ORIGINAL PAPER

Augmentation of antileishmanial efficacy of miltefosinein combination with tuftsin against experimental visceralleishmaniasis

Nishi Shakya & Shraddha A. Sane & Wahajul Haq &

Suman Gupta

Received: 29 June 2011 /Accepted: 16 February 2012 /Published online: 6 March 2012# Springer-Verlag 2012

Abstract Current drugs for the treatment of visceral leish-maniasis are inadequate, and their efficacies are also com-promised due to suppression of immune function during thecourse of infection. Miltefosine is the only promising orallyactive antileishmanial drug, but due to its long half-life,there is risk of development of resistance. To overcomethese problems, efforts are needed to develop combinationtherapy of miltefosine with effective immunostimulatingagents where a decrease of parasitic burden and simulta-neous enhancement of adaptive immunity can be achieved.In the present study, we have explored the antileishmanialefficacy of a subcurative dose of miltefosine in combinationwith free as well as liposomal palmitoyl tuftsin (p-tuftsin)using a Leishmania donovani/BALB/c mouse model. Whenmiltefosine (2.5 mg/kg for 5 days) was given with free p-tuftsin, the inhibitory effect was significantly increased from49.6% to 66% (P<0.01), which was further enhanced up to81% (P<0.001) when given after liposomal encapsulationof p-tuftsin. Significant enhancement in parasitic inhibition(93%, P<0.01) was witnessed when animals were co-administered with liposomal p-tuftsin+5 mg/kg×5 daysdose of miltefosine (72.1%). Enhancement in the productionof Th1 cytokines (IL-12, TNF-α, and IFN-γ), reactive ox-ygen, and nitrogen metabolites was witnessed in the combi-nation group. A remarkable increase in phagocytosis index

was also observed indicating overall immunological en-hancement to antileishmanial activity of miltefosine byp-tuftsin.

Introduction

Visceral leishmaniasis (VL) is a chronic, potentially fatal,and vector-borne parasitic disease caused by different spe-cies of the Leishmania parasite. It is characterized by fever,weight loss, hepatosplenomegaly, anemia, and depression ofthe immune system (den Boer et al. 2009). After realizing itsseverity, the WHO included this illness in the list of neglectedtropical diseases targeted for elimination by 2015 (Maltezou2010).

In the absence of an antileishmanial vaccine, control ofVL relies exclusively on chemotherapy that includes penta-valent antimonials (SbV), pentamidine, amphotericin Bdeoxycholate, lipid formulations of amphotericin B, paro-momycin, and orally effective miltefosine (MF). All thesedrugs are limited to some extent by their toxicity, variableefficacy, inconvenient treatment schedules, and requirementfor hospitalization or cost. On the other hand, emergence ofresistance against all drugs except amphotericin B is proneto develop as Leishmania has already developed resistanceto two commonly used antileishmanials, viz. SbV and pent-amidine in India (den Boer et al. 2009). Although MF(hexadecylphosphocholine) is a promising orally activeantileishmanial drug and is currently used as a first-lineregimen in the Indian subcontinent, its long half-life (Seifertand Croft 2006) and uncontrolled provision to patients mayincrease the likelihood of development of parasite resis-tance. Even when monitored, patient compliance is notoptimal and the risk remains that women in childbearing

N. Shakya : S. A. Sane : S. Gupta (*)Division of Parasitology, Central Drug Research Institute (CSIR),Chattar Manzil Palace, M.G. Road,Lucknow 226001 Uttar Pradesh, Indiae-mail: suman_gupta@cdri.res.in

W. HaqDivision of Medicinal and Process Chemistry, Central DrugResearch Institute (CSIR),Lucknow, India

Parasitol Res (2012) 111:563–570DOI 10.1007/s00436-012-2868-z

age receiving MF do not (or only partially) take contra-ceptives (Meheus et al. 2010).

Efficiency of antileishmanial chemotherapy is also im-paired due to suppression of immune function during thecourse of infection (Bogdan 2008). It is usually associatedwith a depression of Th1 cells and preferential expansion ofTh2 cells. Keeping the current status of leishmaniasis treat-ment in mind, use of low dose or a short course of aneffective drug in combination with an immunomodulatorhas been a successful approach for effective treatment ofleishmaniasis (Musa et al. 2010). Several studies have al-ready been reported emphasizing the benefits of a combina-tion of antileishmanial drugs with immunostimulants. Wehave recently reported enhancement of therapeutic efficacyof MF in combination with CpG oligodeoxynucleotides(CpG ODN) as an immunomodulator in rodent models(Sane et al. 2010).

MF is a major milestone in chemotherapy of VL butassociated with the risk of the development of drug resis-tance against MF as a monotherapeutic regimen (Banerjee etal. 2011). To address this issue, we have explored the low-dose treatment of MF in combination with single dose ofliposomal palmitoyl tuftsin (p-tuftsin) which is a relativelymore accessible, stable, and a potential immunomodulatorfor the treatment of experimental VL in a mouse model.Tuftsin, a tetrapeptide (Thr–Lys–Pro–Arg), is an integralpart of the Fc portion of the heavy chain of the leukophilicimmunoglobulin G (residues 289–292) and is released phys-iologically as the free peptide after enzymatic cleavage.Tuftsin is known to augment the phagocytic response ofnormal and stimulated macrophages assessed both for phago-cytosis mediated via nonspecific receptors (Bar-Shavit et al.1979). These interesting features of tuftsin, coupled withits low toxicity, make the peptide an attractive candidatefor immunotherapy (Fridkin and Najjar 1989). It was alsoable to activate murine peritoneal macrophages to expressnitric oxide (NO) synthase and to produce NO which is ableto kill the amastigotes of the intracellular protozoan parasiteLeishmania major (Cillari et al. 1994).The immunostimula-tory activity of tuftsin and its effect against microorganismsare further improved upon its incorporation in liposomes, afterattaching a sufficiently long hydrophobic anchor of fatty acylresidue (palmitoyl) with the help of a spacer at its C-terminus.This lipopeptide is commonly referred as palmitoyl tuftsin(p-tuftsin; Fig. 1) and has been shown to express potentimmunostimulatory activity both in vitro as well as in vivomodels. The liposomal formulation of p-tuftsin has been

found to be superior in comparison to the free form.The incorporation of p-tuftsin into liposomes is alsoquantitative due to its lipophilic nature to form stable lip-osomes. The importance of tuftsin-bearing liposomes inthe treatment of macrophage-based infections is due toits targeted delivery (Guru et al. 1989; Agrawal and Gupta2000).

In view of these interesting features of p-tuftsin, we haveexplored the potential of a combination therapy using free aswell as liposomal tuftsin with a subcurative dose of MF. Thelower or subcurative dose of MF is used to minimize thetoxicity of the drug during treatment. To establish the effectof this combination therapy on the immune functions of thehost, immunological parameters, viz. Th1/Th2 cytokines,production of reactive oxygen species (ROS), reactive ni-trogen species (RNS), hydrogen peroxide (H2O2), andphagocytosis were also monitored.

Materials and methods

Parasite culture

The WHO reference strain of Leishmania donovani(MHOM/IN/80/Dd8) was obtained from Imperial College,London (UK) and maintained in this laboratory as promas-tigotes in vitro and as amastigotes in golden hamsters.Promastigotes of L. donovani were cultivated in medium199 (Sigma-Aldrich) supplemented with 0.1% gentamycin(Biovaccines Private Ltd., Chevella, India) and 10% fetalcalf serum (Gupta et al. 2005).

Animals

BALB/c mice (18–20 g) of both sexes were used for thestudy. All the experiments were conducted in compliancewith the Institutional Animal Ethics Committee guidelinesfor the use and handling of animals. Throughout the study,the animals were housed in climate- (23±2°C; relative hu-midity, 60%) and photoperiod-controlled (12 h light–darkcycles) animal quarters. They were fed standard rodentpellet supplemented with grain and had free access to drink-ing water.

Palmitoyl tuftsin

Palmitoyl tuftsin [modified form of tuftsin at the C-terminus(Thr–Lys–Pro–Arg–NH–(CH2)2–NH–COC15H31)] wasobtained from the Medicinal Chemistry and Process divi-sion of CDRI, Lucknow, India. For in vivo experiments, p-tuftsin was dissolved in 100% dimethyl sulfoxide (DMSO)and further diluted with deionized water to a final DMSOconcentration of 8–10% prior to administration mice.

Thr-Lys-Pro-ArgHN

NH

O

CH3

Fig. 1 Structure of palmitoyl tuftsin

564 Parasitol Res (2012) 111:563–570

Miltefosine

MF was purchased from SynphaBase AG (Switzerland). Forthe in vivo part of the study, MF was dissolved in deionizedwater.

Liposome preparation

Unilamellar liposome of p-tuftsin was prepared from phos-phatidylcholine (15 μM), cholesterol (7.5 μM), dissolved inchloroform/methanol (2:1, v/v) in a flat bottom tube. Thesolvent was removed by slow evaporation under nitrogen indeposition of a thin film of lipid on the tube surface. Thetube was dried to remove any trace of solvent. The lipid filmwas then hydrated and dispersed in a solution of appropriatequantity of p-tuftsin (7–8% by PC weight) in Tris-bufferedsaline (10 mM Tris containing 150 mM NaCl, pH 7.4) andprocessed by probe sonication (W-220) to get the desiredpreparation of liposomal tuftsin (Singhal et al. 1984).

Antileishmanial efficacy evaluation in mice

BALB/c mice were infected via the lateral tail vein with 2×107 L. donovani (MHOM/IN/80/Dd8) amastigotes and ran-domly sorted into groups of five to six animals each. Samenumbers of mice were kept as an untreated control group.Mice were dosed by ip (intraperitoneal) and po (per os)routes at 7 days pi (post-infection) according to individualdrug schedules. Animals were sacrificed on day 3 posttreat-ment (day 14 pi). Impression smears of livers were prepared,methanol fixed, and stained with 10% Giemsa stain inphosphate-buffered saline. The number of amastigotes per500 liver cell nuclei was determined. The percent inhibition(PI) was calculated for all drug-treated groups in relation tountreated group (Sane et al. 2010).

Optimization of p-tuftsin and MF dose regimens against L.donovani/BALB/c model

Each group consisted of five to six infected animals in tworeplicates. The p-tuftsin was administered in doses, viz. 30,60, and 120 μg/animal, single dose by ip route. The dose of60 μg/animal for p-tuftsin was found to be the most appro-priate because the best antileishmanial efficacy was wit-nessed at this dose (34.0±2.69% inhibition in parasitemultiplication), which was gradually decreased to 21.56±3.96% and 10.0±5.8% parasitic inhibition at 120 μg/animaland 30 μg/animal doses, respectively. Therefore, liposomalencapsulation of 60 μg/animal of p-tuftsin was carried out,and when administered, it delivers 60 μg of p-tuftsin peranimal. MF was given at doses ranging from 1.25 to 20 mg/kg by po route to select the subcurative doses. The subcu-rative doses selected were 2.5 and 5 mg/kg for further

experiments of combinations, and 2.5 mg/kg dose was usedfor biochemical and immunological assays.

Evaluation of free p-tuftsin and liposomal form aloneand in combination with two subcurative doses of MF(2.5 and 5.0 mg/kg)

Nine groups of mice each consisting of five to six animals intwo replicates were used for these experiments. Mice ofgroup I received free p-tuftsin (60 μg/animal) by the iproute, group II received lipo p-tuftsin (60 μg/animal of p-tuftsin), group III received 2.5 mg/kg dose of MF, group IVreceived MF (2.5 mg/kg)+p-tuftsin (60 μg/animal), group Vreceived MF (2.5 mg/kg)+lipo p-tuftsin (60 μg/animal of p-tuftsin). Group VI received 5.0 mg/kg dose of MF, group VIIreceived MF (5.0 mg/kg)+lipo p-tuftsin (60 μg/animal of p-tuftsin), and group VIII received curative dose (20 mg/kg) ofMF. Group IX receiving PBS served as controls for all thegroups. The percent inhibition was calculated for all drug-treated groups in relation to a non-treated group.

Biochemical analysis for measuring production of ROS,RNS, and H2O2

For fluorometric detection of toxic oxygen (ROS and H2O2)and nitrogen (RNS) metabolites, 2-7-dichlorofluorescin(DCFH) and diaminofluorescein-2-diacetate (DAF2DA)dyes were used, respectively. 4α-Phorbol 12-myristate13-acetate (PMA) was used as an inducer for productionof oxygen metabolites. DCFH is reported to oxidize intofluorescent compound 2-7-dichlorofluorescein (DCF).DAF2DA diffuses into cells and tissue where nonspe-cific esterases hydrolyze the diacetate residues therebytrapping diaminofluoroscein-2 (DAF-2) within the intra-cellular space. NO-derived nitrosating agents such asN2O3, nitrosate DAF-2 and yields its highly fluorescentproduct, DAF-2 triazole. Inhibitors used were (1) pen-toxifylline (PTX) which inhibits NADP oxidase requiredfor superoxide ion production, (2) N-nitro-L-argininemethyl ester (L-NAME) which inhibits nitric oxide syn-thase essential for production of nitric oxide ion, and(3) sodium azide (NaN3) inhibiting catalase for produc-tion of H2O2 (Tarpey et al. 2004; Van Assche et al.2011). Maximum drop in fluorescence is an indicator ofthe concentration of its substrate in cell suspension.

The fluorescence drop of treated peritoneal exudate cells(PECs) was measured and compared with that of untreatedPECs. PECs were extracted from animals of all the experi-mental groups and layered at 1×106 cells/ml per well in 24-well tissue culture plates and incubated at 37°C, 5% CO2 for24 h. The non-adherent macrophages were removed bywashing with complete RPMI medium. Cells were incubat-ed with inhibitors (L-NAME, 10 μM; PTX, 10 μM; and

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NaN3, 10 μM) for 1 h at 37°C, 5% CO2 followed byinduction with PMA (20 μM) and incubation of 1 h. Finally,10 μM of dyes (DCFH or DAF2DA) was added to each welland incubated for 30 min. Each step was followed byappropriate washings. Free radicals generated from perito-neal macrophages oxidized non-fluorescent forms of dyes tofluorescent forms. The fluorescent signal from the dye wasread on Cell Quest FACS Calibur (Becton Dickinson) withFL1 UV band pass filter (excitation at 488 nm and emissionat 510 to 513±30 nm) (Sane et al. 2010).

Phagocytosis assay

A flow cytometry-based method was used to study thephagocytic activity of macrophages (Sharma et al. 2004).PECs of each experimental group were seeded at 1×106

cells/ml per well in 24-well tissue culture plates and incu-bated at 37°C, 5% CO2 for 24 h. The non-adherent macro-phages were removed by washing and incubated with10 μM fluorescein isothiocynate-labeled bacteria (1:10 ra-tio) for 30 min at 37°C except control wells. After incuba-tion, excess non-phagocytized bacteria were removed bywashing. The cells were collected in tubes and phagocytosisobserved by FACS Calibur (Becton Dickinson) with FL1UV band pass filter excitation at 488 nm and emission at510 to 513±30 nm. Results were represented as a phago-cytic index (PI) which was the ratio of mean optical density(OD) of stimulated cells to mean OD of unstimulated cells.

Evaluation of cytokine production

Serum samples from animals of treated and untreated con-trol groups were analyzed for various cytokines [tumornecrosis factor-α (TNF-α), interferon gamma (IFN-γ), in-terleukin (IL)-12, and IL-10] by BD OptEIA™ ELISA Kits(BD Biosciences) in accordance with the manufacturer’sinstructions, where TNF, IFN-γ, and IL-12 are Th1 cyto-kines and IL-10 in the category of Th2 cytokine (De Larcoet al. 2001). Each kit contains captured antibody and detec-tion antibody (specific for each cytokine). The wells coatedwith specific capture antibody were incubated overnight at4°C. Thorough washing was done at each step. Coated wellswere blocked with assay diluent and incubated at 22°C for1 h. The standard and serum sample were added to theirrespective wells. After incubation for 2 h at 22°C, a workingdetector (detection antibody + conjugate) was added to eachwell and incubated for 1 h at 22°C. In the next step, afteraddition of the substrate solution to each well, incubationwas done for 30 min in the dark at 22°C. Finally, a solution(5 N H2So4) was added (to each well to stop the reaction).Optical densities of samples were read at 450 nm within30 min with λ correction 570 nm on a microplate

spectrophotometer (Power Wave™ X52, BioTek Instru-ments, USA).

Statistical analysis

Results are presented as the mean±SD of two experiments,and analysis of data is carried out by Bonferroni’s andDunnett’s multiple comparison tests. Differences withP<0.05 were considered significant.

Results

Dose optimization of MF

MF was evaluated at various doses ranging from 1.25 to20 mg/kg for 5 days by the po route to select the subcurativedoses. Parasite inhibition observed at 20 mg/kg was 97.20±2.10% followed by 88.60±3.70%, 72.10±1.20%, 49.60±2.90%, and 33.50±4.10% at 10, 5, 2.5, and 1.25 mg/kgdoses, respectively. Subcurative doses for combination trialswere selected as 2.5 and 5 mg/kg.

Combination therapy of p-tuftsin with two subcurativedoses of MF (2.5 and 5.0 mg/kg)

Results of combination therapy of free and liposomal p-tuftsin with two different doses of MF (2.5 and 5.0 mg/kg)are presented in Fig. 2. Free p-tuftsin showed an efficacy of34%, which was moderately enhanced to 48% by liposomalencapsulation (P<0.05). Parasitic inhibition of MF alone at2.5 and 5.0 mg/kg was 49.6% and 72.1%, respectively.However, when free p-tuftsin was given with 2.5 mg/kgdose of MF, parasite inhibition increased from 49.6% to66% (P<0.01), and liposomal p-tuftsin further enhancedthe efficacy up to 81% (P<0.001). Parasitic inhibition wasmarkedly increased to 93% when liposomal p-tuftsin wasadministered with 5 mg/kg of miltefosine. The efficacy ofthis combination was comparable with the efficacy of acurative dose (20 mg/kg for 5 days) of miltefosine (98%).

Biochemical assays

The results are shown in Fig. 3a, b. Peritoneal exudate cells(PECs) of mice administered with free p-tuftsin showed amoderately significant NO, ROS, and H2O2 production (P<0.05, P<0.05, and P<0.05, respectively). Cells of the trea-ted group also showed significant NO production (P<0.01)and a moderate ROS and H2O2 production (P<0.05 and P<0.05, respectively). PECs of the group treated with thecombination of free p-tuftsin with MF showed enhancedproduction of ROS, NO, and H2O2 (P<0.01, P<0.001,and P<0.01, respectively). Cells of the liposomal p-tuftsin-

566 Parasitol Res (2012) 111:563–570

treated group also showed significant production of all themetabolites (P<0.01). However, when it was combined with

MF, a remarkable upgradation in the production of NO,ROS, and H2O2 was observed in the cells treated with this

I II III IV V VI VII VIII0

25

50

75

100 Free p-Tuftsin

Lipo p-Tuftsin

MF (2.5 mg/kg)

MF(2.5 mg/kg) + Free p-Tuftsin

MF(2.5 mg/kg) + Lipo p-Tuftsin

MF(5.0 mg/kg)

MF(5.0 mg/kg) + Lipo p-Tuftsin

MF (20 mg/kg)

**$$

*

###

$$$@ @

Experimental GroupsP

erce

nt I

nhib

itio

nM

ean

± S

D

Fig. 2 Mean percent inhibition± SD was calculated bycomparing parasitic burden oftreated groups to controlanimals. Significance amongdifferent groups was calculatedby Bonferroni’s multiplecomparison tests. Significance (Ivs II, *P<0.05; I vs IV, **P<0.01); (IV vsV, ###P<0.001); (IIIvs IV, $$P<0.01; III vs V, $$$P<0.001); (VI vs VII, @@P<0.01)

I II III IV V VI0

20

40

60

Dye use d-DCFHInducer-PM ANADPH oxidase Inhibitor-PTxCatalas e Inhibitor-NaN3

Untre ate d ControlA-PM AB-PM A+PTxC-PM A+NaN3

M FA-PM AB-PM A+PTxC-PM A+NaN3

Free p-Tufts inA-PM AB-PM A+PTxC-PM A+NaN3

Fre e p-Tufts in+M FA-PM AB-PM A+PTxC-PM A+NaN3

Liposom al p-Tufts inA-PM AB-PM A+PTxC-PM A+NaN3

Liposom al p-Tufts in + M FA-PM AB-PM A+PTxC-PM A+NaN3

A

B

C

Expe rime ntal Groups

Me

an

Flu

ore

sc

en

t S

ign

al

(a) Biochemical assay for production of ROS and H2O2

I II III IV V VI0

25

50

75

Dye used-DAF2DAInducer-PMANitric Oxide Synthase Inhibitor-L-NAME

A

B

Untreated ControlA-PMAB-PMA+L-NAME

MFA-PMAB-PMA+L-NAME

Free P-TuftsinA-PMAB-PMA+L-NAME

Free P-Tuftsin+MFA-PMAB-PMA+L-NAME

Liposomal P-TuftsinA-PMAB-PMA+L-NAME

Liposomal P-Tuftsin + MFA-PMAB-PMA+L-NAME

Experimental Groups

Flu

ore

sc

en

t S

ign

al

OD

(b) Biochemical assay for production of RNS

Fig. 3 Inhibition of reactiveoxygen species and nitric oxiderelease in response to differenttreated groups against untreated(control) animals. Significantdifference in inhibition wasassessed by Dunnett’s multiplecomparison tests. Significancefor ROS (I vs II, *P<0.05; I vsIII, *P<0.05; I vs IV, **P<0.01; I vs V, **P<0.01; I vs VI,***P<0.001). Significance forH2O2 (I vs II,

#P<0.05; I vs III,#P<0.05; I vs IV, ##P<0.01; Ivs V, ##P<0.01; I vs VI, ###

P<0.001). Significance forRNS (I vs II, $$P<0.01; I vs III,$P<0.05; I vs IV, $$$P<0.001; Ivs V, $$P<0.01; I vs VI,$$$P<0.001)

Parasitol Res (2012) 111:563–570 567

combination (P < 0.001, P < 0.001, and P < 0.001,respectively).

Phagocytosis study

Results of phagocytosis are presented in Fig. 4. Peritonealexudate cells of untreated control mice showed the lowestphagocytic index (5.74±2.3). Cells of the group treated withMF exhibited a moderately increased phagocytic index[17.75±1.30 (P<0.01)]. Cells of mice treated with free p-tuftsin also showed phagocytic index of 11.9±1.2 (P<0.05).Free p-tuftsin, when co-administered with MF, enhanced theindex to 20.60±1.5 (P<0.001) of the cells. Cells of thegroup treated with liposomal p-tuftsin gave a phagocyticindex of 16.75±1.5 (P<0.01), and when given with MF,there was a remarkable increase in index up to 26.06±1.9(P<0.001), which was almost equal to the phagocytic indexof cells of normal uninfected animals (29.32±1.8).

Cell-mediated immune response

Results are shown in Fig. 5. Serum samples of differenttreated groups were used for examining the cell-mediatedimmune response. A twofold rise in TNF-α and IL-12production and a moderate rise in IFN-γ production were

observed after liposomal encapsulation of p-tuftsin. Free p-tuftsin, when combined with MF, resulted in a twofold risein IFN-γ and a moderate rise in TNF-α. Significant en-hancement in the production of IFN-γ and TNF-α waswitnessed in animals co-administered with liposomal p-tuftsin and MF. The level of IL-10 was highest in theuntreated control group. Briefly, we can say that the combi-nation therapy involving free and liposomal p-tuftsin withMF increased Th1 (TNF-α, IFN-γ, and IL-12) cytokinelevels and downregulated Th2 (IL-10) cytokine. Animalstreated with MF alone showed moderately enhanced Th1response. The untreated control group, however, showed noincrease in the Th1 response, whereas production of IL-10was increased.

Discussion

The use of immunomodulators in combination with conven-tional chemotherapy to enhance host immune responsesmay have several advantages as a means of improvingcurrent therapeutic regimens. A low dose or short courseof an effective drug (possibly one dose of amphotericin B) isgiven with an immunomodulator to quickly induce theeffector immune response. Tuftsin is capable of stimulating

I II III IV V VI VII0

10

20

30

40Untreated Control

MF

Free p-Tuftsin

Free p-Tuftsin + MF

Liposomal p-Tuftsin

Liposomal p-Tuftsin + MF

Normal Uninfected Mice

**

*

****

******

Experimenal Groups

Ph

agoc

ytic

Inde

x

Fig. 4 The fluorescence ofstimulated and unstimulated cellsof each group was compared andsignificance of activity ofdifferent treated groups wasassessed against untreated(control) animals by Dunnett’smultiple comparison tests.Significance (I vs II, **P<0.01; Ivs III, *P<0.05; I vs IV, **P<0.01; I vs V, **P<0.01; I vs VI,***P<0.001; I vs VII, ***P<0.001)

I II III IV V VI0

50

100

150

200

250

300

Free p-Tuftsin

Free p-Tuftsin + MF

Liposomal p-Tuftsin

Liposomal p-Tuftsin + MF

MF

Untreated ControlTNF-α

IFN-γ

IL-12

IL-10

Experimental Groups

Op

tical

Den

sity

(OD

)

Fig. 5 Serum samples fromanimals of treated and untreatedcontrol groups were analyzedfor various cytokines (TNF-α,IFN-γ, IL-12, and IL-10) byusing anti-mouse monoclonalantibodies (specific for respec-tive cytokines). Absorbancewas measured on microplatereader

568 Parasitol Res (2012) 111:563–570

white blood cells (monocytes, macrophages, and neutro-phils) and exhibits a wide spectrum of biological activities;notably enhances phagocytosis, the immune response, bac-tericidal, tumoricidal, and antifungal activities (Wardowskaet al. 2007).

It is well documented that the immune system synergis-tically aids the therapeutic efficacy of antiparasitic drugs(Doenhoff et al. 1991; Berger and Fairlamb 1992). Keepingthis in mind, we have explored the adjunct effect of p-tuftsin(free and liposomal) on the efficacy of subcurative doses ofMF using the L. donovani/BALB/c model. Results clearlyshowed that liposomal encapsulation enhanced the antileish-manial efficacy of p-tuftsin. Co-administration of liposomalp-tuftsin with subcurative doses of MF showed a betterinhibitory effect than free p-tuftsin + MF, free p-tuftsin, orMF alone. The efficacy of combination of 5 mg/kg dose ofMF with single dose of lipo p-tuftsin was comparable withthe efficacy of the curative dose of the MF (20 mg/kg).

In our previous study, we reported the effective combi-nation of CpG ODN (Sane et al. 2010) with a subcurativedose of MF (2.5 mg/kg dose) whose efficacy is nearly thesame with the efficacy of combination of MF (2.5 mg/kgdose) with lipo p-tuftsin used in this study. In both thestudies, the dose of MF is the same, but the immunomodu-lators are different. The use of CpG ODN has been veryencouraging, but there are issues related to availability andstability of these phosphorothiated oligonucleotides for clin-ical development, whereas tuftsin is a natural peptide andcan easily be synthesized in laboratories. Even tuftsin hasalso been successfully used in anti-tumor clinical trials(Wleklik et al. 1987).

The results of immunological assays suggested that p-tuftsin potentiated cell-mediated immunity, as evidencedfrom the significant rise in Th1 cytokines and downregula-tion of Th2 cytokine, IL-10. This is in agreement with theearlier reports of Dey et al. (2007) and Bhattacharjee et al.(2009) which stated that Th1 responses from cytokines areprotective for VL, whereas expression of IL-10 increasesduring Leishmania infection. In biochemical assays, liposo-mal p-tuftsin combined with MF resulted in remarkableproduction of NO, ROS, and H2O2. A significant increasein the phagocytosis index was also observed, which revealsthat the cells treated with this combination group deviatetowards normal behavior. Thus, this work clearly demon-strates the application of liposomal p-tuftsin in combinationwith MF as a novel therapeutic approach for a safer treat-ment of VL.

Acknowledgments The authors thank Dr. T. K. Chakraborty, Direc-tor, CDRI, Lucknow, for his encouragement and facilities. Thanks arealso due to Mr. M.S. Negi, Biometry Division, CDRI, for statisticalanalysis and Mr. A. L. Vishwakarma, Flowcytometry Lab for FACSanalysis. This work was supported by the Council of Scientific andIndustrial Research (CSIR), New Delhi, India. Senior research

fellowship given to NS and SAS by CSIR is greatly acknowledged.This paper has CDRI communication number 8183.

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