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Biomedicine & Pharmacotherapy 63 (2009) 643e649

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Inhibitory activity of limonene against  Leishmania  parasites  in vitro and  in vivo Denise C. Arruda, Danilo C. Miguel, Jenicer K.U. Yok oyama-Yasunaka, oyama-Yasunaka, Alejandro M. Katzin, Silvia R.B. Uliana*  Departamento de Parasitologia, Instituto de Cieˆncias ˆncias Biome´´dicas, dicas, Universidade de Sao ~ao Paulo, Av. Prof. Lineu Prestes, 1374, ICB2 - Cidade Universitaria, S~ ao Paulo, SP 05508-900, Brazil  ao

Received 16 October 2008; accepted 24 February 2009 Available online 10 March 2009

Abstract

Limonene is a monoterpene that has antitumoral, antibiotic and antiprotozoal activity. In this study we demonstrate the activity of limonene against  Leishmania  species  in vitro  and  in vivo. Limon Limonene ene killed   Leishmania amazonensis   promastigotes and amastigotes with 50% inhibitory concentra conc entrations tions of 252.0  49.0 and 147.0  46.0  m M, respectively. Limonene was also effective against   Leishmania major ,  Leishmania braziliensis  and  Leishmania chagasi  promastigotes. The treatment of  L.   L. amazonensis-infected macrophages with 300  m M limonene resulted in 78% reduction in infection rates.  L. amazonensis-infected mice treated topically or intrarectally with limonene had significant reduction of lesion sizes. A significant decrease in the parasite load was shown in the lesions treated topically with limonene by histopathological examination. The intrarectal treatment was highly effective in decreasing the parasite burden, healing established lesions and suppressing the dissemination of  ulcers. Limonene presents low toxicity in humans and has been shown to be effective as an agent for enhancing the percutaneous permeation of  drugs. Our results suggest that limonene should be tested in different experimental models of infection by  Leishmania.   2009 Elsevier Masson SAS. All rights reserved.  Keywords: Leishmaniasis; Chemotherapy; Terpene

1. Introduction

Parasites of the  Leishmania  genus are the etiological agents of leishmaniasis, a widely distributed protozoal disease with a prevalence of 12e14 million people and a population at risk  [1].. of 350 million people in 88 different countries  countries   [1] Fir First st choice choice treatm treatment ent of lei leishm shmani aniasi asiss still still rel relies ies on the par parent entera erall adm admini inistr strati ation on of highly highly tox toxic ic antimo antimonia niall comp compou ound nds. s. Th Ther erap apy y with with pent pentav aval alen entt anti antimo moni nial alss is commonly associated with high rates of non-compliance and parasite resistance to these drugs is rising alarmingly   [2] [2].. Therapeutic alternatives have been sought for a long time and some progresses have been achieved with new formulations of amphotericin B in liposomes and, more recently,

* Corresponding author. Tel.:   þ55 11 30917334; fax:   þ55 11 30917417.  E-mail address:   [email protected] [email protected] (S.R.B.  (S.R.B. Uliana). 0753-3322/$ - see front matter     2009 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biopha.2009.02.004

with the use of miltefosine as an oral-administered drug for the treatment of kalazar   [3]  and testing of paromomycin as [4].. However, the need for alternative drugs a topical agent   [4] is still very clear. Reports Repor ts on the antileishm antileishmanial anial activi activity ty of a variety of plant extracts containing terpenoid compounds have been published [5e8] 8].. We have recently shown that nerolidol   e  a sesquiterpene present in essential oils of several citrus plants e is active against promastigotes and amastigotes of  Leishmania   Leishmania in vitro and, applied topically, is partially effective in the treatment of  experimental leishmaniasis  leishmaniasis   [9] [9].. Limone Lim onene ne is a 1010-Car Carbon bon cyc cycloh lohex exano anoid id mo monot noterp erpene ene found fou nd in a va varie riety ty of plants, plants, particul particularl arly y in oils oils of lem lemon, on, orange, dill and bergamot. Limonene’s insecticide and antimicrob mic robial ial pro proper pertie tiess are rep repres resent entati ative ve of plants plants’’ nat natura urall defe defens nsee mech mechan anis isms ms.. Bo Both th qu quir iral al form formss RR-((þ)- and S()-limonene are used in the cosmetic industry as flavoring agents. This terpene has also been shown to be effective as an

 

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agent for enhancing the percutaneous permeation of drugs   in vitro   and  in vivo  [10,11]  [10,11].. Se Seve vera rall st stud udie iess repo report rted ed that that limo limone nene ne ha hass an anti anti-proliferative effect in a variety of cell types, such as melanoma noma,, gast gastri ric, c, and and pros prosta tate te ca canc ncer er ce cell llss   [12e14] 14].. The ther therap apeut eutic ic prop proper ertie tiess of lim limon onen enee in anim animal al mode models ls of  tumorigenesis were used as the basis for Phase I clinical trials in breast and colon cancer patients. The drug was considered

least least three three ind indepe epende ndent nt exp experi erimen ments. ts. The 50% inh inhibi ibitor tory y concentration (IC50) was determined from sigmoidal regression sion of the con concen centra tration tionerespon response se curve curvess using Scientific Graphing and Analysis Software ORIGIN 7.5. Efficacy of limonene against intracellular amastigotes was assessed by counting the number of infected cells in J774.A1 macrophage monolayers. Macrophages were plated in round glass coverslips inside the wells of a 24-well culture dish at 

well toleratedGoulart and resulted resulte in partial partial determined clinical clinical respon response  [15,16].. Rodrigues anddcoworkers thatse [15,16] limonene was active  in vitro  against   Plasmodium falciparum. The antiplasmodial effect was associated with inhibition of dolichol and ubiquin ubiquinone one biosyn biosynthe thesis sis and reduct reduction ion in pro protein tein iso iso- [17].. prenylation [17] prenylation In this this paper paper we describ describee the ant antile ileish ishman manial ial act activi ivity ty of  limonene against   Leishmania amazonensis   promastigotes and amastigotes and its efficacy in the treatment of experimentally induced cutaneous leishmaniasis. 2. Materials and methods

 2.1. Parasites  Leishmania   promast promastigo igotes tes were were grown grown in M19 M199 9 supple supple-mented with 10% fetal bovine serum (FBS) as described mented described [18]  [18].. The strains used were:   L. (Leishmania) amazonensis  MHOM/  BR/1973/M2269,  L. (Leishmania) chagasi  MHOM/BR/1974/  M2682,   L. (Viann (Viannia) ia) bra brazili ziliensi ensiss   MHOM/BR/1975/M2903 and   L. (Lei (Leish shma mani nia) a) majo major  r    (MHOM/IL/1981/Friedlin). Amastigotes of   L. amazon amazonensis ensis   were obtained from experimentally infected BALB/c mice as described  [19].  [19].  2.2. Drugs

(R)-(þ)-limonene, was purchased from Sigma eAldrich (St. Louis, MO, USA). Limonene was diluted in methanol. Topical formulations containing 10% (wt/wt) limonene were prepared in lanovaseline lanovaseline (L (LV) V) (70% lanoline, 30% vase vaseline, line, wt/wt). Ointments were freshly prepared every 20 days and kept at Intrarect ectal al doses doses of 100 mg of lim limone onene/ ne/kg/ kg/day day wer weree 4   C. Intrar delivered by instilling 20  m l of 10% limonene in 20% ethanol/  0.01 0.01 M ph phos osph phat atee bu bufffer fer (pH (pH 7.4) 7.4).. Stoc Stock k solu soluti tion onss of  amphotericin ampho tericin B (Sigma (SigmaeAld Aldrich rich)) were were prepar prepared ed in DMSO DMSO   (5 mM final concentration) and stored at   20 C.  2.3. Antileishmanial in vitro assays

Activity again Activity against st parasites parasites was tested  in vitro  by cultivating promastigotes (1  106) or amastigotes (5  106) in the presence of increasing concentrations of drug in 24-well culture dishes (Corning Life Sciences, NY, USA) for 2, 24 and 48 h. Cell viability was assessed by measuring the cleavage of 3(4,5-dimeth (4,5-d imethylthiaz ylthiazol-2ol-2-yl)-2,5 yl)-2,5-diphe -diphenyl nyl tetraz tetrazolium olium brom bromide ide (MTT;; SigmaeAld (MTT Aldric rich, h, St. Louis, Louis, MO, USA USA)) as des descri cribed bed  [9].. Assays were performed in triplicate and results previously [9] previously are are expr expres esse sed d as th thee mean mean perce percent nt re redu ducti ction on of para parasi site te numbers numbe rs compared compared to untreated untreated control wells calculated calculated for at

5

a concentrationwith of 510%10fetal cells per coverslip in RPMI 1640   Lsupplemented bovine serum (FBS), 2 mM glutamine and 50 mg/ml gentamicin. After 2 h of incubation at 37    C in an atmosphere of 5% CO 2,  L. amazonensis  stationary phase promastigotes were added to the wells (2.5  106 per well) and the cultures were incubated at 33   C in a 5% CO 2 atmosphere. After 3 h, free promastigotes were removed by ext extens ensive ive washi washing ng wit with h RPMI RPMI wit withou houtt FBS and infect infected ed cultures were treated with the different drug concentrations for 48 h. The monolayers were washed, fixed and stained with the Instan Instantt Pro Prov v kit (Ne (Newpr wprov ov,, PR, Bra Brazil zil). ). The per percen centag tagee of  infecte infe cted d ma macro cropha phages ges was assess assessed ed by light light micros microscop copy y observation by counting 100 cells in triplicate coverslips. Cytotoxicit Cytot oxicity y was evaluate evaluated d by culti cultivati vating ng 1  106 LLCMK2 or HEK-293 epithelial kidney cells in 24-well plates for 24 h in the presence of increa increasing sing concentrations concentrations of limon limonene. ene. Cell viability was assessed by the MTT assay as described above and results are expressed as percent reduction in cell viabil viability ity com compar pared ed to unt untrea reated ted con control trol cul culture tures. s. The 50% cytotoxic concentration (CC50) was determined as described above for IC50  values.  2.4. In vivo assays

Group roupss of 5e7 fema female le C5 C57B 7BL/ L/6 6 mice mice we were re infe infect cted ed 6 subcutaneously at the basis of the tail with 10 amastigotes of   L. amazonensis. After 5e6 weeks, swelling at the inoculation site developed. Infected animals were randomized according to lesions sizes and treatment was initiated. Lesion size was recorded once a week by measuring the thickness of the tail in two dimensions ( D   and   d ) at right angles to each other with a caliper. The size of the lesion (S) was estimated by calculating the mean diameter of the tail using the formula  S ¼ ( D þ d )/2. All animal experiments were appr approv oved ed by the the Ethi Ethica call Comm Committ ittee ee.. Da Data ta on the the lesi lesion on progression were analyzed for statistical significance by using the two-tailed Student’s   t -test -test for unpaired samples. A result was considered significant at   P < 0.05.  2.5. Histopathology

Fragments Fragme nts of lesion lesionss were were rem remov oved ed and fixe fixed d in neu neutra trall buffered formalin for subsequent paraffin embedding. Sections (5  m m thick) were stained with HematoxylineEosin (HE).  2.6. Limiting dilution

The tissue at the lesion site was removed and separated from from the bon bone, e, wei weighe ghed d and hom homoge ogeniz nized ed in 1 ml cul cultur turee

 

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medium. Parasites from tissue were quantified as described [20]. [20].

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Incubation of  L.   L. amazonensis promastigotes in the presence

control animals presented clear evidence of disease dissemination to snout, footpad, and neck (Fig. ( Fig. 1B). 1B). Parasite burden was quantified in intrarectally-treated mice 15 weeks after the interruption of the treatment by limiting dilution assay (Fig. (Fig. 2 2). ). A reduction of more than 99.9% in the parasite load was noted in 80% of the treated mice. Topi opical cal tre treatm atment ent was admini administe stered red to   L. amazonensis amazonensisinfected mice with preparations of limonene in a LVointment. LV ointment.

of increasing concentrations of limonene resulted in activity a dosedependent decrease in the viability of parasites. Drug in 24-h assays, expressed as the concentrations that killed 50% of the parasites (IC50) was 252.0  49.0  m M. The volumes of  methanol meth anol used to deliver deliver the highest highest drug concentrations concentrations used in the tests were not responsible for parasite toxicity. Limonene had a leishmanicidal effect as demonstrated by microscopic examination of treated parasites, which revealed cells with wit h disrup disrupted ted str struct ucture ure,, and by the abs absenc encee of gro growth wth in cult culture uress trea treated ted wi with th conc concen entr trat atio ions ns abov abovee the the IC90   and recultured in media without drug (data not shown). The IC50 of  amphotericin B against   L. amazonensis   promastigotes determined in parallel assays was 0.15  0.06  m M, compatible with previously reported data [21] data  [21].. Amastigotes of   L. amazonensis   purified from lesions and

Treatment was initiated 6 weeks after infection controlinfected groups treated 5with the basis alone wereand included in all experiments. Treatment with 10% limonene in LV resulted in a significant reduction on the average lesion size.   Fig. 3 shows the follow up of individual limonene-treated mice, in a representative experiment, up to 19 weeks after the start of  trea treatm tmen ent. t. Co Comp mple lete te heal healin ing g at the the inoc inocul ulati ation on site site wa wass detected in 67e86% of the treated mice. No side effects were detected detec ted in   L. amazonensis amazonensis-infec -infected ted and contro controll uninfe uninfected cted mice treated with ointments containing 10% limonene in LV. Thirteen weeks after the interruption of treatment, tails from mice treated with limonene were prepared for tissue analysis and showed parasites to be absent or very scarce (Fig. (Fig. 4 4d def). In com compar pariso ison n to con contro troll unt untrea reated ted animal animals, s, the lesi lesions ons fro from m mice mice receiv receiving ing LV alon alonee had red reduce uced d siz sizes es whi while le the

grown  in vitro  for 24 h were also killed by limonene with an IC50  of 147.0  46.0  m M. The susceptibility of  L.  L. major  promastigotes   promastigotes to this terpene was also tested and proved to be similar to that of   L. amazonensis   with IC50   of 354.0  33.0  m M. Limonene was also active acti ve against against   L. braziliensis braziliensis   and   L. chagasi chagasi   promastigotes with IC50  of 185.0  19.0 and 201.0  17.0  m M, respectively. Limonene was also able to inhibit the growth of intracellular amastigotes. amastigotes. The treatment treatment of   L. amazonensis-infected macrophages for 48 h with 200 and 300  m M limonene resulted in sign signifi ifica cant nt redu reduct ctio ions ns of intra intrace cellu llula larr para parasi sitis tism, m, by 52.0  1.7 and 78.0  4.2%, respectively.

oint ointme ment nt wa wass bein being g appli applied ed but but soon soon reve reverte rted d to sizes sizes comp compar arab able le to the the untr untrea eate ted d cont contro rols ls wh when en ther therap apy y wa wass interrupted (data not shown). Moreover, lesions on LV treated mice did not heal and the histopathological examination of the inoculation site in control untreated mice or in tissue taken from the mock treated lesions revealed the classical picture of  a cuta cutane neou ouss leis leishm hman ania iasi siss lesi lesion on wi with th a larg largee numb number er of  parasitized macrophages (Fig. (Fig. 4a 4aec). Amastigotes recovered from treated mice were transformed in vitro  and grown as promastigotes. The sensitivity of these cultures to limonene was the same as the parental line used for infect infection ion (da (data ta not sho shown) wn),, indica indicating ting that that the remain remaining ing parasites were not resistant to limonene.

3. Results

 3.1. Activity of limonene against Leishmania in vitro

e

 3.2. In vitro cytotoxicity activity of limonene 4. Discuss Discussion ion

The toxic effect of limonene was tested using epithelialderived cell lines. The results of  in   in vitro  experiments showed that toxicity of limonene to mammalian cells was lower than that obtained for the parasites. The CC 50   for human (HEK293) and  Rhesus  monkey epithelial kidney cells (LLC-MK2) were higher than 1012  m M.  3.3. In vivo efficacy of limonene in L. amazonensisinfected C57BL/6 mice

Limonene was administered to C57BL/6 mice infected with  L. amazonensis  by topical or intrarectal routes. The treatment with limonene by the intrarectal route was initiated 5 e6 weeks after infection and performed for 2 weeks. No toxic effects were observed in treated mice. A significant reduction on the average lesion size was achieved in 80% of the treated animals and B). B). Th Thee after after adm adminis inistra tration tion of this this terpen terpenee (Fig. Fig. 1A and therap therapeut eutic ic respon response se was sustain sustained ed and 18 wee weeks ks afte afterr the onset of treatment treatment responsive responsive mice remained healthy while

In this study we have evaluated the activity of limonene against  Leishmania. Our interest in this terpene was raised by previous reports on its effect against tumor cells. Limonene has been shown to suppress the growth of neoplastic cells   in vitro   and   in vi vivo vo   [22,12,13] [22,12,13].. Fur Furthe thermo rmore, re, the safety safety and chemopreventive or chemotherapeutic activity of limonene’s system sys temic ic adm admini inistra stration tion has bee been n sho shown wn in sev severa erall   in viv vivo o models and for several types of cancer   [22,23]. [22,23]. LongLong-term term analysis of limonene’s toxicity in mice and rats has not found evidences of severe side effects in animals receiving from 250 [16].. Lim Limone onene ne to 1000 1000 mg mg/k /kg/d g/day ay limo limone nene ne for for 2 year yearss   [16] application was tested in Phase I clinical trials, resulting in a very satisfactory tolerance [15,23] tolerance  [15,23].. In addition,   D-limonene does not pose a mutagenic, carcinogenic, or nephrotoxic risk  to huma humans ns and and low low toxi toxicit city y wa wass obse observ rved ed afte afterr sing single le or repeated doses for up to 1 year  [24].  [24]. Antileishmanial properties of terpenoids have been previously reported. Triterpen Triterpenoid oid sapon saponins ins isolated isolated from   Maesa

 

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Fig. 1. Evaluation of disease development in mice treated with limonene by the intrarectal route. (A) Intrarectal treatment with 100 mg/kg/day limonene for 2 weeks (indicated by the horizontal bar) starting 40 days after infection (week 0). The results shown are the individual measurements for each treated animal (grey symbols, dashed lines) and the mean and standard deviation for a group of five control animals (black diamonds, solid line) (** P: 0.03). Results are representative of two independent experiments. (B) Pictures were taken 15 weeks after the interruption of treatment. Control untreated mice (aed) and limonene-treated animals (eeh). Disseminated ulcers in control untreated animals are indicated by arrows.

balansae   reduced liver parasite burden in  Leishmania infantum-in -infec fected ted BALB/c ALB/c mice mice   [6] [6].. Th Thym ymol, ol, a mono monote terpe rpene ne phenol derivative of cymene, presented partial   in vivo   leishmanicidal activity decreasing by 46% the parasite burden in  Leishmania panamensis   infected golden hamsters  [25].  [25].

 L. amazonensis-infected mice initially develop swelling at the ino inocul culatio ation n sit sitee that that pro progre gress ss into an ulcera ulcerated ted lesion lesion,, even eventu tual ally ly lead leadin ing g to the the loss loss of the the tail tail and and to le lesi sion on dissemination. Intrarectal or topical treatment with limonene inhibited inhibi ted the progre progression ssion of lesion lesionss and suppressed metast metastasis asis

 

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metabolism. Taken together, our results suggest that limonene could be considered a template compound to be chemically modifi mo dified ed to increas increasee act activi ivity ty and red reduce uce metab metabolic olic qui quick  ck  removal. In neoplastic cells, limonene’s mechanism of action was initia initially lly associ associated ated wit with h inhibi inhibitio tion n of pro protein tein pre prenyl nylati ation, on, particularly of small G-proteins such as Ras. Early data suggested that limonene was in fact an inhibitor of the enzymes

Fig. 2. Parasite burden at the inoculation site in two control untreated (C1 and C2) and from five limonene-treated mice (L1 eL5) taken 15 weeks after the interruption of treatment. Parasites were quantified by limiting dilution.

development. Limonene also led to a reduction of more than 99.9% in the parasite load observed by limiting dilution and histopathological examination. Reason Rea sonss why response response to tre treatm atment ent was not uni unifor form m in treated groups, varying from 67 to 86%, are not clear. Genetic variation in C57Bl/6 littermates has been detected due to copy number numb er variations variations   [26] [26].. On the the othe otherr ha hand nd,, be beha havi viou oura rall differences in topically treated mice could result in reduced abso absorp rptio tion n of th thee drug drug,, wi with th ea early rly re remo mova vall by frict frictio ion n or licking. Similarly, gut intolerance in some mice could explain early expulsion of the instilled drug. In an attempt to control these variables, variables, both intraperiton intraperitoneal eal and intravenous intravenous routes were also employed to administer limonene. In both cases, treatment treatm ent was ineffecti ineffective ve (data not shown). Since limon limonene ene is ra rapi pidly dly meta metabo boli lize zed d in th thee live liverr to peril perilly lyll al alco coho holl   [27] [27],, these these results results sugges suggestt tha thatt act activ ivity ity is lost lost after after firs first-p t-passa assage ge

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Weeks amazonensis ensis-infe Fig. 3. Topica opicall treatm treatment ent of   L. amazon -infected cted mice with limonene. limonene. Treatment of C57BL/6 mice infected at the basis of the tail and treated with a cream containing 10% (wt/wt) limonene in LV basis for 6 weeks (indicated by the horizontal bar) starting 40 days after infection. The results shown are the individual measurements for each treated animal (grey symbols, dashed lines) and the mean and standard deviation for a group of six control animals (black diamonds, solid line) (* P: 0.01). Results are representative of three independentt experiments. independen

protein prote farnes farnesyl transfe transferase rase and gerany geranylgeran lgeranyl yl transfe transferase rase [28]. . in There areylalso evidences suggesting that limonene may [28] ac actt by decre decreas asin ing g the the stea steady dy state state le leve vels ls of Ra Ras, s, by tran tran-scription script ion or transla translation tion inhibition or stimu stimulation lation of specifi specificc pathways of protein degradation  [29].  [29]. Other possible mechanisms underlying the tumor-suppressive activity of limonene include inclu de induct induction ion of apopto apoptosis sis and reduc reduction tion of hydro hydroxy-3xy-3methylglutaryl coenzyme A reductase activity [23] activity  [23].. In  P. falciparum   limonene was shown to have wider effects on the isoprenoid biosynthesis pathway, inhibiting the synthesis of  dolichol, ubiquinone and reducing levels of small prenylated proteins [17] proteins  [17].. There are also reports on the effect of limonene modulating the imm immune une respon response se in mic micee wit with h increa increased sed del delaye ayed-ty d-type pe hypers hyp ersens ensiti itivit vity y reactio reactions, ns, pha phagoc gocyto ytosis sis and mic microb robici icidal dal activity,, higher total antib activity antibody ody production production and bone marrow  [30,31].. The discrepancy between limonene’s weak  cellularity [30,31] cellularity antileishmanial activity   in vitro, with IC50  values in the high micromolar range, and its effectiveness in a highly susceptible experimental model  in vivo  might be related to these immune enhancing properties.   In vitro   studies indicated that   D-limonenee increa nen increased sed NO produc productio tion n in per periton itoneal eal mac macrop rophag hages es obtained obtai ned from tumor-beari tumor-bearing ng mice mice   [30] [30].. We did not detect increased accumulation of nitrate on supernatants of   L. amazonensis-infec -infected ted macro macrophages phages treated with limonene, suggesting that increased production of NO is not operating as the killin killing g mec mechan hanism ism in this this cas case. e. We also inves investiga tigated ted the pattern of humoral immune responses on infected mice treated or not with this this terpen terpenee and did not detec detectt increa increased sed anti Leishmania   antibody titres or a shift on   Leishmania-specific antibody subtypes produced (data not shown). At present, we ca cann nnot ot excl exclud udee othe otherr effe effect ctss of the the drug drug on the the im immu mune ne response of infected mice. In summary, we showed that topical treatment of   L. amazonensis-infec -infected ted mice with limonene induced healing in 67e 86% of the tre treate ated d animal animals. s. Intrar Intrarect ectal al adm admini inistra stratio tion n of  limone lim onene ne was also also effect effective ive in the resolu resolution tion of cut cutane aneous ous lesions. Topi opical cal tre treatm atment ent of cutane cutaneous ous leishm leishmani aniasi asiss is a ver very y desirable goal. Patients with this disease often live in areas with poor access to medical care and where injections are difficult to obtain and use. Our best results were obtained by the intrarectal instillation of limonene. Different vehicles or formulations for the topical administration of limonene can potentially increase absorption and effective concentration in the tissue, while chemical modifications of limonene deserve to be inves investig tigate ated. d. The Theref refore ore,, this this terpen terpenee ma may y hav havee gre great at potential in the development of new antileishmanial chemotherapeutic agents.

 

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  L. amazonensis-infected C57BL/6 mice with limonene. Fragments were taken from lesions or from the inoculation site 13 weeks after Fig. 4. Topical treatment of  L. the end of treatment and submitted to histopathological analysis. Samples from control (a), placebo (LV) (b, c) and limonene-treated (def) mice are shown. Figures are representative of at least two animals analyzed in each group. The outcome of footpad lesion of control (a) and placebo-treated mice (b, c) is marked by numerous nume rous parasi parasitized tized macrophage macrophagess in the dermis. In limonenelimonene-treat treated ed mice a fibro fibrotic tic scar is observed observed at the inoculat inoculation ion site (d), which is surr surroun ounded ded by a mononuclear inflammatory reaction with sparse (arrowheads) or absent infected macrophages (e, f and data not shown). HE staining; (a, c, e, f)   400; (b, d) 100. Bars represent 20  m m.

Acknowledgments

We thank Dr. Tania Bijovsky de Katzin, Emilia Kimura and Ma´ rcia Ribeiro Pinto for valuable suggestions and Dr. Maria Irma Seixas Duarte, Roosecelis Arau´ jo Brasil and Bernardo Paulo Albe for help with the histopathology. D.C.A. (2008/  51256-7) and D.C.M. (2005/59881-0) held studentships from Fund Fu ndac ac¸~ao a o de Am Ampa paro ro a`   Pesqu Pesquisa isa do Es Esta tado do de S~ aao o Paulo Paulo (FAPESP). This work was supported by FAPESP and CNPq and was carried out according to the rules and regulations of  animal experimentation in Brazil.

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