pl. injection of carra-
geenan (300 lg/paw) signiﬁcantly increased the levels of KC, IL- 1b and TNF-a (Fig. 4B–D) in the mouse paw tissue. Systemic treat- ment with SB225002 (0.1–3 mg/kg, i.p.) markedly and dose-depen-
dently reduced the enhancement of KC levels, with a maximal inhibition of 90 ± 5%, at the dose of 1 mg/kg, i.p. (Fig. 4B). The same treatment with SB225002 (0.1–3 mg/kg, i.p.) signiﬁcantly reduced the production of IL-1b (Fig. 4C) and TNFa (Fig. 4D), in a dose- dependent manner. The observed inhibitions at the dose of 3 mg/ kg were 25 ± 1% and 58 ± 3%, for IL-1b and TNFa, respectively.

Fig. 2. Effect of SB225002 administered by (A) i.p. (0.1–3 mg/kg, 30 min prior), (C) i.pl. (35–106 lg/paw, co-administrated), (D) i.t. (1–35 lg/site, 10 min prior) or (E) i.c.v. (3.5–35 lg/site, 10 min prior) routes on mechanical hypernociception induced by i.pl. injection of carrageenan (300 lg/paw) in mice. Panel B represents areas under the curves of panel A. Signiﬁcantly different from control values *p < 0.05, **p < 0.01, (two-way ANOVA followed by Bonfferroni’s post-hoc test).

Fig. 3. (A) Response frequency of the right hindpaws injected with complete Freud’s adjuvant (CFA – 20 ll/paw) in control group and in mice treated with
SB225002 (1 mg/kg, i.p., administered twice a day) at different time intervals after the treatment with these drugs. (B) Response frequency of the right hindpaw in sham-operated and operated (PLSN) mice treated with saline (10 ml/kg) or SB225002 (1 mg/kg, i.p., administered twice a day) at different intervals of time after the treatment. Signiﬁcantly different from control values *p < 0.05, **p < 0.01, (two-way ANOVA followed by Bonfferroni’s post-hoc test).

The administration of SB225002 (1–3 mg/kg, i.p.), given 30 min before, did not signiﬁcantly affect the locomotor activity of the ani- mals in the open-ﬁeld test (Table 2). Moreover, SB225002 (1–3 mg/ kg, i.p., 30 min) did not signiﬁcantly change the motor coordina- tion of the animals in the rotarod test or the latency responses for thermal stimuli, in comparison to the control group (Table 2).

⦁ Discussion

This study evaluated the effects of the non-peptide, competitive and selective CXCR2 antagonist SB225002 in different models of nociception in mice. SB225002 signiﬁcantly diminished nocicep- tion in either acute or chronic models, corroborating the notion that chemokine receptors display a crucial role in pain.
SB225002 produced a dose-related and marked reduction of acetic acid-induced writhes. Ribeiro et al. (2000) previously dem- onstrated that acetic acid-evoked writhes are dependent on TNFa, IL-1b and IL-8 generation, from resident peritoneal macrophages and mast cells. Additionally, Duarte et al. (1988) evidenced the
chemokine involvement in the sympathetic component of this model. Present data extend previous evidence on the relevance of chemokines, and demonstrate for the ﬁrst time the role of CXCR2 in the acetic acid test.
The formalin model presents a clear biphasic proﬁle, with two distinct components: neurogenic and inﬂammatory. Chichorro et al. (2004) demonstrated that orofacial nociception caused by formalin in rats can either be increased by IL-8, or even reduced by anti-IL-8. Surprisingly, mouse treatment with SB225002 did not affect either of the phases of formalin test. Some distinctions between our study and that conducted by Chichorro et al. (2004), such as the use of different animal species and the anatomical site, might explain this discrepancy. SB225002 also failed to signiﬁ- cantly affect capsaicin- or glutamate-elicited nociception. Similar to the formalin model, nociceptive responses induced by capsaicin or glutamate represent useful tools for investigating the involve- ment of neurogenic pathways (Sakurada et al., 1992; Beirith et al., 2002). Therefore, the antinociceptive effects of SB225002 do not seem to be related to an interference with neuropeptide re- lease from sensory peripheral ﬁbres.
PKC activation is an important step for the nociception caused by numerous stimuli (Ji and Woolf, 2001; Ferreira et al., 2005). The upregulation and activation of PKCc and PKCb in the spinal cord appear to exert a key role in chronic pain (Ji and Woolf,
2001). Furthermore, the stimulation of peripheral PKCe is probably involved in the acute nociception evoked by epinephrine or TNFa, and in chronic models of pain (Dina et al., 2001; Parada et al.,
2003). SB225002 failed to affect the spontaneous nociception caused by the PKC activator PMA; thus, we might discard a possible direct effect of SB225002 on PKC stimulation. In fact, PKCe activa- tion does not seem to represent a signalling pathway related to CXCR2 (Nasser et al., 2005).
The administration of SB225002 was capable of inhibiting the licking induced by the PKA activator 8-Br-cAMP, what suggests that CXCR2 activation during pain processing is related to the PKA pathway. This premise is supported by the results demonstrat- ing that SB225002 markedly reduces the mechanical hypernoci- ception induced by PGE2 or epinephrine. Both mediators activate second messenger pathways involving cAMP and PKA, which con- sequently reduce the nociceptor threshold and increase neuronal membrane excitability (Samad et al., 2002). Thus, blocking CXCR2 by SB225002 might result in the inhibition of PKA-cAMP pathways, contributing to its antinociceptive actions.
It has been demonstrated that i.pl. injection of the CXC chemo- kine KC results in a dose- and time-dependent mechanical hypern- ociception in mice, and this hypernociceptive response is reduced by indomethacin (a non-selective COX inhibitor) or guanethidine (a sympathomimetic neuron-blocking agent) (Cunha et al., 2005). Our data revealed that mechanical hypernociception induced by KC is greatly reduced by SB225002. In this regard, we might sug- gest that prostanoids and sympathomimetic amines are implicated in the sensorial sensitization mediated by CXCR2 receptor activa- tion, in a mechanism involving the activation of PKA.
Cunha and co-workers (2008) demonstrated that systemic treatment with DF 2162, a non-competitive allosteric inhibitor of CXCR1/CXCR2 receptors, greatly prevented the hypernociception induced by KC, carrageenan, LPS or zymosan in mice. The treat- ment of mice with SB225002 produced anti-hypernociceptive ef- fects, for up to 4 h, in the carrageenan acute model of pain. Interestingly, i.t. administration of SB225002 markedly inhibited carrageenan-induced mechanical hypernociception (for up to 6 h). Also, the hypernociception caused by carrageenan was signif- icantly reduced when SB225002 was dosed by either i.pl. or i.c.v. routes. Hence, SB225002 probably controls inﬂammatory pain, by interfering with both peripheral and central nociceptive pathways, although we might not afﬁrm the potential of SB225002 in crossing

Fig. 4. Effects of SB225002 (0.1–3 mg/kg, i.p.) on the increase of: (A) MPO, (B) IL-1b, (C) TNFa, or (D) KC production induced by i.pl. injection of carrageenan (300 lg/paw) into the mice hindpaw. Signiﬁcantly different from saline group values ##p < 0.001, and signiﬁcantly different from control values *p < 0.05, **p < 0.01, (ANOVA followed by Newmann–Keuls post-hoc test).

the blood–brain-barrier (BBB). Considering that i.pl. injection of carrageenan is followed by an inﬂammatory response, accompa- nied by cell migration and release of inﬂammatory mediators (such as bradykinin and substance P), it is feasible to believe that the BBB has its function altered. It is generally accepted that neutrophils, through the release of cytotoxic enzymes and reactive oxygen spe- cies (ROS), contribute to the BBB disruption with consequent exac- erbation of inﬂammatory conditions (Koennecke et al., 1999). Of high interest, Mitchell et al. (2008) recently demonstrated that peripheral inﬂammation elicited by carrageenan injection into the rat paw results in increased MPO activity in the spinal cord,

Table 2
Test Control SB 225002 (mg/kg, i.p.)
1 3
Rota-rod Open-ﬁelda Hot plate 54 ± 4 s
68 ± 7
4 ± 1 s 58 ± 1 s
68 ± 6
3 ± 1 s 55 ± 3 s
73 ± 10
4 ± 1 s

Analysis of possible adverse effects induced by SB225002 in mice.

a This data is expressed as the number of crossed squares. All the data represents the mean ± S.E.M. of 5–7 animals.
which carry on the expression of some relevant partners of the inﬂammatory process.
Cunha et al. (2005) have suggested that release of primary mediators responsible for carrageenan-induced mechanical hypernociception is preceded by cytokine production. Likewise, neutrophil migration appears to represent a pivotal step in the events leading to carrageenan-evoked mechanical hypernocicep- tion (Cunha et al., 2008). Relevantly, the systemic treatment with SB225002 signiﬁcantly prevented the neutrophil migration and
production of TNFa, IL-1b and KC induced by carrageenan. Aug-
mented KC levels can lead to an increased production of other cytokines and inﬂammatory mediators, including KC itself; this might explain the effects of SB225002 in the production of KC in the mouse paw. The effects of SB225002 on neutrophil inﬂux to the mouse paw might also have consequences in the generation of inﬂammatory mediators at distant sites (peripheral or central), contributing to the resultant antinociception.
I.pl. injection of CFA produces an inﬂammatory response that develops within a few hours, an effect that is associated with a striking modiﬁcation in the activity of superﬁcial (I and II) and deep (V and VI) laminal dorsal horn neurons receiving noxious in- puts (Chan et al., 2000). The present results show that therapeutic treatment with SB225002 produces a prominent inhibition of the

mechanical hypernociception induced by CFA, for up to 5 days. Supporting our ﬁndings, a recent report has shown that allosteric inhibition of CXCR1/CXCR2 receptors by DF 2162 signiﬁcantly pre- vents CFA-induced arthritis in rats (Barsante et al., 2008).
Notably, the repeated treatment with SB225002, at a low dose such as 1 mg/kg, markedly reduced the neuropathic pain-like behaviour induced by PLSN. Chemokines have been implicated in the pathophysiology of neuropathic pain, by mechanisms involving changed expression of chemokine receptors, the modulation of neuronal electrical activity, and interference with other neuro- transmitters (White et al., 2007). Recent evidence has demon- strated the effectiveness of CCR2 and CXCR4 selective antagonists in reducing hypernociception in animal models of neuropathy (Bhangoo et al., 2007; White et al., 2007). However, to our knowl- edge, this is the ﬁrst study showing the ability of a selective CXCR2 antagonist to prevent the long-lasting hypernociceptive alterations induced by PLSN. Certainly, the use of CXCR2 knockout mice could be a valuable instrument to extend the experimental evidence ob- tained with SB225002. In the PLSN model, the antihypernocicep- tive effects of SB225002 might be allied to an indirect effect on the mechanisms of neuronal plasticity observed in neuropathies. There are compelling pieces of evidence indicating the relevance of neutrophil migration in pain mechanisms (Cunha et al., 2008; Saab et al., 2008). In this regard, Shaw et al. (2008) have shown that activated neutrophils can aggravate neuronal injury and cause functional changes to peripheral sensory neurons, when co-cul- tured with DRG cells. These pieces of evidence conﬁrm the notion that anti-neutrophil strategies, such as CXCR2 antagonists, might be useful for treating neuropathic pain-like states.
The antihypernociceptive actions of SB225002 do not seem to
be associated with non-speciﬁc central actions, as this drug did not signiﬁcantly inﬂuence the performance of mice on the rota- rod or the open-ﬁeld tests, and it did not interfere with the thermal latency. This is quite favourable to a possible therapeutic applica- tion of SB225002 for the treatment of long-lasting pain, where chronic treatment is required.
In this study, we show that SB225002 displayed marked antin- ociceptive effects in acute and persistent mouse models of pain. It is well known that either CXCR2 ligands or receptors are expressed by several cell types, including neutrophils, monocytes, endothelial and neuronal cells (Charo and Ransohoff, 2006). The chemokines produced by these cells, in the different experimental models eval- uated in this work, might well reach sensory nerve endings, caus- ing nociceptor sensitization. Alternatively, these chemokines might also induce the stimulation of additional pathways implicated in pain generation (White and Wilson, 2008), such as PKA-cAMP activation, or the release of prostanoids and sympat- homimetic amines. Finally, the induction of inﬂammation and/or damage at peripheral sites has been associated to the up-regula- tion of chemokines and their receptors at the central nervous (Gosselin et al., 2008). It is reasonable to propose that SB225002 could block the generation of pain, by inhibiting CXCR2 activation at any of these levels. Taken together, our ﬁndings extend the idea on the pivotal role played by chemokine receptors in painful alter- ations, and point out selective CXCR2 antagonists as promising therapeutic choices.

Acknowledgements

This work was supported by grants from the Conselho Nacional de Desenvolvimento Cientíﬁco e Tecnológico (CNPq), the Coorde- nação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), the Programa de Apoio aos Núcleos de Excelência (PRONEX) and the Fundação de Apoio à Pesquisa do Estado de Santa Catarina (FAPESC). M.M.N. is a Pharmacy & Biochemistry undergraduate
student supported by CNPq. N.L.M.Q. and P.C.L. are Ph.D. students in Pharmacology and Chemistry, respectively, and are recipients of CNPq grants.

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MiRna21

MicroRNA 21 known as hsa-mir-21 or miRNA21 is a mammalian microRNA

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