Antinociceptive, Anti-Inflammatory and Antipyretic Properties of the Aqueous Extract of Bauhinia purpurea Leaves in Experimental Animals

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LFS-13815; No of Pages 8

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Antinociceptive, anti-inflammatory and antipyretic effects of1.5-diphenyl-1H-Pyrazole-3-carbohydrazide, a newheterocyclic pyrazole derivative

David do Carmo Malvar a,d,⁎, Raquel Teixeira Ferreira a, Raphael Andrade de Castro a, Ligia Lins de Castro a,Antonio Carlos Carreira Freitas b, Elson Alves Costa c, Iziara Ferreira Florentino c, João Carlos Martins Mafra b,Glória Emília Petto de Souza d, Frederico Argollo Vanderlinde a

a Universidade Federal Rural do Rio de Janeiro, Instituto de Biologia, Departamento de Ciências Fisiológicas, Seropédica, RJ, Brazilb Universidade Federal do Rio de Janeiro, Núcleo de Pesquisas de Produtos Naturais, Cidade Universitária, Rio de Janeiro, RJ, Brazilc Universidade Federal de Goiás, Instituto de Ciências Biológicas, Departamento de Ciências Fisiológicas, Goiânia, GO, Brazild Faculdade de Ciências Farmacêuticas de Ribeirão Preto, USP, Departamento de Física e Química, Ribeirão Preto, SP, Brazil

⁎ Corresponding author at: Universidade Federal RuraBiologia, Departamento de Ciências Fisiológicas, Br 465,30. 23.890-000, Seropédica, RJ, Brazil. Tel./fax: +55 21 26

E-mail address: [email protected] (D.C. Malvar).

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Please cite this article as: Malvar DC, etcarbohydrazide, a new heterocyclic pyrazole

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Received 15 September 2013Accepted 4 December 2013Available online xxxx

Keywords:1.5-Diphenyl-1H-Pyrazole-3-carbohydrazideAnti-inflammatory activityAntinociceptive activityAntipyretic activityCyclooxygenasePGE2TNF-α

Aims: Heterocyclic pyrazole derivative has been described for the treatment of pain and inflammatory diseases.This study evaluated the in vivo, antinociceptive, anti-inflammatory and antipyretic effects of 1.5-diphenyl-1H-Pyrazole-3-carbohydrazide (1.5-DHP) and the in vivo or in vitro mechanism of action.Mainmethods:Acetic acid-inducedwrithing, hot-plate and formalin-induced nociception testswere used to eval-uate the antinociceptive effect, while the rota-rod test was used to assess the motor activity. Croton oil-inducedear edema and carrageenan-induced peritonitis tests were used to investigate the anti-inflammatory effect of1.5-DHP. The antipyretic effect was assessed using the LPS-induced fever model. The mechanism of action wasevaluated by PGE2 and TNF-α measurement and cyclooxygenase inhibition assay.Key findings: Oral administration (p.o.) of 1.5-DHP (1, 3, 10 mg/kg) caused a dose-related inhibition of the aceticacid-inducedwrithing, however the highest dosewasnot effective on the hot-plate and rota-rod. In the formalin-induced nociception, 1.5-DHP (10 mg/kg, p.o.) inhibited only the late phase of nociception. This samedose of 1.5-DHP also reduced the croton oil-induced ear edema. 1.5-DHP (3, 10, 30 mg/kg, p.o.) produced a dose-related

reduction of leukocyte migration on the carrageenan-induced peritonitis. 1.5-DHP (60 mg/kg, p.o.) reducedthe fever and the increase of PGE2 concentration in the cerebrospinal fluid induced by LPS. 1.5-DHP inhibitedboth COXs in vitro. Finally, 1.5-DHP (10 mg/kg, p.o.) reduced the TNF-α concentration in peritoneal exudatesafter carrageenan injection.Significance: These results indicate that 1.5-DHP produces anti-inflammatory, antinociceptive and antipyreticeffects by PGE2 synthesis reduction through COX-1/COX-2 inhibition and by TNF-α synthesis/release inhibition.

© 2013 Elsevier Inc. All rights reserved.

Introduction

Pyrazole compounds are synthetic molecules that have, in their mo-lecular structure, a pyrazoline ring, which is a five-membered heterocy-cle with two adjacent nitrogen and three carbon atoms (Borne, 1995;Gursoy and Demirayak, 2000). The discovery of this class of drugsallowed the development of several agents widely used in many coun-tries (Rahman and Siddiqui, 2010) such as analgesics, antipyreticsand/or anti-inflammatory drugs, which include dipyrone, antipyrine,aminopyrine and phenylbutazone (Brogden, 1986; Rainsford, 2007).

l do Rio de Janeiro, Instituto deKm 7/Pavilhão de Química/sala823222.

ghts reserved.

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Molecular modifications of pyrazole compounds can lead to variousdrug prototypes with a wide range of pharmacological activity, such asantipyretic, analgesic, anti-inflammatory, soothing, muscle relaxant,anti-epileptic, anti-depressant, antimicrobial and antihypertensiveactivities (Rahman and Siddiqui, 2010). Therefore, the development ofnew pyrazole derivatives aims to maintain the desired effects of theold pyrazole derivatives, such as antipyrine or dipyrone, but with lesstoxicity (Borne, 1995).

These compounds have some structural analogy as they areacylhydrazone pyrazole and N-phenylpyrazole derivatives (arylamines,arylhydrazones and thioaryl). Among these, DuP 697, a diaryl heterocy-cle, which has a key structural feature described as critical to its analge-sic and anti-inflammatory activity, has been proposed as a prototypeinhibitor of COX-2 (Gans et al., 1990; Pinto et al., 1996). This featureresides on its 1.2-diaryl substitution in the pyrazole ring and gives rise

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to a tricyclic structural system that is present in the structure ofcelecoxib (SC58125), a selective COX-2 inhibitor drug (Prasanna et al.,2005).

Moreover, some heterocyclic pyrazoles have also been shown toinhibit the synthesis of pro-inflammatory cytokines, such as TNF-α(Keche et al., 2012; Townes et al., 2004). There is not enough data inthe literature to establish a definitive structure-relationship betweenheterocyclic pyrazole and its effect on TNF-α synthesis inhibition.Townes et al. (2004) showed that a 2-methoxypropyl amine substitu-tion at the 2-position of the pyrimidine ring of bicyclic heterocyclicpyrazole resulted in a modest improvement in potency while sec-butylamine substitution demonstrated a pronounced improvementin potency against TNF-α formation as compared to the other 2-substituted pyrimidyl bicyclic pyrazole analogs.

Based on this background, we synthesized a new heterocyclicpyrazole derivative 1.5-diphenyl-1H-Pyrazole-3-carbohydrazide(1.5-DHP) following the synthetic pathway shown in Fig. 1. Theantinociceptive, anti-inflammatory and antipyretic effects of1.5-DHP were evaluated in this work. Also, the involvement of PGE2and TNF-α synthesis inhibition in the 1.5-DHP mechanism of actionwas investigated.

Material and methods

Animals

Adult male Swiss mice (25–35 g) and maleWistar rats (180–200 g)were housed in plastic cages, with food and tap water available adlibitum in the colony room at 24 ± 1 °C under a 12:12 h light–darkcycle (lights on at 06:00 AM). Mice were acclimatized in the laboratoryfor at least 60 min prior to any test procedures and left without food for12 h before the gavages. Rats were acclimatized in the experimentalroom for at least 15 h prior to the test procedures. All experimentswere carried out in accordance with current guidelines for the care oflaboratory animals and the ethical guidelines on the use of animals inpain research (Zimmermann, 1986; National Research Council, 1996).Experimental protocols were approved by the local Animal Care andUse Committee (011/2007/CEPEB/UFRuralRJ) and the minimum num-ber of animals and duration of observation required to obtain consistentdata were employed.

Fig. 1. Steps involved in synthesis of the compound 1.5-diphenyl-1H-Pyrazole-3-carbohydrazide (1.5-DHP).

Please cite this article as: Malvar DC, et al, Antinociceptive, anti-inflcarbohydrazide, a new heterocyclic pyrazole derivative, Life Sci (2013), h

Drugs and reagents

The following drugswere used: Acetic acid and formalin fromMerckAG (Darmstadt, Germany), croton oil, indomethacin, carrageenan andLPS (Escherichia coli 0111:B4) from Sigma Chemical Co. (St. Louis, MO,USA), fentanyl from Janssen Pharmaceutical (Ewing, NJ, USA), dexa-methasone from Prodome (Campinas, SP, Brazil) and diazepam fromCristália (São Paulo, SP, Brazil). 1.5-DHP was dissolved in the DMSOsolution (3% in water), while the other drugs were diluted in saline.The drugswere dissolved to such a concentration as to allow the admin-istration of constant volumes of 10 mL/kg for each dose given to mice.For rats, the drugswere dissolved at a concentration to allow the admin-istration of constant volumes of 0.5 mL/rat, in accordancewith the aver-age weight of the animals. Control animals received similar volumes ofthe vehicle only. Oral (p.o.) and subcutaneous (s.c.) pre-treatmentswere always done 60 and 30 min, respectively, before injection of theinflammatory, pyrogenic or nociceptive stimuli.

Acetic acid-induced writhing

Groups of six mice were treated with vehicle (3% DMSO in water;10 mL/kg), 1.5-DHP (1 to 10 mg/kg) or indomethacin (10 mg/kg) orally60 min before acetic acid injection (1.2%, 0.1 mL/10 g). The number oftimes the mice writhed during the following 30 min was counted(Koster et al., 1959).

Hot-plate

The latency (seconds) of heat stimulus (55.0 ± 0.5 °C) was mea-sured every 30 min, starting 30 min before and for up to 2.5 h aftertreatment of the mice (n = 8) with 1.5-DHP (10 mg/kg, p.o.), fentanyl(200 μg/kg, s.c.) or vehicle (3% DMSO in water, p.o.). The animalswhose basal flick responses were longer than 9 s were discharged anda cut-off time of 30 s was maintained throughout the experiment, toprevent tissue damage (D'amour and Smith, 1941).

Rota-rod test

Mice (n = 8) were treated with vehicle (3% DMSO in water, p.o.),1.5-DHP (10 mg/kg, p.o.) or diazepam (3 mg/kg, s.c.). After 60 min,the animals were placed on the rota-rod apparatus (12 rpm), whichwas an anti-slip plastic rod located 28 cm over the base. Results areexpressed as the number of falls and the time (seconds) in which ani-mals remained on the rota-rod over a period of 1 min (Dunham andMiya, 1957).

Formalin-induced nociception

Groups of 6–10micewere treatedwith vehicle (3% DMSO inwater),1.5-DHP (10 mg/kg) or indomethacin (10 mg/kg) orally 60 min prior tothe intraplantar injection of formalin solution (3%; 20 μL/paw) into thehind paw (i.pl. injection). The time that animals spent licking the forma-lin stimulated pawwas measured with a chronometer and was consid-ered as an index of pain. The initial nociceptive response peaked atabout 5 min (early phase) and was followed by a second peak (latephase) that occurred at 15–30 min post injection (Hunskaar et al.,1986; Hunskaar and Hole, 1987).

Croton oil-induced ear edema

One hour after oral administration of vehicle (3% DMSO in water),1.5-DHP (10 mg/kg) or 30 min after dexamethasone (2 mg/kg, s.c.) in-jection, each animal (n = 8)was treatedwith 20 μL of freshly preparedcroton oil (2.5% in acetone) on the inner surface of the right ear. The leftear was treated with the same volume of acetone (control). Four hoursafter treatments, mice were killed by cervical dislocation and a plug

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(6 mm in diameter) was taken from both the inflamed and control earswith a punch. The inflammatory response (edema) was recorded byweighing (mg) both plugs and calculating the difference (Δ) as de-scribed by Tubaro et al. (1986) and Zanini et al. (1992).

Carrageenan-induced peritonitis

Groups of mice (n = 10) were treated with a vehicle (3% DMSO inwater) or 1.5-DHP (3 to 30 mg/kg) orally 60 min prior to an injectionof carrageenan (1% in saline solution; 250 μL/mouse) into the peritonealcavity. The positive control group was pre-treated (30 min) with dexa-methasone (2 mg/kg, s.c.). The animals were anesthetized with etherand sacrificed 4 h later. The peritoneal cavities were washed with2 mL of PBS-heparin (10 UI/mL) and the peritoneal exudates were col-lected and then diluted (Türk solution 1:20). The leukocytes werecounted in a Neubaüer chamber and the results were expressed ascells × 106/mL or percentage of inhibition of leukocyte migration com-pared to control groups as described by Ferrándiz and Alcaraz (1991).

LPS-induced fever

Groups of 6–11 rats were treated orally with 1.5-DHP (30–60 mg/kg,p.o.) or vehicle (3% DMSO in water) 30 min before intravenous (i.v.)injection of LPS (5 μg/kg) or sterile saline (0.2 mL, i.v.). The LPS dosewas selected based on previous studies (de Souza et al., 2002;Kanashiro et al., 2008). The rectal temperature (Tr)wasmeasured in con-scious and unrestrained rats every 30 min for 6 h by gently inserting avaseline-coated thermistor probe (model 402 coupled to a model 46telethermometer, Yellow Springs Instruments, Ohio, USA) 4 cm into therectum,without removing them from their cages. The experimentalmea-surements were conducted in a room with a controlled temperature of27 ± 1 °C, the thermoneutral zone for rats (Gordon, 1990). Baseline tem-peratures were determined 3–4 times and at 30 min intervals prior toany injection treatment (and always before 10:00 am). Only animalsdisplaying mean basal rectal temperatures between 36.8 and 37.2 °Cwere selected for the study. The rectal temperatures were expressed asthe changes from the mean basal value (ΔTr). Mean baseline tempera-tures did not differ significantly among the groups included in the exper-iments. In order to minimize core temperature changes due to handling,animals were habituated to this environment and procedure twice onthe preceding day (Malvar et al., 2011).

Determination of CSF PGE2 concentration

A single CSF samplewas collected from each animal according to themethod described by Consiglio and Lucion Consiglio and Lucion (2000).Briefly, just prior to CSF collection, each rat was anesthetized asdescribed before and fixed to a stereotaxic apparatus, with its bodyflexed downward. The top and back of the head were trichotomizedand moistened with a cotton swab soaked in ethanol to facilitate thevisualization of the small depression between the occipital protuber-ance and the atlas. A 25-gauge needle connected to a 1 mL syringewas then inserted vertically and centrally through this depression intothe cisterna magna and a gentle aspiration caused the CSF to flowthrough it, resulting in 50 to 100 mL samples. Gentle movements ofthe needle are necessary during collection in order to prevent bleeding.The collected CSF samples were placed in Eppendorf tubes containingindomethacin (10 mM) to prevent prostaglandin production, ex vivo.Samples were maintained in the dark and on ice until centrifugationat 1300 g for 15 min at 4 °C, and the supernatants were immediatelyfrozen at −70 °C until analysis. Samples contaminated with bloodwere discarded.

The CSF PGE2 levels of rats (n = 6) treated orally with 1.5-DHP(60 mg/kg) or vehicle (3% DMSO in water) were measured 30 minbefore i.v. injection of LPS or sterile saline using ELISA kits fromCayman Chemical (Ann Arbor, MI, USA) following the manufacturer's

Please cite this article as: Malvar DC, et al, Antinociceptive, anti-inflcarbohydrazide, a new heterocyclic pyrazole derivative, Life Sci (2013), h

instructions with a detection limit of 7.8 pg/mL. Cross-reactivity datawere as follows: 17.5% with PGE3, 11.9% with PGE1, 7% with PGF1α, 6%with PGF2α, 2.5% with 6-oxo-PGF1α and less than 0.1% with all otherprostanoids tested. Intra- and inter-assay coefficients of variation wereb11%. All samples were assayed according to the manufacturer'sinstructions.

In-vitro cyclooxygenase (COX) inhibition assay

The inhibitory effect of 1-5-DHP on ovine COX-1/COX-2 enzymaticactivity was determined using a colorimetric COX (ovine) inhibitorscreening assay kit (Cayman Chemical, Catalogue No. 760111) accord-ing to the protocol recommended by the supplier (Dussossoy et al.,2011). The range of 1-5-DHP concentrations used in this experimentwas from 3.9 to 250 μg/mL.

Determination of TNF-α concentration in peritoneal exudate

In another experimental session, groups of animals (n = 8) weretreated (p.o.) with 1.5-DHP (10 mg/kg) or vehicle (3% DMSO in water)60 min prior to carrageenan injection (1% in saline; 500 μL/mouse)into the peritoneal cavity. Four hours after carrageenan administration,the peritoneal exudate was collected with 1 mL of heparinized PBS.TNF-α concentration in peritoneal exudates were evaluated using animmunosorbent assay kit (ELISA) (Ebioscience) as described previouslyby Nicoletti et al. (2010). Results were expressed as means ± SEM ofTNF-α concentration (pg/mL).

Statistical analysis

Results were expressed as means ± standard error of the mean(SEM). Differences between two means were determined using theStudent t test. Differences between more than 2 means were calculatedusing one-way analysis followed by Tukey test. In the experimental setsfor thermal antinociceptive and antipyretic evaluation, the changes inthe latency of heat stimulus and in the rectal temperature werecompared across treatments and timepoints by two-way ANOVA for re-peated measurements followed by the Bonferroni test. All data were an-alyzed using Prism computer software (Graph-Pad, SanDiego, CA, USA).Differences were considered significant when p b 0.05.

Results

Antinociceptive effect of 1.5-DHP

The antinociceptive effect of 1.5-DHP was assessed using the aceticacid-induced writhing model. Fig. 2 shows that the pre-treatmentwith 1.5-DHP (1, 3 and 10 mg/kg, p.o.) produced a dose-related inhibi-tion of acetic acid-induced writhing (40.5, 62.1 and 76% respectively)compared to the control group (37.5 ± 4.5 writhings). The inhibitorydose 50% (ID50) calculated was 1.68 mg/kg. As expected, the pre-treatment with indomethacin also decreased the numbers of writhingsinduced by acetic acid (11.7 ± 3.6 writhings).

In the hot-plate test, the basal latency of the control group was9.4 ± 1.6 s and the 1.5-DHP (10 mg/kg, p.o.) did not alter the nocicep-tive behavior latency of the animals even at a dose level high enough toinduce maximal response in the acetic acid test. The positive controlfentanyl increased the latency of heat stimulus by 3.1-fold (30 minand 60 min), 2.1-fold (90 min) and 1.6-fold (120 min) when comparedto the control group (data not show).

In the formalin-induced nociception, pre-treatment with 1.5-DHP(10 mg/kg, p.o.) did not modify the nociceptive behavior during theearly phase (neurogenic pain) of nociceptive response (154.5 ± 10 s).However, 1.5-DHP significantly inhibited by 41.1% the late phase(inflammatory pain) of nociception (145.9 ± 14.6 s) compared to thecontrol group (142.4 ± 15.2 s and 247.4 ± 32.9 s, early and late

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Fig. 2. Antinociceptive effect of 1.5-DHP on acetic acid-inducedwrithing. Vehicle, 1.5-DHP(1, 3 and 10 mg/kg) or positive control indomethacin (2 mg/kg) were orally administrated60 min before intraperitoneal injection of acetic acid injection (1.2%, 0.1 mL/10 g). Thenumber of writhings was counted for 30 min after acetic acid injection. The columns andvertical bars represent the mean ± SEM of 6–10 mice. Significantly different from controlgroup: **p b 0.01; *** p b 0.001 compared with control group.

Fig. 3. Antinociceptive effect of 1.5-DHP on early phase (A) and the late phase (B) offormalin-induced nociception. Vehicle, 1.5-DHP (10 mg/kg) or positive control indometh-acin (2 mg/kg) were orally administrated 60 min before intraplantar injection of formalin(3%; 20 μL/paw). Reactivity, represented by the time that animals spent licking the forma-lin injected paw, was evaluated during the early phase (0–5 min) and the late phase(15–30 min) after formalin injection. The columns and vertical bars represent themean ± SEM of 8 mice. Significantly different from control group: **p b 0.01;*** p b 0.001 compared with control group.

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phases respectively). As expected, indomethacin only reduced thereactivity by 62.8% (92 ± 36.5 s) during the late phase (Fig. 3).

In the rota-rod test, mice of the vehicle group remained on the rota-rod apparatus for 53.9 ± 2.6 s, with 1.6 ± 0.5 falls in a period of 1 min.This performance was not affected by the administration of 1.5-DHP(10 mg/kg, p.o.), when the animals remained on the rota-rod for54.8 ± 1.5 s with 1.9 ± 0.3 falls, while diazepam (3 mg/kg, s.c.)pretreatment changed significantly the motor response of the animals,decreasing the time on the rota-rod by 39.1% (32.8 ± 6.1 s) andincreasing the falls (7.2 ± 2.1).

Anti-inflammatory effect of 1.5-DHP

To assess the antiedematogenic effect of the 1.5-DHP, the croton oil-induced mice ear edema method was used. The difference of right andleft ear weight (Δ) obtained from the control group (treated with vehi-cle) was 8.5 ± 1.0 mg. Pre-treatment with 1.5-DHP (10 mg/kg, p.o.) ordexamethasone (2 mg/kg, s.c.) inhibited the edematogenic responseafter topical application of croton oil by 55.3% (Δ = 3.8 ± 0.7 mg)and 54.1% (Δ = 3.9 ± 0.8 mg), respectively (Fig. 4).

Using carrageenan as a stimulus, it was possible to produce an acuteinflammatory response after 4 h in the peritoneal cavity of mice, with alarge number of leukocytes in the exudates. Compared to the vehicle-treated animals (13.6 ± 0.2 leukocytes × 106/mL), the 1.5-DHP-treatedanimals (3, 10 or 30 mg/kg) exhibited a dose related reduction of leuko-cyte migration by 11.8, 39 and 54.4% respectively (Fig. 5). The positivecontrol dexamethasone (2 mg/kg, s.c.) also effectively reduced the leuko-cyte migration by 72.8%.

Antipyretic effect of 1.5-DHP

The antipyretic effect of 1.5-DHP was investigated on the LPS-induced fever model. Under our experimental conditions, the i.v. injec-tion of LPS (5 μg/kg) elicited a marked increase of rectal temperaturethat started 2 h after the injection and persisted up to 6 h in rats.Pre-treatment with 1.5-DHP (30–60 mg/kg, p.o.) produced a dose-dependent reduction of the LPS-induced fever. At the highest dose,1.5-DHP reduced the rectal temperature by 47.5% on average from 2to 6 h after i.v. injection of LPS when compared to the vehicle/LPSgroup, whereas it did notmodify the basal rectal temperature of controlrats (Fig. 6).

Please cite this article as: Malvar DC, et al, Antinociceptive, anti-inflcarbohydrazide, a new heterocyclic pyrazole derivative, Life Sci (2013), h

Evaluation of antinociceptive, anti-inflammatory and antipyreticmechanismof 1.5-DHP

In order tomeasure the PGE2 content in cisternal CSF of rats, sampleswere collected 3 h after LPS. PGE2 levels in the CSF of control animalstreated with vehicle and saline were below the detection limit ofthe assay. As expected, the LPS injection (5 μg/kg, i.v.) inducedfever (ΔTr: 1.5 ± 0.1 °C) and increased the CSF PGE2 concentration(769.9 ± 60.8 pg/mL). Pre-treatment with 1.5-DHP (60 μg/kg, p.o.) re-duced the increase of rectal temperature by 51.1% (ΔTr: 0.7 ± 0.1 °C)and the CSF PGE2 concentration by 31.9% (523.9 ± 71.8 pg/mL) 3 hafter LPS injection. However 1.5-DHP did not change these parametersin rats injected with saline (ΔTr: 0.01 ± 0.02 °C; PGE2 concentrationwas not detectable) (Fig. 7).

Corroborating the results above, the in-vitro COX inhibition assayshowed that 1.5-DHP inhibited both COX-1 and COX-2 (Table 1). Theinhibitory concentrations 50% (IC50) calculated for COX-1 and COX-2inhibition were 20.8 μg/mL and 50.0 μg/mL, respectively, and the selec-tivity index (SI; COX-1 IC50/COX-2 IC50) was 0.42.

Finally, we investigated the effect of 1.5-DHP on TNF-α concentrationafter intra-peritoneal injection of carrageenan in mice. Pre-treatment

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Fig. 4. Antiedematogenic effect of 1.5-DHP on croton oil-induced ear edema. Vehicle or1.5-DHP were orally administrated 60 min, while the positive control dexamethasone(2 mg/kg, s.c.) was administrated 30 min before application of croton oil (2.5% in acetone)or acetone on the inner surface of the right or left ear, respectively. Ear edema, representedby the difference (Δ) between right and left ear weight, was evaluated 4 h after croton oilor acetone administration. The columns and vertical bars represent the mean ± SEM of 8mice. Significantly different from control group: **p b 0.01 compared with control group.

Fig. 6. Antipyretic effect of 1.5-DHP on LPS-induced fever. Rats received oral administra-tion of 1.5-DHP (30 and 60 mg/kg) or vehicle 30 min prior to LPS (5 μg/kg, i.v.) or sterilesaline (0.2 mL, i.v.—control) injection. Values represent the means ± SEM of the changesin rectal temperatures (ΔTr, °C) of 6–11 rats per group. *, p b 0.05 compared to the groupstreated with vehicle/LPS. Basal rectal temperatures of each group were as follows:○ = 36.96 ± 0.07; Δ = 37.00 ± 0.04; ● = 36.99 ± 0.05; ■ = 36.95 ± 0.04;▲ = 37.00 ± 0.05.

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with 1.5-DHP (10 mg/kg, p.o.) reduced the TNF-α concentration by 27.6%(6.2 pg/mL) in peritoneal exudates 4 h after carrageenan injection (1% insaline; 500 μL/mouse) into the peritoneal cavity when compared to thegroup treated with the vehicle (8.6 pg/mL) (Fig. 8).

Discussion

The antinociceptive effect of 1.5-DHP was tested in three analgesiamodels: acetic acid-induced abdominal writhing, hot-plate test and

Fig. 5. Effect of 1.5-DHP on the number of leukocytes migrated to peritoneal cavity withcarrageenan-induced peritonitis. Vehicle or 1.5-DHP (3, 10 and 30 mg/kg) were orallyadministrated 60 min,while positive control dexamethasone (2 mg/kg, s.c.) was adminis-trated 30 min before intraperitoneal injection of carrageenan (1% in saline solution;250 μL/mouse). The leukocytes (cells × 106/mL) were counted 4 h after carrageenaninjection. The columns and vertical bars represent the mean ± SEM of 6–10 mice. Signif-icantly different from control group: **p b 0.01; *** p b 0.001 compared with controlgroup.

Please cite this article as: Malvar DC, et al, Antinociceptive, anti-inflcarbohydrazide, a new heterocyclic pyrazole derivative, Life Sci (2013), h

formalin-induced nociception. The writhing induced by acetic acid inmice is a result of a chemically induced acute peripheral inflammatoryreaction (Zakaria et al., 2006a). The treatment with 1.5-DHP promoteda dose-related antinociceptive effect in the acetic acid-inducedwrithing. This test is normally used for screening synthetic and naturalcompounds to investigate central and/or peripheral antinociceptiveactivity, because it is sensitive to nonsteroidal anti-inflammatorydrugs (NSAIDs) and opioids (Zakaria et al., 2006b; Fischer et al., 2008).The nociception induced in this model has been associated withprostanoids, such as PGE2, PGF2α and PGI2 as well as lipoxygenase prod-ucts (Parveen et al., 2007; Ballou et al., 2000).

The hot plate test is a good model to investigate the central effect ofanalgesic drugs and has selectivity for opioid-like drugs. This assay isthought to involve the supraspinal reflexes (Oliveira Fde et al., 2008)and produces an acute non-inflammatory nociception (Zakaria et al.,2006a, 2006b; Fischer et al., 2008). 1.5-DHP did not affect the nocicep-tive behavior latency time from the thermal stimulus, suggesting that1.5-DHP has no central action.

In the formalin-induced nociception, the early phase (neurogenicpain) is short-lived and commences immediately after injection, beingcharacterized by the release of mediators such as kinins, histamine, se-rotonin, substance P and CGRP, and can be suppressed by opioid-likedrugs, such as morphine (Oliveira Fde et al., 2008; Parada et al., 2001;Goncalves et al., 2008). On the other hand, the late phase (inflammatorypain) is a longer and more persistent period of pain mainly caused bythe local formation of inflammatory mediators such as cytokines(TNF-α and IL-1β), prostaglandins (PGE2), glutamate, kinins and nitricoxide (Chichorro et al., 2004; Cunha et al., 2005; Tassorelli et al., 2006)and has been reported to be sensitive to the action of the majority ofNSAIDs, including acetylsalicylic acid, indomethacin and naproxen(Goncalves et al., 2008). 1.5-DHP reduced the nociceptive behavior dur-ing the late phase but not the early phase in the formalin-inducednociception, suggesting that 1.5-DHP produces antinociception byinterfering in the inflammatory process.

The antinociceptive effect of 1.5-DHP was evidenced by a change inthe nociceptive behavior of the mice. Therefore, the rota-rod test was

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Fig. 7. Effect of 1.5-DHP on changes in rectal temperatures (A) and CSF (B) PGE2 concen-tration after LPS injection. 1.5-DHP (60 mg/kg, p.o.) or vehicle was administered 30 minprior to LPS (5 μg/kg, i.v.) or sterile saline (0.2 mL, control) injection. The CSF was harvested3 h after LPS or saline injection. PGE2 concentration was determined by ELISA. Values repre-sentmeans ± SEMof the variation in rectal temperature (ΔrT, °C) and the PGE2 levels in theCSF (pg/mL) of 6 rats per group. *,# p b 0.05 compared to the groups treated with vehicle/saline or vehicle/LPS, respectively. Basal rectal temperatures of each group were as follows:▭ = 37.01 ± 0.04; = 36.89 ± 0.03; ■ = 36.87 ± 0.03; = 36.98 ± 0.04.

Fig. 8.Effect of 1.5-DHP on TNF-α concentration in the peritoneal exudate after carrageenaninjection. 1.5-DHP (10 mg/kg, p.o.) or vehiclewas administered 60 min prior to intraperito-neal injection of carrageenan (1% in saline; 500 μL/mouse). The peritoneal exudate wascollected 4 h after carrageenan injection. TNF-α concentration was determined by ELISA.Values represent means ± SEM of the TNF-α concentration in the peritoneal exudate(pg/mL) of 6 mice per group. *** p b 0.001 compared to the control group.

6 D.C. Malvar et al. / Life Sciences xxx (2013) xxx–xxx

used to investigate if the treatments could influence the motor activityof the animals, in order to avoid false positives in the nociception testswhich could impair the assessment of these results. Our data showedthat 1.5-DHP did not change the number of falls and the time on the

Table 1Effect of 1.5-DHP on COX-1 and COX-2 activity.

1.5-DHP Concentration (μg/mL) Inhibition (%)

COX-1 COX-2

0 0 03.9 ND 2.177.8 ND 14.615.6 45.1 30.431.2 52.9 46.762.5 76.5 50.0125.0 82.3 73.9250.0 92.2 ND

Please cite this article as: Malvar DC, et al, Antinociceptive, anti-inflcarbohydrazide, a new heterocyclic pyrazole derivative, Life Sci (2013), h

rota-rod, which validates the specificity of its antinociceptive effect(Costa et al., 2013).

The anti-inflammatory effects of 1.5-DHP were assessed on thecroton oil-induced ear edema and on carrageenan-induced leukocytemigration. The croton oil causes constant irritation of the mouse ear,which leads to fluid accumulation as well as edema characteristic ofan acute inflammatory response. The edema formation is initially medi-ated by histamine and serotonin and later by the release of prostaglan-dins (Parveen et al., 2007). 12-O-Tetradecanoylphorbol-13-acetate, akind of phorbol ester present in croton oil, has been reported to stimu-late phospholipid-dependent protein kinase C and overexpression ofinducible nitric oxide synthase and cyclooxygenase-2 (Nakadate,1989; Castagna et al., 1982; Lee et al., 2013). 1.5-DHP reduced thecroton oil-induced ear edema by a significant 55.3%, suggesting ananti-inflammatory effect.

The anti-inflammatory effect of 1.5-DHP was also evidenced by thereduction, in a dose-related manner, of the total leukocyte migrationto the peritoneal cavity induced by carrageenan, which is dependenton the synthesis/release of chemoattractant mediators such as leukotri-enes (LTB4) (Afonso et al., 2012; Samuelsson, 1983), cytokines (IL-1 andTNF-α) (Frode et al., 2001; Mazzon and Cuzzocrea, 2007) andchemokines (Kobayashi, 2008; Sanz and Kubes, 2012).

Our results also demonstrated that 1.5-DHPproduced a dose-relatedantipyretic effect on LPS-induced fever in rats. There are several studiesdemonstrating that the LPS-induced fever is mediated by several cyto-kines, such as TNF-α, IL-1β and IL-6, and prostaglandins, such as PGE2and PGF2α (Roth and de Souza, 2001; Blatteis, 2007).

PGE2 is well known to be involved in the establishment of severalcardinal signs of inflammation such as pain, swelling, heat and redness(Mancini and Di Battista, 2011). Moreover, several studies have report-ed the involvement of brain PGE2 in the febrile response induced byseveral stimuli, such as LPS, zymosan and Staphylococcus aureus(Malvar et al., 2011; Kanashiro et al., 2009; Martins et al., 2012; Futakiet al., 2009). Our results demonstrated that 1.5-DHP inhibited bothCOX-1 and COX-2 in vitro. Its SI was 0.42, indicating that 1.5-DHP isonly slightly more effective against COX-1 than COX-2. Moreover, theantipyretic effect of 1.5-DHP was followed by a reduction of CSF PGE2concentration after LPS injection, demonstrating that this drug also in-hibits brain PGE2 synthesis in vivo. Altogether, these results suggestthat 1.5-DHP produces anti-inflammatory, antinociceptive and

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antipyretic effects by cyclooxygenase inhibition, such as non-selectivenon-steroidal anti-inflammatory drugs.

Moreover, our results also demonstrated that 1.5-DHP reduced theTNF-α concentration in peritoneal exudates by 27.6% after carrageenaninjection. TNF-α is a pro-inflammatory cytokine involved in inflamma-tory hypernociception (Chichorro et al., 2004; Cunha et al., 2005),cellular chemotaxis (Frode et al., 2001; Mazzon and Cuzzocrea, 2007),and febrile response induced by several stimuli, such as LPS (Roth andde Souza, 2001; Blatteis, 2007). Therefore, this result suggests thatTNF-α inhibition may also be involved in the anti-inflammatory,antinociceptive and antipyretic effects of 1.5-DHP. It is important tonote that TNF-α is upstream in the cascade synthesis of several pro-inflammatory mediators involved in inflammation, nociception andfever processes such as the cytokines (IL-1β and IL-6), adhesion mole-cules (ICAM-1 and VCAM-1) and COX-2-derivated prostaglandins(PGE2) (Cunha et al., 2005; Fabricio et al., 2006; Byeon et al., 2012).Therefore, we cannot rule out the possibility that 1.5-PHP could alsoproduce effects by inhibiting the synthesis of these pro-inflammatorymediators. However this proposal still needs to be evaluated.

Conclusion

This study describes the anti-inflammatory, antinociceptive and an-tipyretic properties of a new heterocyclic compound: 1.5-diphenyl-1H-Pyrazole-3-carbohydrazide. These effects involve the reduction of PGE2synthesis through COX-1 and COX-2 inhibition aswell as by TNF-α syn-thesis/release inhibition. These data show potential for the develop-ment of new effective drugs for treating fever, pain and inflammatorydiseases.

Conflict of interest

The Authors declare that they have no conflicts of interest.

Acknowledgments

The authors thank the CNPq, FAPERJ, FAPESP and PostgraduateCourse in Veterinary Medicine/UFRRJ for financial support.

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