Review

Anti-inflammatory effects of diethylcarbamazine: A review

Christina Alves Peixoto n, Bruna Santos SilvaLaboratório de Ultraestrutura, Centro de Pesquisas Aggeu Magalhães, Fundação Oswaldo Cruz, CidadeUniversitária, Av. Moraes Rego s/n, Recife CEP 50670-420, PE, Brazil

a r t i c l e i n f o

Article history:Received 6 January 2014Received in revised form19 March 2014Accepted 24 March 2014Available online 12 April 2014

Keywords:DiethylcarbamazineCyclooxygenaseLipoxygenaseNF-κB

a b s t r a c t

Diethylcarbamazine (DEC) interferes with cyclooxygenase and lipoxygenase pathways, reducing theproduction of thromboxane, prostacyclin, prostaglandin and leukotrienes. Recent studies using differentexperimental models of inflammation have indicated that DEC, in addition to inhibiting cyclooxygenaseand lipoxygenase pathways, also inhibits nuclear transcription factor kappa B (NF-κB) activation, which isa key regulator of proinflammatory genes such as TNF-α, IL-1β, inducible nitric oxide synthase (iNOS) andeven cyclooxygenase 2 (COX-2). The aim of the present study is to provide a comprehensive summaryof DEC, including a description of filaricidal action, inhibition of synthesis and secretory pathways,immunomodulatory activity, and specific inhibition of lipoxygenase and cyclooxygenase pathways.

& 2014 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 352. DEC as a filaricidal drug . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 353. DEC acting as an inhibitor of synthesis and secretory pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 364. Immunomodulatory activity of DEC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 375. DEC acting as specific lipoxygenase pathway inhibitor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 376. DEC acting as cyclooxygenase pathway inhibitor. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 387. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

1. Introduction

The well-known drug diethylcarbamazine (DEC) is used through-out the world against lymphatic filariasis. However, in recent yearsmany studies have described other pharmacological activities of DEC.Some preliminary clinical studies have found DEC to be reasonablyeffective in asthmatic conditions, and a number of experimentalstudies have used DEC as a potent leukotriene inhibitor. It has nowbeen established that DEC interferes with the cyclooxygenase andlipoxygenase pathways, reducing: eicosanoid production. The aim of

this review is to provide a brief perspective of research into the roleof DEC in inflammatory dysfunction.

2. DEC as a filaricidal drug

Diethylcarbamazine (DEC) has been used successfully by publichealth authorities as a key tool in the elimination of lymphaticfilariasis in several countries. Its filaricidal activity was discoveredin a study by Hewitt et al. (1947) of wild cotton rats infectedwith Litomosoides sigmodontis. Afterwards, Santiago-Stevensonet al., (1948) demonstrated the effectiveness of the drug againstWuchereria bancrofti in human patients. Although DEC is the drugof choice for the treatment of lymphatic filariasis, its mode ofaction is still controversial.

One of the most frequent findings is that DEC increases micro-filarial adhesion to endothelial cells and granulocyte (Johnson et al.,

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European Journal of Pharmacology

http://dx.doi.org/10.1016/j.ejphar.2014.03.0460014-2999/& 2014 Elsevier B.V. All rights reserved.

n Correspondence to: Laboratório de Ultraestrutura Centro de PesquisasAggeu Magalhães (CPqAM-FIOCRUZ)Av. Moraes Rego s/n, CEP: 50670-420, CidadeUniversitária, Recife PE, Brazil. Tel. þ55 81 2101 2557; fax. þ55 81 2101 2500.

E-mail address: peixoto.christina@gmail.com (C.A. Peixoto).

European Journal of Pharmacology 734 (2014) 35–41

1988; Piessens and Beldekas, 1979; Rácz et al., 1982). Furtherbiochemical studies found this adhesion to be the result of theinhibition of cyclooxygenase and lipoxygenase pathways (Mathewsand Murphy, 1982; Ogletree, 1987; Razin et al., 1984). From theseresults it was suggested that DEC stimulates the innate arm of theimmune system. In contrast, evidence indicates that the microfilar-icidal effect of DEC is not dependent on a specific humoral response.Weiner and Soulsby (1982) found that DEC reduced microfilariaelevels by 95.8%, even when microfilariae of L. sigmodontis releasedin vitro were transfused into a naive animal, suggesting thatan adaptive immune response was not a sufficient condition forDEC effectiveness. Similarly, Vickery et al. (1985) showed thatDEC mediates the clearance of Brugia pahangi microfilariae inimmunodeficient nude mice.

Several other proposals have suggested that DEC has nodirectly effect on microfilarial surface, as exposure of the micro-filariae to high concentrations of DEC left them unharmed. Theseresults led to the idea that DEC has no direct effect on filarialparasites (Hawking, 1979; Hawking et al., 1950; Hawking andLaurie, 1949; Johnson et al., 1988). Barranco et al. (1962) describedthe paralysis of microfilariae treated in vitro, but unfortunately themethodology of this study used imprecise DEC dilutions. However,Rathaur et al. (2009) incubated adult female worms and micro-filariae of Setaria cervi, a bovine filarial parasite, with 100 mM ofDEC, aspirin or indomethacin for 4 hours and observed that aspirinirreversibly affected the motility of both microfilariae and adultworms, while indomethacin and DEC were effective only at themicrofilarial stage, and had no significant effect on adult parasites,even at higher concentrations. DEC treated microfilariae werestraight, immobile and had a wrinkled surface.

Ultrastructural studies developed in the laboratory of the authorsof the present study showed drastic morphological damage tomicrofilariae of W. bancrofti after in vitro and in vivo treatmentwith DEC, indicating a possible direct mode of action. DEC causedsevere damage of microfilarial cells, including the presence of largevacuoles, lysis of the cytoplasm and chromatin and bodies extrudingfrom the plasma membrane, features indicative of an apoptoticprocess (Florêncio and Peixoto, 2003a, 2003b; Peixoto et al., 2004)which were confirmed using molecular testing such as ligation-mediated polymerase chain reaction and in situ terminal deoxynu-cleotidyl transferase mediated dUTP nick end-labeling at light andtransmission electron levels (Peixoto et al., 2008).

Pharmacological studies showed that DEC interferes witharachidonic acid metabolism, acting as an anti-inflammatory drug.It has been found that DEC blocks a number of steps in both thecyclooxygenase (COX) and lipoxygenase pathways, including theinhibition of leucocyte chemotaxis, granulocyte degranulation, andperipheral vasodilation (Maizels and Denham, 1992).

Filarial parasites also synthesize and release prostanoids, parti-cularly prostacyclin and PGE2, which are vasodilators and potentplatelet anti-aggregatory factors (Brattig et al., 2006; Liu et al.,1990; Liu and Weller, 1992, 1990; Sommer et al., 2003). Further-more, it has also been reported that DEC is a potent inhibitor ofparasitic prostaglandin H synthase (PGHS) (cyclooxygenase),which is the first and rate-limiting enzyme in the transformationof polyunsatured fatty acids into prostaglandins in filarial parasites(Kanesa-thasan et al., 1991). Accordingly to Rathaur et al. (2009),Setaria cervi contains significant amounts of prostaglandin Hsynthase-like enzyme (Sc-like-PGHS). The same authors alsoprovided experimental evidence that DEC, indomethacin andaspirin inhibit Sc-like-PGHS at very lower concentrations, andhypothesized that the microfilaricidal activity of DEC may partiallydepend on the inhibition of the parasitic PGHS.

Recently, Sankari et al. (2013) observed a significant reduction inPGE2 and 6-keto-PGF1α concentrations in microfilaraemic sera after12 h of DEC treatment, suggesting that the mechanism by which DEC

lowers the level of microfilariae in the circulation may in part involveits effects on host endothelial and parasite eicosanoid production.

Some studies demonstrated that nitric oxide (NO) plays animportant role in host defense against filarial parasites in vitro(Rajan et al., 1996; Taylor et al., 1996), but no evidence was foundthat DEC itself induces NO synthesis in murine macrophages andrat endothelial cells in vitro (Rajan et al., 1998). However, McGarryet al. (2005) confirmed a lack of activity of DEC in mice deficient iniNOS infected with B. malayi, in addition to a lower loss of COX-1protein in peritoneal exudate cells. According to these authors, itseems that inducible NO is essential for rapid sequestration ofmicrofilariae, and DEC probably stimulates its secretion via inter-action with cyclooxygenase pathways.

Recently, Singh and Rathaur (2010) demonstrated that exposurein vitro of the filarial parasite Setaria cervi to a combination of DEC plusaspirin (100 mM) decreased PGHS activity leading to an increase in NOlevel. NO inhibits tyrosine phosphatases, increasing mitochondrialpermeability through Bax, which allows the release of cytochrome cinto cytosol and activate caspases. Some apoptosis markers such asDNA fragmentation and ladder formation, upregulation of Bax anddecrease of Bcl-2 suggested that adult Setaria cervi worms were killeddue to apoptosis. However, these effects were not observed when theworms were incubated with DEC alone or aspirin. In conclusion,taking into account existing data, DEC may directly affect filarialwormsmediated by the inhibition of cyclooxygenase pathways (PGHS)leading to an increase in NO levels, which in high concentrations, areknown inductors of mitochondrial-mediated apoptosis.

3. DEC acting as an inhibitor of synthesis and secretorypathways

DEC appears to inhibit synthesis and excretory activities insome cells. Ridge et al. (1980) showed that macrophages in culturesynthesize and secrete a soluble factor(s) that induces the synth-esis of collagenase in primary cultures of rabbit chondrocytes.Since macrophages are often present in inflammatory sites thiswould provide a possible mechanism of local connective tissuedestruction. Other studies have indicated that a lipoxygenasepathway of arachidonic metabolism is critical in activating varioustypes of cells. The incubation of chondrocytes with MCM (Macro-phage Conditioned Medium) and low doses of indomethacin(1–10 μM) had no effect on collagenase synthesis. However, theuse of lipoxygenase inhibitors such as NDGA (a nonspecificinhibitor) and DEC inhibited the synthesis of collagenase inchondrocytes. These inhibitors did not affect collagenase activitynor did they interfere with the activation of latent collagenase.This data indicated that although cyclooxygenase plays no role inthe MCM dependent induction of collagenase in chondrocytes,lipoxygenase activity may be essential (Nolan and Pickett, 1985).

Similarly, in Stevens et al., (1985) performed biochemical andmorphological studies and demonstrated that DEC inhibited thesynthesis and exocytosis of proteoglycan in rat chondrocytes.Treatment of chondrosarcoma chondrocytes with DEC alteredthe transport of molecules from the reticulum to the Golgiapparatus and the transport of molecules from the Golgi to thecell surface. After treatment with DEC, chondrocytes presentedlarge vacuoles and a distended Golgi apparatus. Upon removal ofDEC, the vacuoles disappeared and distended organelles returnedto their normal appearance, which was coincident with the startof exocytosis of S-proteoglycan and β-D-xyloside-bound 35S-glycosaminoglycan. Other studies confirmed that DEC disturbsthe traffic of vesicles to and from the Golgi apparatus. Spiroet al. (1986) demonstrated that DEC altered vesicular transportfrom the endoplasmic reticulum to the Golgi, and from the Golgito the plasma membrane, inhibiting the surface expression of

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melanoma associated chondroitin sulfate proteoglycan. DECinduced a complete disorganization of the Golgi apparatus andthe appearance of large vacuoles within the cytoplasm in fetalmouse cerebellar neurons in culture, suggesting that the varioussynthetic and secretory activities involving these organelles areimpaired (Bird, 2000).

Fujimaki et al., (1990) studied the effect of DEC on microtubulesusing microtubule proteins prepared from porcine brains. DECinhibited assembly of microtubules and disassembled preformedmicrotubules in vitro. DEC also inhibited the proliferation of LLC-MK2, and cells grown in the presence of DEC were likelyto separate from each other and become round in shape. Immuno-fluorescence microscopy revealed that LLC-MK2 exposed to DEClacked the delicate pattern of the cytoplasmic microtubule com-plex. Although these studies revealed possible DEC activity as aninhibitor of some secretory pathways, it must be considered thatthose were obtained with different kinds of cell lineages and drugconcentrations. Further studies in vivo and/or ex vivo are necessaryto confirm these observations.

4. Immunomodulatory activity of DEC

Different animal models and experimental approaches havebeen used for the assessment of DEC as a potential immunomo-dulator. Kitchen (1987) described the immunomodulatory effectof the drug on cats given feline leukemia virus vaccine. Whenadministered with an oral retroviral vaccine DEC increasedthe titers and duration of serum antibodies, as well as resultingin a 10-fold increase in peripheral monocyte counts of treated-vaccinated cats, when compared to cats that received a vaccinewithout DEC. More recently, Kitchen and Cotter (1988) andKitchen et al. (1988) confirmed and expanded on these earlierobservations, demonstrating that treatment with DEC was asso-ciated with decreased levels of circulating virus and prevented ordelayed the development of lymphopenia in feline leukemia virus-infected cats.

Using another line of investigation, Medina-De la Garza et al.,(1990) described the enhancing effect of DEC on the adherence ofeosinophils to larvae of Onchocerca volvulus in the presence ofimmune serum, but not in the presence of normal serum, indicatinga possible involvement of DEC in modulation of effector cells. In arecent study, Medina-De la Garza et al., (2012) demonstrated thatDEC exerts a dose-dependent immunomodulatory effect. Theseauthors evaluated the effect of DEC (50 mg/day and 500 mg/day)on antibody production, cytokine response and respiratory burst inBALB/c mice challenged with a thymus-dependent antigen (tetanustoxoid) and a thymus-independent antigen (lipopolysaccharide).These authors concluded that DEC treatment enhanced antibodyproduction in tetanus toxoid immunized mice, and cytokineresponse in LPS immunized animals. Furthermore, the increaseddose of DEC (500 mg) induced a respiratory burst of polymorpho-nuclear leukocytes and monocytes. According to these authors, DECcan act directly as a potential immunomodulator or adjuvant for usein the treatment of human diseases.

5. DEC acting as specific lipoxygenase pathway inhibitor

Diethylcarbamazine performs a further therapeutic role as ananti-inflammatory drug for use in asthmatic conditions. In SalazarMallén (1965) reported very promising results in a preliminary DECtrial in 14 out of 15 patients with intractable asthma, withouttropical eosinophilia. In Benner and Lowell (1970) performed adouble-blind study of 18 patients with perennial asthma and didnot find any significant differences between groups. In contrast,

Srinivas and Antani (1971) found that DEC was moderately effectivein the symptomatic treatment of 40 randomly selected patientswith an acute attack of bronchial asthma, reducing, and in somecases eliminating altogether, the amount of corticosteroids some ofthe patients had been taking. Confirming the findings of this laterstudy, Thiruvengadam et al. (1974) conducted a double-blind studyof 50 patients with intractable bronchial asthma and concluded thatDEC may be used in symptomatic relief, and could be a worthwhileaddition to the armamentarium of drugs used in this disorder, evenif it could not by itself provide total symptom relief.

DEC, like other drugs which inhibit antigen-induced mediatorrelease in vitro such as sodium cromoglycate (Cox, 1967), and thebeta-adrenoceptor agonists (Assem and Schild, 1969), preventsexercise induced-asthma in a significant proportion of asthmaticpatients (Anderson et al., 1981, 1979; Li, 1991; Sly and Matzen, 1974).González et al. (1979) examined the pharmacological activity of thisdrug in several in vivo and in vitro experimental anaphylaxismodels. They stated that DEC at doses of 25 and 50 mg/kgadministered intraperitoneally significantly reduced the mortalityrate in sensitized guinea pigs during protracted shock phase.

Ultrastructural lung studies demonstrated that 12 days of DECtreatment induced significant alterations in type II pneumocytesand a substantially greater number of mature surfactant secretionvesicles in mice, confirming that DEC exerts a role in the activationof important pulmonary cellular pathways, which is in all like-lihood related to the clinical improvement of asthma symptomsafter DEC treatment (Florêncio et al., 2005).

Leukotrienes (LTs), including cysteinyl-leukotrienes (LTC4, LTD4and LTE4) and LTB4, are potent lipid mediators that have animportant pathophysiological role in asthma and allergic rhinitis.LTs are potent lipid mediators derived from arachidonic acid throughthe 5-lipoxygenase (5-LOX) pathway (Montuschi et al., 2007).Initially, Orange et al. (1968) demonstrated that DEC inhibits therelease of slow-reacting substance of anaphylaxis (SRS-A) in the rat,before being shown to be a mixture of cysteinyl-leukotrienes (CysLT).Mathews and Murphy (1982) demonstrated that DEC inhibitsthe formation of LTB4 and cysteinyl-leukotrienes in mastocytomacells while actually stimulating the formation of 5-hydroxyeicosat-etraenoic acid (5-HETE), suggesting that the site of action of DEC ininhibiting leukotriene formation may be the leukotriene A4 synthe-tase reaction. Similar results were obtained by Bach and Brashler(1986) in rat basophil leukemia cells.

Under experimental conditions DEC also attenuated the forma-tion of LTC4 when macrophages were incubated with LPS, andprevented cellular activation (enhanced phagocytosis) (Schade,1986). Histamine and serotonin are important mediators in theinitiation and development of antigen-induced immediate asth-matic responses including air-way smooth muscle contraction andmicrovascular leakage. Nomura et al., (2001). demonstrated that inresponse to histamine and serotonin alveolar macrophages releasechemotactic factors for inflammatory cells in vitro, which could bemainly attributed to LTB4. As with the other lipoxigenase inhibi-tors used in that study, DEC induced a significant decrease in therelease of chemotatic factors from alveolar macrophages.

In an “elegant” study Stenmark et al. (1985) tested the hypoth-esis that monocrotaline would activate the acid arachidonic meta-bolism in rats, playing a role in the hypertensive monocrotalineinjury. After 3 weeks of monocrotaline administration they found 6-ketoprostaglandin F1α, leukotrienes C4, and thromboxane B2 in lunglavages. Also, lung extracts contained CysCLs, including leukotrieneD4. DEC administration reduced the numbers and activity ofinflammatory cells, blocked pulmonary hypertension, preventedright ventricular hypertrophy, and inhibited CysCLs production.

Similarly, Farber et al. (1990), studying pulmonary hypertensiveresponse to foreign body microemboli, measured pulmonaryhemodynamics and accumulation of arachidonic acid metabolites

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in dogs during the infusion of indomethacin, with cyclooxygenaseinhibitor, DEC as lipoxygenase inhibitor, and FPL 55712, a receptorantagonist for leukotriene C4/D4, and concluded that only DECwas effective in blocking pulmonary hypertension.

Semb et al. (1990), analyzing the role of leukotrienes in thetreatment of functional depression of isolated rat hearts demon-strated that DEC significantly inhibited the cardiodepressiveeffects of oxygen free radicals produced by activated polymorpho-nuclear granulocytes. In Zuo et al., 2004 using DEC as a specific5-LOX blocker also demonstrated that DEC significantly reducesthe release of superoxide anion radical in skeletal muscle.

Several other studies have utilized DEC as a specific leukotrieneinhibitor, such as in acute allergic bronchoconstriction (Vargaset al., 1990), hypoxia pulmonary vasoconstriction (Mayers et al.,1991), acute intestinal inflammation (Woolverton et al., 1989),ethanol-injury to the gastric mucosa (Balaa and Subramony, 1990),pathogenesis studies of bowel necrosis (Hsueh et al., 1986), and inexperimental trauma-sepsis in rats by intravenous infusion of liveE. coli (Hall-Angerås et al., 1986).

More recently, Queto et al. (2010) using BALB/c mice OVA-sensitized by airway demonstrated that DEC effectively preventedthe effects of subsequent challenges, such as airway resistance,Th1/Th2 cytokine production, pulmonary eosinophil accumulationand suppressed eosinophilopoiesis in vivo and ex vivo.

It is interesting to note that DEC treatment of acute TropicalPulmonary Eosinophilia (TPE), an interstitial lung disease thatresults from a heightened immunologic response to the humanfilarial parasites W. bancrofti and B. malayi, leads to a marked rapidreduction of the eosinophilic alveolitis, decrease of peripheralblood eosinophilia and a concomitant increase in lung function.These observations are consistent with the concept that at leastsome of the abnormalities found in the lung in acute TPE aremediated by an eosinophil-dominated inflammatory process inthe lower respiratory tract (Pinkston et al., 1987; Rom et al., 1990;Vijayan, 2007).

6. DEC acting as cyclooxygenase pathway inhibitor

Although DEC is often described as a specific lipoxygenaseinhibitor, several studies have demonstrated that the drug alsoblocks some enzymatic steps of the cyclooxygenase pathways.Hagmann et al. (1984) demonstrated that DEC blocked the lethalaction of endotoxin in mice sensitized against lipopolysaccharide,and concluded that this effect was related to the dual lipoxygenaseand cyclooxygenase inhibitor role of DEC. Other studies showedthat DEC inhibited PGD2 and histamine release from purifiedhuman lung mast cells, indicating that the drug is responsiblefor pharmacologic modulation of arachidonic acid metabolites(Peters et al., 1985).

Piper and Stewart (1986) studying coronary vasoconstriction inrats induced by platelet-activating factor, demonstrated that DEC(7.7 mM), besides acting as a lipoxygenase inhibitor, markedlyattenuated the release of cyclooxygenase products such as 6-keto-prostaglandin F1α (6-keto-PGF1α), PGF2α, PGE2 and thromboxaneB2 (TXB2). Similarly, DEC inhibits PGI2, 12-HETE, and 15-HETEsynthesis in human endothelial cells (Ibe et al., 1989). Agreeingwith such results, El Tahir et al. (1991) demonstrated that DEC hasthe inherent property of inhibiting the synthesis of the potentvasodilator, the platelet antiaggregatory effect, anticonvulsantand anti-inflammatory mediator PGI2 in thoracic aorta of rats,probably by interfering with the action of the PG endoperoxidesynthase (PG cyclooxygenase).

The molecular cross-talk between iNOS and COX may regulatetissue homeostasis and contribute to pathophysiological processes.

Some previous studies had shown that COX-2 up-regulationwas strictly iNOS dependent (Jiang et al., 2006; Li et al., 2003;Shinmura et al., 2002; Virdis et al., 2005). Further studies demon-strated that NF-κB activation is essential for iNOS-dependentupregulation of COX-2 (Li et al., 2007).

McGarry et al. (2005) observed that iNOS and COX pathwaysare essential for DEC activity in vivo. These authors showed thatDEC was ineffective in Brugia malayi infected mice lacking iNOSand also reduced the amount of the host COX-1. Interestingly,Queto et al. (2010), besides demonstrating that DEC has anefficient action as a leukotriene inhibitor in an OVA-sensitizedBALB/c mice model, reported that DEC had no effect on Th1/Th2cytokine production, pulmonary eosinophil accumulation andeosinophilopoiesis suppression in CD95L-deficient gld mice, theligand for the apoptosis-inducing receptor CD95L (Fas), and inmice lacking iNOS activity. These results confirmed that DEC canpossibly act as an apoptosis inductor as previously demonstratedby ultrastructural and molecular studies of the microfilariae ofW. bancrofti (Peixoto et al., 2008).

Few studies have focused on the role of DEC in the pathophy-siology of inflammatory conditions. According to Gonzalez et al.(1994), rats with hepatic inflammation induced by CCl4 had anevident reduction of morphological damage after DEC treatment at25 and 50 mg/kg, presenting well-preserved organelles and hepa-tic membrane system. Using the same doses, Rocha et al. (2012a)prevented hepatic lipid accumulation and the damage caused bymalnutrition after treatment of malnourished mice with DEC.

Recently, a study performed in the laboratory of the authors ofthe present study analyzed the anti-inflammatory effect of DEC onhepatic cells of alcoholic mice. The DEC-treated group, whichreceived 50 mg/kg for 12 days (a dose equivalent to 6 mg/kg givento human preconized by OMS) significantly reduced severalparameters of chronic hepatic inflammation. Histological andultrastructural analysis of the alcoholic group showed evidenthepatocellular damage, which was strikingly reduced in thealcoholic DEC-treated group. Also DEC treatment reduced steato-sis, necrosis and foci of inflammatory infiltrates, and diminishedserum AST levels and inflammatory markers such as malondial-dehyde (MDA), NF-kB p65, tumor necrosis factor-α (TNF-α),interleukin-6 (IL-6), vascular cell adhesion molecule-1 (VCAM-1)and intercellular cell adhesion molecule (ICAM-1), monocytechemotactic peptide-1 (MCP-1) and its functional receptor C2chemokine receptor (CCR2), as well as inducible nitric oxidesynthase (iNOS) in hepatic tissue. This study found that chronicconsumption of ethanol increased NF-kB p65 and targeted severalproinflammatory cytokines, chemokines and oxidases. Moreover,the administration of DEC inhibited NF-kB p65 expression, redu-cing hepatic injury and decreasing inflammatory markers, suggest-ing a potential therapeutic use in chronic inflammation (Rochaet al., 2012b).

There is ample evidence in studies of carrageenan and othermodels of inflammation that the enhanced formation of prosta-noids following the induction of COX-2 contributes to the patho-physiology of local inflammation (Salvemini et al., 1995; Sautebinet al., 1998) and that inhibitors of COX-2 exert potent anti-inflammatory effects. Recently, Ribeiro et al. (2014) evaluated theanti-inflammatory activity of DEC in a mouse model of acute lunginflammation (carrageenan-induced pleurisy). The injection ofcarrageenan into the pleural cavity induced the accumulation offluid containing a large number of polymorphonuclear cells(PMNs) as well as infiltration of PMNs in lung tissues. It enhancedthe production of nitrite in serum and also increased expressionTNF-α, IL-1β, COX-2, iNOS and NF-κB p65. The oral administrationof DEC (50 mg/Kg) three days prior to the carrageenan challengeled to a significant reduction in NF-kB p65 expression and in allother inflammation markers.

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It is interesting to note that DEC treatment of OVA-sensitized/challenged mice significantly reduced cytokines that support thedifferentiation (IL-5, IL-4) and migration (Eotaxin-1) of eosinophils(Queto et al., 2010). Similarly, other authors have shown thatairway eosinophilic inflammation and high levels of eotaxin, IL-4,IL-5, IL-13 and LTC₄ in bronchoalveolar lavage are mediated by NF-κB nuclear translocation, NF-κB DNA-binding activity and RelA/p65 phosphorylation (Xiong et al., 2012). Thus, the remarkableeffects of DEC on murine models of asthma are probably related tothe inhibition of the NF-kB signaling pathway.

Several studies have demonstrated that NF-κB plays a centralrole in the regulation of many genes responsible for the generationof mediators or proteins in chronic inflammation such as TNF-α,IL-1β, nitric oxide synthase inducible (iNOS) and COX-2 (Sunget al., 2013; Xu et al., 2013; Garcia-Garcia et al., 2012). Sung et al.(2013) demonstrated by chromatin immunoprecipitation analysisthat NFκB binds to the COX-2 promoter inducing its expression.Therefore, the inhibition of the expression of cytokines (TNF-α,IL-1β, IL-4, IL-5, IL-6), adhesion molecules (VCAM-1, ICAM-1),chemokines (MCP-1, eotaxin), and enzymes (iNOS and COX-2)observed after DEC treatment could be attributed to the inhibitoryeffects of NF-κBp65 activation (Fig. 1).

7. Conclusion

According to recent data, DEC not only inhibits the activityof important enzymes of arachidonic acid metabolism such aslipoxygenases and cyclooxygenases, but can also inhibit the NF-κBsignaling cascade, a key regulator of proinflammatory genes suchas TNF-α, IL-1β, inducible nitric oxide synthase (iNOS) and evencyclooxygenase 2 (COX-2). However, the exact mechanism bywhich NF-κB is inhibited by DEC is still open to question. Whileit is possible that DEC acts by MAPK/NF-kB signaling, furtherstudies are necessary to elucidate this issue.

Acknowledgments

The study was funded by grants from the Fundação de Amparoà Ciência e Tecnologia de Pernambuco (FACEPE) and the InstitutoNacional de Biologia Estrutural e Bioimagem (INBEB). C.A Peixotois a research fellow of the Conselho Nacional de DesenvolvimentoCientífico e Tecnológico. The authors declare that there are noother conflicts of interest.

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Fig. 1. Schematic diagram showing the hypothetical mechanism of Diethylcarba-mazine (DEC) action on inflammatory pathways. DEC can directly inhibit cycloox-igenase (COX-1, COX-2) enzymatic activity, or can indirectly down-regulate COXexpression by inhibiting the nuclear transcription factor NF-kB. DEC also inhibitsLTB4 and cystenil-leukotrienes synthesis. Enzymes are named in italics. Blockedlines indicate inhibitory activity of DEC. Numbers indicate related references: (1)Kanesa-thasan et al., 1991; Rathaur et al., 2009; (2) Razin et al., 1984; Mathews andMurphy, 1982; (3) Rocha et al., 2012b; Ribeiro et al., 2014.

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