European Journal of Pharmacology 616 (2009) 91–100

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European Journal of Pharmacology

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Neuropharmacology and Analgesia

Effect of 5-trifluoromethyl-4,5-dihydro-1H-pyrazoles on chronic inflammatory painmodel in rats

Patricia D. Sauzem a, Gabriela da S. Sant'Anna a, Pablo Machado b, Marta M.M.F. Duarte d, Juliano Ferreira a,Carlos F. Mello c, Paulo Beck b, Helio G. Bonacorso b, Nilo Zanatta b, Marcos A.P. Martins b, Maribel A. Rubin a,⁎a Laboratório de Neurotoxicidade e Psicofarmacologia, Departamento de Química, Centro de Ciências Naturais e Exatas, Universidade Federal de Santa Maria, 97.105-900, Santa Maria, RS, Brazilb Núcleo de Química de Heterociclos (NUQUIMHE), Departamento de Química, Centro de Ciências Naturais e Exatas, Universidade Federal de Santa Maria, 97.105-900, Santa Maria, RS, Brazilc Departamento de Fisiologia e Farmacologia, Centro de Ciências da Saúde, Universidade Federal de Santa Maria, 97.105-900, Santa Maria, RS, Brazild Departamento de Ciências da Saúde, Universidade Luterana do Brasil, 97.020-001, Santa Maria, RS, Brazil

⁎ Corresponding author. Tel.: +55 55 3220 8053; faxE-mail address: maribel.rubin@gmail.com (M.A. Rub

0014-2999/$ – see front matter © 2009 Elsevier B.V. Adoi:10.1016/j.ejphar.2009.06.008

a b s t r a c t

a r t i c l e i n f o

Article history:Received 22 October 2008Received in revised form 27 May 2009Accepted 8 June 2009Available online 18 June 2009

Keywords:AntinociceptionPyrazole carboxiamide derivativeTactile allodyniaPaw edemaToxicity

Nonsteroidal antiinflammatory drugs (NSAIDs) are commonly used for treatment of arthritis. However, theirlong-term use has been associated with considerable morbidity, limiting their application. Thus, thereremains a need to develop new drugs for the effective and safe relief of chronic inflammatory pain. In thiscontext, the present study was designed to evaluate the antinociceptive and antiedematogenic effects of the5-trifluoromethyl-4,5-dihydro-1H-pyrazole derivatives EPFCA3 and MPFCA4 after acute (1–1000 µmol/kg)and chronic (100 µmol/kg for 15 days) administration in rats submitted to a model of adjuvant-inducedarthritis. We also analyzed some biochemical indicators of toxicity (alanine aminotransferase, aspartateaminotransferase, urea and creatinine levels) after prolonged administration of these compounds. We foundthat acute and chronic subcutaneuous administration of EPFCA3 and MPFCA4 produces an antinociceptive,but not antiedematogenic, effect on the arthritis animal model induced by complete Freund's adjuvant(CFA). No signs of toxicity were observed in the animals chronically treated with EPFCA3 or MPFCA4.Dipyrone (1–1000 µmol/kg) was used as the positive control and its effect was similar to that of the novelpyrazoles. The activity of tissue myeloperoxidase, the tissue TNF-α level and the serum haptoglobin level wasincreased by intraplantar CFA injection. However, chronic administration of EPFCA3, MPFCA4 or dipyronewas not able to alter the relation between these parameters and inflammation. Our results suggest thatEPFCA3 and MPFCA4 are good candidates for the development of new drugs for pain treatment.

© 2009 Elsevier B.V. All rights reserved.

1. Introduction

Chronic pain affects hundreds of millions of people worldwide andalters their physical and emotional functioning, decreases their qualityof life and impairs their ability towork (Goldenberg,1987; Ashburn andStaats, 1999). Osteoarthritis and rheumatoid arthritis comprise twocommon forms of inflammatory joint disease, and the primary featurewhich patients exhibit in the clinic is chronic pain (Wilson et al., 2006).

Nonsteroidal antiinflammatory drugs (NSAIDs) are commonly usedfor the treatment of arthritis. However, their long-term use has beenassociated with considerable morbidity in the form of dyspepsia,gastrointestinal hemorrhage, renal dysfunction, aggravation of hyper-tension, and precipitation of heart failure (Narsinghani and Chaturvedi,2005). Since the currently available therapies for chronic inflammatorydisorders fail to adequately alleviate pain in many patients, and sideeffects of the treatments often limit their use, there remains a need todevelop new drugs for the effective and safe relief of chronicinflammatory pain (Wilson et al., 2006).

: +55 55 3220 8978.in).

ll rights reserved.

Pyrazole compounds and their derivatives are widely known fortheir excellent effectiveness as analgesics and antipyretics (Williamset al., 1999). Moreover, some data have shown that the ulcerogenicactivity of dipyrone, a pyrazole derivative, in rats and humans issubstantially lower than the risk associatedwith other NSAIDs, such asacetylsalicylic acid and diclofenac, commonly used for pain relief(Andrade et al., 1998; Sánchez et al., 2002). These findings suggestbetter tolerability of dipyrone in relation to other NSAIDs.

In this context, we have synthesized new pyrazole derivatives andreported their antinociceptive and antipyretic effects in animal modelsof inflammation, fever and pain (Souza et al., 2001; Souza et al. 2002;Godoy et al., 2004; Tomazetti et al., 2005; Tabarelli et al., 2004; Prokoppet al., 2006; Sauzem et al., 2007;Milano et al., 2008). The compounds 3-ethyl-5-hydroxy-5-trifluoromethyl-4,5-dihydro-1H-1-carboxyamide-pyrazole (EPFCA3) and 4-methyl-5-hydroxy-5-trifluoromethyl-4,5-dihydro-1H-1-carboxyamidepyrazole (MPFCA4) belong to a series often pyrazole derivatives recently synthesized and screened for anti-nociceptive and antiedematogenic activity in mice. These compoundscause antinociception in neurogenic and inflammatory pain induced byformalin, and antiedematogenic action on a carragenin model afteracute administration (Sauzem et al., 2007). However, the action on

Fig. 1. Chemical structure of EPFCA3 and MPFCA4 pyrazole derivatives.

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chronic models of pain and the toxicity of these compounds have notbeen evaluated to date.

This study was designed to determine whether the 5-trifluoro-methyl-4,5-dihydro-1H-pyrazole derivatives EPFCA3 and MPFCA4(Fig. 1) exhibit antinociceptive and antiinflammatory properties afteracute and chronic administration in rats submitted to a model ofadjuvant-induced arthritis. We also analyzed some biochemicalparameters indicative of toxicity after prolonged administration ofthese compounds. Finally, we compared the action of the newpyrazolecompounds with that of dipyrone.

2. Materials and methods

2.1. Animals

All experiments were performed using three-month-old maleWistar rats weighing 180–220 g. Animals were housed in groups of 6per cage, at a controlled temperature (22±1 °C), on a 12 h light/12 hdark cycle and with standard lab chow and water ad libitum. Theanimals were acclimated to the experimental room for at least 2 hbefore the experiments. All experiments were performed in accor-dance with the ethical guidelines established for investigations ofexperimental pain in conscious animals (Zimmermann, 1983) andwere approved by the Committee on the Use and Care of LaboratoryAnimals of our university (no. 23081.018371/2006-94).

2.2. Drugs

The pyrazole derivatives EPFCA3 and MPFCA4 were synthesized aspreviously described (Sauzem et al., 2007), and correspond, respec-tively, to compounds 2c and 2j in this previous paper. All other drugsand reagents were purchased from local suppliers. EPCA3, MPFCA4and dipyrone were suspended in an aqueous vehicle containingTween 80 (5% v/v) and polyethylene glycol 400 (20% v/v). In allexperiments, pyrazole derivatives were injected subcutaneously inthe interscapular area (1 ml/kg).

2.3. Induction of inflammation

Inflammation induced by complete Freund's adjuvant (CFA) waselicited as described by Wilson et al. (2006). Rats were briefly anes-thetized with halothane (for 2–3 min) and 150 µl of the completeFreund's adjuvant (CFA, 1 mg/ml of heat killedMycobacterium tubercu-losis, from SIGMA) was subcutaneously injected into the ventral surfaceof the right hind paw (intraplantar injection).

2.4. Measurement of tactile allodynia

Rats were placed in cages with a wire mesh bottomwhich allowedfull access to the paws. Behavioral habituation was allowed forapproximately 15–30 min, until cage exploration and major groomingactivities ceased. The area tested was the mid-plantar right hind paw.The paw was touched with 1 of a series of 7 von Frey hairs withlogarithmic increments (0.8, 1.5, 3.5, 6.0, 7.5, 10.0 and 15.0 g). The von

Frey hairs were applied perpendicular to the plantar surface withsufficient force to cause slight buckling against the paw, and held forapproximately 6–8 s. Stimuli were presented at intervals of severalseconds, allowing for apparent resolution of any behavioral responsesto previous stimuli. Ambulation was considered an ambiguousresponse, and in such cases the stimulus was repeated (Chaplanet al., 1994). The 50% withdrawal threshold was determined using theup-and-down method of Dixon (1980). In this paradigm, testing wasinitiated with the 10.0 g hair. Stimuli were always presentedconsecutively, whether ascending or descending. In the absence of apaw withdrawal response to the initially selected hair, a strongerstimulus was presented. In the event of paw withdrawal, the nextweaker stimulus was chosen. A total of 6 responses were registeredand the 50% response threshold was calculated using the equation:

50%g threshold = 10½Xf + kδ�

where Xf = value of the final von Frey hair used (in log units); k =tabular value for the pattern of positive/negative responses (Dixon,1980); and δ=meandifference (in log units) between stimuli (0.2122).

2.5. Measurement of paw edema

Edema formation was quantified by changes in paw volumemeasured before and after CFA injection (at the time indicated in eachexperimental protocol). The paw volume evaluation was made by im-mersing the injected paw into a cuvette filled with a solution of 2.5%Extran inwater (v/v). The cuvettewasfixed on the plate of an electronicscale (precision of 0.01 g), and the careful immersion of the paw intocuvette solution, without touching the cuvette, was accompanied by anincrease in the weight displayed. The weight in grams is related to theincrease in the liquid column in the cuvette, but not to the mass of thepaw. The cuvette solution densitywas 1 mg/ml, and the value displayedby the scale was assumed to represent the paw volume (Daher et al.,2005). The paw edema was calculated using the following equation:

%edema = ½ðVfinal−VinitialÞ= V initial� × 100

where V initial is the volume of the paw before CFA or saline injectionand V final is the volume of the paw after CFA or saline injection.

2.6. Open field test

The effect of the subcutaneous injection of the novel pyrazoles onspontaneous locomotor activity of the rats was assessed in the openfield test, as previously reported by Guerra et al. (2006). This test wascarried out to identify motor disabilities, which could inhibit thepawwithdrawal response of the animals when stimulated by von Freyfilaments. The apparatus was a box measuring 56×40×30 cm withthe floor divided into 12 equal areas. The animals treated at the doseof 1000 µmol/kg or vehicles were subjected to the open field test,immediately after allodynia measurement. In this test, the number ofcrossings of the rats in a 5-minute period was registered.

2.7. Measurement of body weight

In the procedure for the chronic administration of the drugs, bodyweight was monitored for the whole treatment time. The percentchange in corporal weight was calculated from the basal weight (beforeCFA or saline paw injection) and was considered as an indicator of toxicor side effects of the drugs (Yu et al., 2006).

2.8. Gastric lesion assessment

Ulcerogenic activity was evaluated after chronic administration ofEPFCA3, MPFCA4 or dipyrone (100 µmol/kg, one dose daily for 15 days;

Fig. 2. Effect of acute administration (1000 µmol/kg; s.c.) of the novel pyrazolederivatives EPFCA3 (A), MPFCA4 (B) and dipyrone (C) on CFA-induced mechanicalallodynia in rats. BL represents the threshold of the mechanical response of the animalsbefore intraplantar CFA or saline injection. Time 0 corresponds to the thresholdimmediately before subcutaneous treatment with the pyrazole compounds (48–72 hafter intraplantar CFA or saline injection). Data are reported as mean±S.E.M.; n=9–14.⁎Pb0.05 compared to the CFA/vehicle (SNK test).

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controls received vehicle) in the rats that received CFA intraplantarinjection. Six hours before sacrifice, an acute dose of indomethacin(30 mg/kg) was administered to a separate group of rats that was usedas the positive control for ulcer presence. Food, but not water, wasremoved 18 h before euthanasia. The evaluationwasmade24 h after thelast dose of the drugs. The animals were euthanizedwith an overdose ofpentobarbital and the stomach was extracted, opened along the smallcurvature to assess the lesion index and the number of ulcers. The lesionindex of each animal was calculated by adding the following valuesobtained in the observation of mucosa, considering the lesion degreeproduced: light (1 point), moderate (2 points) or intense (3 points),evaluating the discoloration of mucosa, the presence of petechialhemorrhages and mucus loss. The number of ulcers was determinedby direct count of small lesions (up to 1 mm). When larger, the lesionswere quantified considering 1.5 points per mm. The observations weremade under a microscope (Djahanguiri, 1969; Ghedini et al., 2002).

2.9. Biochemical analyses

2.9.1. Sample collectionTwenty-four hours after the cessation of the chronic treatment, the

animals were killed by i.p. injection of sodium pentobarbital. Bloodsamples were collected from the abdominal aorta in serum separatortubes (for aspartate aminotransferase, alanine aminotransferase, urea,creatinine and haptoglobin analysis) or in tubes with EDTA (forhematological analyses). The skin of the hind paws was removedand immediately frozen for later analysis of themyeloperoxidase (MPO),N-acetyl-β-D-glucosaminidase activity (NAG), eosinophil peroxidase(EPO) and tumor necrosis factor-alpha level (TNF-α). A gross examina-tion was made of the abdominal organs, which were removed andweighed.

2.9.2. Hematological analysisThe number of erythrocytes, leukocytes, lymphocytes, monocytes,

and platelets and the concentration of hemoglobin and hematocritweremeasured in order to identifyanypossible blooddyscrasias. Quantitativedeterminationswere carriedoutonwhole blood collected in EDTA tubesby use of standard methods with the fully automated PENTRA 120®(ABX Diagnostics, Montpellier, France).

2.9.3. Hepatic and renal function analysisPlasma alanine aminotransferase (ALT) and aspartate aminotrans-

ferase (AST) activities and plasma urea and creatinine levels weremeasured as indicators of hepatic and renal injury, respectively. PlasmaAST and ALT activities were measured using standard enzymaticmethodswith theuseofOrtho-Clinical Diagnostics— Johnson& Johnsonreagents, with a fully automated analyzer (Vitros 950, dry chemistry;Johnson & Johnson, Rochester, NY, USA).

2.9.4. Haptoglobin analysisHaptoglobin, an acute phase protein, the level of which increases

during inflammation (Giffen et al., 2003a,b), was measured by nephe-lometric immunoassay using Dade Behring kits (BN II System AnalyzerDade Behring, Germany).

2.9.5. Myeloperoxidase (MPO), N-acetyl-β-D-glucosaminidase (NAG)and eosinophil peroxidase (EPO) assay in the rat paw

The MPO activity was assayed according to the method of Suzukiet al. (1983), with some modifications. Briefly, the skin paw sampleswere defrosted and homogenized with 20 volumes of 80 mM sodiumacetate buffer (pH 5.4) plus 0.5% hexadecyltrimethylammoniumbromide (HTAB), centrifuged (11,200×g, 4 °C, 20 min) and the super-natants were collected. For assay, 10 µl of supernatant and 220 µl of80 mM sodium acetate buffer (pH 5.4) containing 15% of 0.3 mM H2O2

were added in triplicate to a 96-well plate. The reactionwas initiated bythe addition of 20 µl of 18.4 mMtetramethylbenzidine. Themixturewas

incubated for 3 min at 37 °C and then immersed in an ice bath. Thereaction was stopped by the addition of 30 µl of acetic acid and theabsorbance was monitored at a wavelength 630 nm.

For the NAG and EPO assays, samples were prepared as previouslydescribed for MPO samples. In the NAG assay, the reaction mixturecomprised 10 µl of supernatant, 100 µl of 50 sodium citrate buffer(pH 4.5) and 25 µl of 4-nitrophenyl N-acetyl-β-D-glucosaminide. Thereaction mixture was incubated for 1 h at 37 °C and was stopped with100 µl of 200 mM glycine buffer pH 10.4. The enzyme activity wasevaluated colorimetrically as absorbance at 405 nm. In the measure-ment of EPO activity, 100 µl of substrate solution consisted of 0.1 mMof o-phenylenediamine in 0.05 M Tris–HCl buffer (pH 8.0) containing0.1% Triton X-100 and 1 mM H2O2 was added to 100 µl of supernatant

Fig. 3. Effect of acute administration (1000 µmol/kg; s.c.) of the novel pyrazole derivativesEPFCA3 (A) and MPFCA4 (B) or dipyrone (C) on CFA-induced paw edema in rats. BLcorresponds to pawvolume of the animals before intraplantar CFAor saline injection. Time0 corresponds to paw volume immediately before subcutaneous treatment with thepyrazole compounds (48–72 h after intraplantar CFAor saline injection). Data are reportedas mean±S.E.M.; n=7–11.

Table 1Effect of the 5-trifluoromethyl-4,5-dihydro-1H-pyrazoles EPFCA3, MPFCA4 anddipyrone on spontaneous locomotor activity of rats.

Treatment Crossings

Vehicle 31.1±3.8EPFCA3 1000 µmol/kg 30.1±8.8MPFCA4 1000 µmol/kg 33.6±6.0Dipyrone 1000 µmol/kg 30.0±5.1

Data are reported as mean±S.E.M. for n=8–16 animals per group.

Fig. 4. Dose-response effect of acute administration of the pyrazole derivatives EPFCA3(A) and MPFCA4 (B) or dipyrone (C) on CFA-induced mechanical allodynia in rats. Dataare reported as mean±S.E.M.; n=7–11. ⁎Pb0.05 compared to the CFA/vehicle group(SNK test).

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(Lloret andMoreno,1995; Kang et al., 2008). The reactionmixturewasincubated for 30 min at 37 °C, and then the reaction was stopped bythe addition of 50 µl of 4 M H2SO4. The enzyme activity was evaluatedcolorimetrically as absorbance at 490 nm.

2.9.6. Measurement of TNF-α level in the rat pawTNF-α production in the rat paw was assessed using an ELISA kit,

as described by the manufacturer (DuoSet ELISA DevelopmentSystem, rat TNF-α/TNFSF1A, R&D Systems). Briefly, rats that receivedEPFCA3, MPFCA4, dipyrone or vehicle for 15 days after intraplantarCFA injection were sacrificed 24 h after last administration of thedrugs. The subcutaneous tissue of the paws was defrosted and placedin an 80 mMPBS (pH 7.4) solution containing: 0.5 % Tween 20, 0.1 mMPMSF, 2 mM EDTA, 0.01 µg/ml antitrypsin, 0.1% bacitracin and 0.1%BSA. The tissue was homogenized, centrifuged at 16,000×g for 10 minand the supernatant obtained was stored at −20 °C until furtheranalysis (Souza et al., 2000; Campos et al., 2002).

2.10. Experimental design

2.10.1. Time-course study after acute administration of the novelpyrazole compounds and dipyrone

The basal threshold for mechanical stimulus and paw volume wasevaluated immediately before CFAor saline administration into the righthind paw. On day 2–3 post-CFA or saline, the 50% response thresholdand the paw volume were again analyzed to verify tactile allodyniadevelopmentandpawedema, respectively. This timewasestablishedonthebasis of pilot experiments and consistedof thenecessary timeso that90% of the animals presented significant allodynia and edema in theCFA-injected paw. The CFA-injected animals that presented allodynia

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received subcutaneously in the interscapular area: vehicle (distilledwater, polyethylene glycol 400 and polysorbate 80; 75:20:5), EPFCA3(1000 µmol/kg),MPFCA4 (1000 µmol/kg) or dipyrone (1000 µmol/kg;internal standard). Separate groups of the animals received saline inthe pawandwere also treatedwith vehicle and evaluated together withthe other groups. The response threshold and the paw edema weremeasured from 30 min to 24 h after treatment.

2.10.2. Dose-response study after acute administration of the novelpyrazole compounds and dipyrone

The procedures for allodynia induction were identical to thosedescribed above. The CFA-injected animals that developed allodyniawere injected with vehicle, EPFCA3 (1–1000 µmol/kg, s.c.), MPFCA4(1–1000 µmol/kg, s.c.) or dipyrone (1–1000 µmol/kg, s.c.) and theresponse threshold was evaluated after 1 h.

2.10.3. Effect of chronic administration of the novel pyrazole compoundsand dipyrone on pharmacological and biochemical parameters

Before CFA or saline intraplantar injection, the baseline threshold formechanical stimulus, the volume of both paws was assessed. Approxi-

Fig. 5. Effect of chronic administration (100 µmol/kg; s.c.; daily for 15 days) of the pyrazolinduced ipsilateral mechanical allodynia in rats. A, B and C show the thresholds before dailrepresents the threshold of mechanical response of the animals before intraplantar CFA or sCFA/vehicle group (SNK test).

mately 2–3 days after CFA or saline administration, these parameterswere again evaluated and the animal treatment with vehicle, EPFCA3,MPFCA4ordipyronebegan.All drugswere administered subcutaneouslyin the interscapular area at a dose of 100 µmol/kg and dipyrone wasincluded as the internal standard. The animals received one injectiondaily for 15 days. On days 1, 2, 3, 6, 9, 12 and 15, the 50% responsethreshold for the von Frey hairs test and the volume of both paws wereevaluated before and 1 h after treatments with EPFCA3, MPFCA4,dipyrone or vehicle (day 1 corresponds to the first day of the treatment,2–3 days after CFA or saline intraplantar injection). The measurementstaken before the daily injection of the drugs were used to verify thecumulative effect of the treatment and those taken 1 h after treatmentswere used to identify the development of tolerance. Twenty and fourhours after the end of the pharmacological treatment, the animals weresacrificed under deep thiopental anesthesia and blood and tissuesamples were collected for biochemical analyses.

2.10.4. Statistical analysisThe results were expressed as mean±S.E.M. The ED50 values were

reported as geometric means accompanied by their respective 95%

e derivatives EPFCA3 (A and D) and MPFCA4 (B and E) or dipyrone (C and F) on CFA-y injection of the drugs and D, E and F show the responses 1 h after administration. BLaline injection. Data are reported as mean±S.E.M.; n=7–9. ⁎Pb0.05 compared to the

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confidence limits. Data were analyzed by one-way analysis of variance(ANOVA) and post hoc tests (Student–Newman–Keuls test—SNK)were carried out when appropriate. The level of significancewas set atPb0.05.

3. Results

3.1. Time-course study after acute administration of EPFCA3, MPFCA4and dipyrone on mechanical allodynia and paw edema

Prior to induction of inflammation by CFA, the mean thresholdvalue was around 15.0 g. By post induction day 2–3, the threshold fellto around one forth of this value, characteristic of the development ofallodynia (Fig. 2). For comparison, the mean value for saline-injected(intraplantar) rats remained at around 15.0 g (Fig. 2). The subcuta-neous treatment of the animals with EPFCA3, MPFCA4 or dipyroneproduced an increase in the response threshold of the von Frey hairstest in the CFA-injected (intraplantar) animals (Fig. 2A–C). Theresponse onset occurred 30 min after injection of the drugs and hada long duration (12 h) for all treatments. The compound MPFCA4 hadreversed the allodynia by 85.6±12.2 % (Fig. 2B) 4 h after administra-tion. The compound EPFCA3 presented similar effectiveness (70.8±8.1 %, Fig. 2A), but its effect occurred earlier than that of MPFCA4. The

Fig. 6. Effect of chronic administration (100 µmol/kg; s.c.; daily for 15 days) of the pyrazolinduced ipsilateral paw edema in rats. A, B and C show the paw volume before daily injectionthe threshold of mechanical response of the animals before intraplantar CFA or saline injec

effectiveness of the internal standard dipyrone did not differ from thatof EPFCA3 and MPFCA4, however, its maximum effect was delayed,occurring 6h after injection (72.7±9.5 %, Fig. 2C).

In the same group of animals the paw volume was evaluated inorder to verify the development of edema after intraplantar CFA orsaline injection. It was found that CFA, but not saline, caused anincrease in the paw volume (around 100 %), characteristic of thedevelopment of edema. However, the treatments of the animals withEPFCA3, MPFCA4 or dipyrone, 48–72 h after CFA injection, had noeffect on paw edema induced by CFA (Fig. 3). The animals thatreceived saline intraplantar injection and were treated with vehiclepresented no alterations in the response threshold or paw edema(Figs. 2 and 3).

The animals were evaluated in the open field test 1 h afterreceiving subcutaneous treatments. The number of crossings was notmodified by EPFCA3, MPFCA4 or dipyrone treatment compared withthe animals that had received vehicle (Table 1).

3.2. Dose-response study after acute administration of EPFCA3, MPFCA4or dipyrone

In the Fig. 4 the effect of EPFCA3, MPFCA4 and dipyrone (1–1000 µmol/kg) on CFA-induced allodynia is shown.Doses of 10 µmol/kg

e derivatives EPFCA3 (A and D) and MPFCA4 (B and E) or dipyrone (C and F) on CFA-of the drugs and D, E and F show the paw volume 1 h after administration. BL representstion. Data are reported as mean±S.E.M.; n=5–6.

Fig. 7. Effect of chronic administration (100 µmol/kg; s.c.; daily for 15 days) of thepyrazole derivatives EPFCA3, MPFCA4 and dipyrone on the serum haptoglobin level(A) and TNF-α level (B) in rat paw tissue. Data are reported as mean±S.E.M.; n=3–5.⁎Pb0.05 compared to the saline/vehicle group (SNK test).

Table 2Effect of chronic administration of the novel pyrazole derivatives EPFCA3 and MPFCA4and of dipyrone on gastric mucosa of rats.

Lesion index Number of ulcers N

Saline/vehicle 0.75±0.25 0 4CFA/vehicle 0.71±0.18 0 7CFA/EPFCA3 1.00±0.26 0.67±0.67 6CFA/MPFCA4 1.00±0.36 0 6CFA/dipyrone 1.40±0.25 0.40±0.40 5Salinea 0.67±0.33 0 4Indomethacina 6.25±0.83⁎ 32.00±7.82⁎ 3

N is the number of animals per group. Data were analyzed by ANOVA (post hoc SNK test).a Single dose.⁎ Pb0.05 compared with saline group.

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of EPFCA3, MPFCA4 and dipyrone caused anti-allodynic effect in theanimals as demonstrate by an increase in the threshold response to vonFrey hairs after drug treatment. All drugs had similar antinociceptivepotency, with ED50 values of 9.9 (0.9–114.2), 11.3 (1.9–65.9) and 10.8(2.6–44.0) µmol/kg for EPFCA3, MPFCA4 and dipyrone, respectively.However, EPFCA3 was more efficacious than MPFCA4, reverting com-pletely the allodynia at a dose of 1000 µmol/kg (Fig. 4A and B).

3.3. Effect of chronic administration of EPFCA3, MPFCA4 or dipyrone ontactile allodynia, edema and biochemical parameters

3.3.1. Tactile allodyniaComplete Freund's adjuvant injection produced a profound and

long-lasting decrease in the threshold response to von Frey hairs inthe ipsilateral paw. Fig. 5A–C show the cumulative effect of chronictreatment with EPFCA3, MPFCA4 and dipyrone on CFA-induced tactileallodynia. The day 1 value corresponds to the threshold immediatelybefore treatment, and the values for days 2, 3, 6, 9, 12 and 15 representthe thresholds 24 h after drug administration onwards. It is possible tonote, in Fig. 5A–C, that from the third day of treatment, all drugsalready had a stable anti-allodynic effect. EPFCA3 and MPFCA4(100 µmol/kg) showed antinociceptive action from day 3 up to thelast day of observation, while dipyrone (100 µmol/kg) produced aneffect from day 2 of the treatment.

In Fig. 5D–F the threshold responses of the animals 1 h aftertreatment at days 1, 2, 3, 6, 9, 12 and 15 are shown. These observationswere performed in order to identify the possible development oftolerance. The results obtained demonstrate no decrease in effective-ness of the drugs during the observation period.

The animals injected with saline (intraplantar) and treated withEPFCA3, MPFCA4 and dipyrone did not show different thresholdvalues compared with the vehicle-treated rats (data not shown).

Furthermore, we did not detect allodynia in the contralateral pawduring the15 days of evaluationof the threshold response tomechanicalstimulus of the rats that received CFA or intraplantar saline injection(data not shown).

3.3.2. Paw edemaThe injection of CFA into the right hind paw of the rats produced an

increase in the ipsilateral paw volume when compared to the animalsthat received intraplantar saline injection (Fig. 6). The treatments ofthe rats with EPFCA3, MPFCA4 or dipyrone were not able to decreasethe CFA-induced edema (Fig. 6). The contralateral paw of the animalsdid not show an increase in the volume (data not shown).

3.3.3. Weight of whole body and some internal organsThe body weight was evaluated before CFA administration and

from the beginning of the treatment with EPFCA3, MPFCA4, dipyroneor vehicle (supplemental Table S1). It was observed that the animalshad a reduction inweight gain only on days 1 and 2 of the experiment(3–4 days after CFA). This effect was not observed in the rats thatreceived intraplantar saline injection.

Twenty-four hours after the last injection of drugs, the animals weresacrificed and the liver, spleen, right kidney and stomachwere removedand weighed. The organ/body weight ratio (in percentage) wascalculated, and is shown in supplemental Table S2. The treatment withEPFCA3, MPFCA4 or dipyrone did not alter organ/body weight ratiowhencomparedwithvehicle-treatedanimals. Likewise, the intraplantarCFA injection did not modify this parameter (supplemental Table S2).

3.3.4. Gastric lesion assessmentChronic administration of EPFCA3, MPFCA4 or dipyrone (100 µmol/

kg daily for 15 days) did not induce significant gastric lesions as shownin Table 2. On the other hand, indomethacin (30 mg/kg/single dose),used as the internal standard, produced severe damage to the stomachmucosa as demonstrated by a high lesion index (Table 2).

3.3.5. HaemogramThe haemogram demonstrated changes in the leukocyte levels for

the rats injected with CFA (intraplantar) and chronically treated withEPFCA3, MPFCA4 or dipyrone (100 µmol/kg daily for 15 days), but notfor those injected with vehicle. The number of erythrocytes andplatelets, and the hemoglobin and hematocrit concentrations in theblood samples of the animals that received CFA (intraplantar injection)were not modified (supplemental Table S3).

3.3.6. AST, ALT, urea and creatinine levelsThe AST, ALT, urea and creatinine levels were assayed in blood

samples of the animals injected with CFA (intraplantar) andchronically treated with EPFCA3, MPFCA4, dipyrone or vehicle, 24 hafter the last injection. The serum levels of the hepatic injury

Table 3Effect of chronic administration of the novel pyrazole derivatives EPFCA3 and MPFCA4and of dipyrone (100 µmol/kg) on the MPO, NAG and EPO levels of paw tissue of ratssubmitted to chronic inflammation induced by intraplantar CFA injection.

MPOa NAGb EPOc

(absorbance/mg protein)

(absorbance/mg protein)

(absorbance/mg protein)

Saline/vehicle 0.02±0.03 0.14±0.13 0.14±0.10CFA/vehicle 0.40±0.21⁎ 0.14±0.08 0.42±0.24CFA/EPFCA3 0.61±0.24⁎ 0.16±0.12 0.45±0.17CFA/MPFCA4 0.43±0.13⁎ 0.15±0.08 0.51±0.34CFA/dipyrone 0.23±0.05⁎ 0.11±0.04 0.32±0.20

Data are reported as mean±S.E.M. Data were analyzed by ANOVA (post hoc SNK test).a Myeloperoxidase.b N-acetyl-β-D-glucosaminidase.c Eosinophil peroxidase.⁎ Pb0.05 compared with saline/vehicle group.

98 P.D. Sauzem et al. / European Journal of Pharmacology 616 (2009) 91–100

indicators (AST and ALT) and renal injury indicators (urea andcreatinine) were not modified by chronic treatment with the novelpyrazoles or dypirone when compared with vehicle-treated animals(supplemental Table S4).

3.3.7. Haptoglobin levelsThe serum haptoglobin level was measured at the end of the

experiment (18th day after CFA injection) in blood samples of rats thatreceived chronic treatment with EPFCA3, MPFCA4 or dipyrone. Noneof the tested compounds reversed CFA-induced increase of plasmahaptoglobin levels (Fig. 7A).

3.3.8. MPO, NAG and EPO activityThe MPO activity increased in the rat paw tissue after intraplantar

CFA administration when compared to animals that receivedintraplantar saline (Table 3). However, chronic administration of thenovel pirazole derivatives EPFCA3 and MPFCA4 or dipyrone did notaffect the CFA-induced increase in MPO activity. On the other hand,NAG and EPO activities were not affected by CFA injection (Table 3).

3.3.9. Tissue level of TNF-α in the rat pawThe CFA injection into the rat paw produced an increase in TNF-α

levels when compared to animals that received intraplantar injectionof saline. This increase was not reverted or attenuated by dailyadministration, for 15 days, of EPFCA3, MPFCA4 or dipyrone (Fig. 7B).

4. Discussion

In the present study, we evaluated the antinociceptive andantiedematogenic effect of the acute and chronic administration oftwo novel pyrazole derivatives, EPFCA3 and MPFCA4, against mechan-ical allodynia and paw edema induced by the intraplantar injection ofCFA. Additionally, some toxicity parameters were evaluated anddipyrone was included as an internal standard, in order to compare itseffect with those of the novel pyrazole derivatives. As previouslyreported (Chaplanet al.,1994;Wilsonet al., 2006), intraplantar injectionof CFA produced intense and long-lasting mechanical allodynia andedema in the injected paw (Figs. 2–6). The contralateral paw was notaffected, suggesting that inflammation was restricted to the CFAinjection site. The acute systemic treatment with EPFCA3, MPFCA4 ordipyrone produced rapid (30 min after treatment) and long-lastingantinociception (up to 12 h after treatment) for both novel pyrazoles(Fig. 2). The latency for maximum antinociceptive effect differedbetween drugs. Analysis of time–effect curves revealed that while themaximum antinociceptive effect of EPFCA3 occurred 1 h after admin-istration, the maximal effect of MPFCA4 took 4h to develop. The long-lasting antinociception induced by the novel fluorine-substitutedpyrazole compounds is in agreement with reports that indicate thatthe carbon―fluorine bond is stable, probably resulting in metabolically

stable compounds (Parck et al., 2001; Karthikeyan et al., 2007). In thisregard, it has been suggested thatfluorine substitutionmakesmoleculesmore resistant to cytochrome P450 (Parck et al., 2001), but additionalstudies must be carried out to elucidate the pharmacokinetics of thesenovel pyrazolines and their elimination routes. Regardless the mechan-ism, the long-lasting action of EPFCA3 and MPFCA4 is an interestingpharmacological characteristic, because it allows long dose intervals, aclear advantage in the clinics.

It is important to point out that while the pyrazole EPFCA3completely reverted the mechanical allodynia at a dose of 1000 µmol/kg (Fig. 4A), MPFCA4 and dipyrone only partially reduced CFA-inducedtactile hypersensitivity (Fig. 4B and C, respectively). These results revealthat EPFCA3 is more efficacious than MPFCA4 and dipyrone.

The paw edema induced by CFAwas not reverted by acute or chronictreatment with EPFCA3, MPFCA4 or dipyrone (Figs. 3 and 6). Previousstudies have demonstrated that dipyrone prevents the development ofedema induced by agents such as endotoxin (Fracasso et al., 1996),carrageenan (Brune and Alpermann, 1983) and CFA (Weithmann andAlpermann, 1985; Tatsuo et al., 1994). However, previous studies dem-onstrating an antiedematogenic effect for dipyrone in a CFA model differfrom ours in that they used different procedures of induction andevaluation of the inflammation. Tatsuo et al. (1994) induced inflamma-tion using a subcutaneous injection of CFA in the dorsal root of the rat tailandmeasured the pawedema 14 days after CFA injection. In thismodel, asystemic reaction induced by CFA occurs, whereby injection in the tailproduces inflammation in the paw of the animal. In this study, CFA wasinjected into the right hind paw and the inflammation was restricted tothe injection site (ipsilateral paw), since no allodynia or edema in thecontralateral paw were detected. Such differences in the site of CFAinjection can produce different degrees of inflammation and differentsensitivity to thepharmacological treatment.WeithmannandAlpermann(1985) used CFA fromMycobacterium butyricum and the experimentwascarried out with Lewis rats, in contrast to our study in which CFA fromMycobacterium tuberculosis and Wistar rats were used. In fact there is awide variation in the incidence and severity of CFA-induced arthritis,depending on the rat and Mycobacterium strains used (Cai et al., 2006;van Eden et al., 1994; Swingle et al., 1969; Banik et al., 2002). Therefore,the lack of antiedematogenic action of dipyrone here described seems tobe related to the experimental procedure adopted. Such a view is alsosupported by the findings that EPFCA3 and MPFCA4 did not decreaseinflammation in the CFA model (Figs. 3 and 6), while previous resultshave indicated a possible antiinflammatory action for these pyrazolederivatives in the carrageenan model (Sauzem et al., 2007). A furtherpossible explanation is the use of different experimental designs. In theprevious study, the compounds were administered before intraplantarcarrageenan injection, therefore preventing the development of inflam-mation. In the present study, the compounds were administered afterinflammation has developed, a proceduremore similar towhat occurs inmedical treatments.

The chronic administration of the pyrazole derivatives EPFCA3,MPFCA4 and dipyrone decreased CFA-induced allodynia (Fig. 5).Moreover, EPFCA3 andMPFCA4, did not seem to cause tolerance, sincetheir antinociceptive effect maintained upon repeated administration(one daily dose for 15 days). In this experiment, the animals showed asignificant increase in the threshold to tactile stimulus (Fig. 5A–C)after two doses of the novel compounds, suggesting some degree ofpain relief at early stages of the pharmacological treatment. However,pharmacological treatment did not abolish CFA-induced allodynia. Itis possible that increasing doses and/or decreasing dose intervalsmay produce a better response, and this will be the subject of futurestudies.

We also evaluated whether pharmacological treatment altered thelevels of haptoglobin, an acute phase protein that increases duringinflammation, which has been regarded an adequate marker of theprogression of the disease (Giffen et al., 2003a,b). It was found that theintraplantar CFA injection increased plasma haptoglobin levels, but

99P.D. Sauzem et al. / European Journal of Pharmacology 616 (2009) 91–100

the treatment of the animals with EPFCA3, MPFCA4 or dipyrone, for15 days, did not alter haptoglobin levels, suggesting that thesecompounds did not alter the course of the inflammation. These resultsare in agreement with the findings from paw edema experiments, inwhich the novel pyrazole derivatives and dipyrone were ineffective.

The signs and symptoms of inflammation include, besides pain andedema, cellmigration (Marchand et al., 2005). The type of immune cellthat contributes to inflammatory pain depends on the inflammatorycondition but, in general, various cell types are recruited andcontribute to abnormal pain sensitivity, albeit to different degrees(Marchand et al., 2005). In this regard, the activities of MPO, NAG andEPO, in samples of paw tissue, were analyzed as indicators of therecruitment of neutrophils, macrophages and eosinophils at theinjection site, respectively (Suzuki et al., 1983; Lloret and Moreno,1995; Kang et al., 2008). Only the MPO activity was increased at18 days after CFA injection, suggesting accumulation of neutrophils inthe inflamed paw (Table 3). The treatments for 15 days with EPFCA3,MPFCA4 or dipyrone did not reduce theMPO activity, possibly becausethey did not alter the recruitment of immune cells at the lesion site.However, it is important to point out that, to date, the time course ofimmune cell recruitment has not been described in the literature forpaw inflammation induced by CFA. Levy et al. (2006) have shown thatthe number of total leukocytes increased considerably 28 days afterinduction ofmonoarthritis in the knee joint by CFA injection, while thenumber of macrophages was only modestly increased at theinflammation site. If the infiltration of immune cells into the CFA-injected paw occurs similarly to the case demonstrated for mono-arthritis in the knee joint, it is possible that our experimental designdid not detect the increase in macrophage number or activity.Activated macrophages have been reported to contribute to experi-mental pain states. They can release many inflammatory mediators,including TNF-α, a pro-inflammatorycytokine (Marchand et al., 2005).In this study, we verified that the TNF-α level increased in inflamedpaw tissue for rats that received intraplantar CFA injection (Fig. 7B).Moreover, the chronic treatment of the animals with EPFCA3, MPFCA4or dipyrone was ineffective in reducing the TNF-α level. These data,together with the lack of effect in the other biochemical parametersevaluated and the lack of effect of EPFCA3 andMPFCA4 on paw edema,support that these drugs do not have antiinflammatory activity in theCFA model. Therefore, it is possible that EPFCA3 and MPFCA4 causeantinociception by mechanisms other than inflammation decrease. Infact, MPCA, a pyrazole very similar to EPCA3 and MPFCA4, does nothave antiinflammatory effect (Souza et al., 2001) and seems to causeantinociception by serotoninergic and noradrenergic mechanisms(Godoy et al., 2004). Opiod mechanisms also have been described forstructurally similar pyrazoles (Milano et al., 2008), but the involve-ment of such mechanisms in the antinociceptive effect of EPCA3 andMPFCA4 needs to be investigated to clarify this point.

Previous studies have shown that intraplantar CFA injectiondecreases weight gain and produces signs of polyarthritis throughoutthe experiment (Walz et al., 1971; Franch et al., 1994; Nagakura et al.,2003; Yu et al., 2006). In this study, body weight was measured dailythroughout the treatment and a significant reduction in weight gainwas verified only in the first days after CFA intraplantar injection. Onthe other hand, no signs of polyarthritis development were detected.It is possible that these discrepancies in relation to data previouslypublished by other researchers are related to different experimentaldesigns, and the rat strains and adjuvant used.

The plasma AST and ALT activities, and urea and creatinine levelswere analyzed in order to identify possible hepatic and renal lesionsdue to chronic treatment with EPFCA3 or MPFCA4, a critical point forthe development of novel analgesic or antiinflammatory drugs. Noneof the treatments altered these parameters, suggesting that EPFCA3and MPFCA4 are not nephro- or hepatotoxic in our test conditions.

The effect of chronic administration of EPFCA3, MPFCA4 on hema-tological parameters was determined to detect possible effects on

hematopoiesis. Only a significant increase in the number of leukocytesdue to intraplantar injection of CFA was observed, when compared toanimals that received intraplantar saline (supplemental Table S3).EPFCA3, MPFCA4 and dipyrone had no effect on the number ofleukocytes of animals that received intraplantar saline (data notshown). Thus, leukocytosis seems occur due to CFA-induced inflamma-tion, and a direct effect of pyrazole drugs on hematopoiesis soundsunlikely. In this context, the currently reported increase in thenumberofleukocytes due to CFA injection is in agreement with previous reports(Mikolajew et al., 1969; Walz et al., 1971). One important side effectassociated with the classical NSAIDs is development of ulcers, gastro-intestinal bleeding perforation of the gastrointestinal mucosa, since asignificant percentage of those experiencing such effects are at riskof death (Vane et al., 1998). In this study, we showed that chronicadministration of EPFCA3 and MPFCA4, at effective antinociceptivedoses, does not lesion the stomach mucosa (Table 2).

In conclusion, in this study we show the antinociceptive action ofEPFCA3 and MPFCA4 against the chronic inflammatory pain inducedby CFA in rats. The antinociceptive effect occurred in the absence ofadverse effects, indicating that these compounds may be interestingfor the development of new drugs for chronicpain management.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi: 10.1016/j.ejphar.2009.06.008.

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