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International Journal of Antimicrobial Agents 31 (2008) 142–146

Novel chemotherapy for tuberculosis: chemotherapeutic potential ofeconazole- and moxifloxacin-loaded PLG nanoparticles

Zahoor Ahmad 1, Rajesh Pandey 2, Sadhna Sharma, G.K. Khuller ∗Department of Biochemistry, Postgraduate Institute of Medical Education and Research, Chandigarh 160012, India

Received 10 August 2007; accepted 5 October 2007

bstract

The potential of econazole (ECZ) and moxifloxacin (MOX) individually against tuberculosis (TB) caused by multidrug-resistant and latentycobacterium tuberculosis has been demonstrated. In this study, poly-(dl-lactide-co-glycolide) (PLG) nanoparticle-encapsulated ECZ andOX were evaluated against murine TB (drug susceptible) in order to develop a more potent regimen for TB. PLG nanoparticles were prepared

y the multiple emulsion and solvent evaporation technique and were administered orally to mice. A single oral dose of PLG nanoparticlesesulted in therapeutic drug concentrations in plasma for up to 5 days (ECZ) or 4 days (MOX), whilst in the organs (lungs, liver and spleen)t was up to 6 days. In comparison, free drugs were cleared from the same organs within 12–24 h. In M. tuberculosis-infected mice, eightral doses of the formulation administered weekly were found to be equipotent to 56 doses (MOX administered daily) or 112 doses (ECZ

dministered twice daily) of free drugs. Furthermore, the combination of MOX + ECZ proved to be significantly efficacious compared withndividual drugs. Addition of rifampicin (RIF) to this combination resulted in total bacterial clearance from the organs of mice in 8 weeks.LG nanoparticles appear to have the potential for intermittent therapy of TB, and combination of MOX, ECZ and RIF is the most potent.2007 Published by Elsevier B.V. and the International Society of Chemotherapy.

eywords: Econazole; Moxifloxacin; Tuberculosis; Chemotherapy; Drug delivery

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. Introduction

To combat tuberculosis (TB) in a better and quicker way,mphasis has been given to evaluating the clinically evaluatedntibiotics (used against other infectious diseases) againstycobacterium tuberculosis. In recent years, azole antifun-

als have received great attention and have been shown tonhibit the growth of Streptomyces coelicotar, Mycobac-erium smegmatis and M. tuberculosis H37Ra under in vitroonditions [1]. More recent studies have shown the bacterici-

al potential of econazole (ECZ) and clotrimazole against M.uberculosis under in vitro and ex vivo conditions at very lowoncentrations as well as their synergism with conventional

∗ Corresponding author. Tel.: +91 172 2755 175;ax: +91 172 2744 401/2745 078.

E-mail address: gkkhuller@yahoo.co.in (G.K. Khuller).1 Present address: Center for Tuberculosis Research, Johns Hopkins Uni-

ersity School of Medicine, Baltimore, MD 21231, USA.2 Present address: Department of Biochemistry, MM Institute of Medicalciences and Research, Mullana, Ambala, Haryana, India.

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924-8579/$ – see front matter © 2007 Published by Elsevier B.V. and the Internatoi:10.1016/j.ijantimicag.2007.10.017

ntitubercular drugs (ATDs) [2]. In addition, azoles havehe potential to replace isoniazid (INH) and rifampicinRIF) in murine TB and are effective against latent TB [3].he antimycobacterial potential of ECZ against multidrug-

esistant (MDR) isolates of M. tuberculosis has also recentlyeen demonstrated [4]. Simultaneously, the fluoroquinoloneoxifloxacin (MOX) has shown potent antimycobacterial

ctivity at very low concentrations compared with otheruoroquinolones [5]. MOX exhibits sterilising activity and isffective against a RIF-tolerant population of M. tuberculosis.urther experiments have shown that substitution of frontlineTDs with MOX shortens the treatment duration againsturine TB [6]. MOX has been shown not to exhibit any cross-

eactivity with conventional frontline or second-line drugs7]. These facts have lead to the consideration of this drug asfrontline ATD. It is well known that monotherapy results

n the selection of drug-resistant mutants, thus multiple drugdministration has been recommended for TB. Therefore, it isorthwhile to evaluate the effect of MOX and ECZ combina-

ion with or without other ATDs against TB. Since azole drugs

ional Society of Chemotherapy.

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Z. Ahmad et al. / International Journa

ave poor oral bioavailability and MOX is cleared from theody within 24 h, these drugs will require multiple or once-aily administration [8]. It is possible to reduce drug dosagess well as dosing frequency of any drug by employing drugelivery technologies. Our recent study has demonstratedhat poly-(dl-lactide-co-glycolide) (PLG) nanoparticlesave the potential to enhance the bioavailability of azoles8]. Hence, the use of PLG nanoparticle-encapsulated MOXnd ECZ with or without other ATDs may be a noveltrategy to combat TB. In this study we have evaluated thehemotherapeutic as well as pharmacokinetic potential ofOX and ECZ alone or in combination against murine TB.

. Materials and methods

.1. Chemicals and drugs

ECZ, MOX, RIF, INH, pyrazinamide (PZA), ethambutolEMB) and polyvinyl alcohol (PVA) (molecular weight (Mr)3 000–19 000, 85% hydrolysed) were obtained from Sigmahemical Co. (St Louis, MO). Poly lactide-co-glycolide

50:50 Resomer RG 506, Mr 97 400) was purchased fromoehringer Ingelheim GmbH (Ingelheim, Germany). High-erformance liquid chromatography (HPLC)-grade solventsnd water were obtained from Merck Ltd. (Mumbai, India).ll other chemicals used in the study were of analytical grade.

.2. Culture

The culture of M. tuberculosis H37Rv originally obtainedrom the National Collection of Type Cultures (NCTC, Lon-on, UK) was maintained on Youman’s modified medium.

.3. Animals

LACA mice of either sex weighing 20–25 g obtained fromhe Central Animal House, Postgraduate Institute of Medicalducation and Research, Chandigarh, India, were used in

he study. Animals were housed in biosafety cabinets (NU05-600E, Series 6; NuAire Instruments, Plymouth, MN) androvided with pellet diet/water ad libitum.

.4. Preparation of ATD-loaded PLG nanoparticles

Drug-loaded PLG nanoparticles were prepared by theultiple emulsion and solvent evaporation technique as

escribed previously [8]. Briefly, MOX was dissolved inistilled water, which was then added to dichloromethaneDCM) containing PLG (total drug:polymer 1:1 w/w;ater:DCM 1:10 v/v). The mixture was sonicated for 1 min

o form the primary emulsion, which was poured into 1%

/v aqueous PVA and re-sonicated for 3 min (sonicationower 20 000 KHz). The secondary emulsion so formedas stirred overnight to remove DCM and then centrifuged

8000–10 000 rpm for 15 min) to harvest the nanoparticles,

GAE

imicrobial Agents 31 (2008) 142–146 143

hich were washed with distilled water and vacuum dried.anoparticles of ECZ and other ATDs were prepared simi-

arly with slight modifications as described previously [8,9].mpty drug-free nanoparticles were prepared by substitutingodium chloride in place of drugs.

.5. Characterisation of ATD-loaded PLG nanoparticles

The particles were characterised for their size and polydis-ersity index on a Zetasizer 1000 HS (Malvern Instruments,alvern, UK). The formulations were lysed in 5% w/v

odium dodecyl sulphate (SDS) in 0.1N NaOH to releasehe drugs. The percentage drug encapsulation efficiency wasetermined by the formula: [amount of drug (mg) releasedrom the nanoparticles/amount of drug (mg) initially taken torepare the nanoparticles] × 100. The drugs were analysedy a HPLC system comprising of a dual-piston reciprocatingump, an online de-gasser, an ultraviolet visual dual wave-ength detector (each of Series 200) and a 600 Series Linknterface for data acquisition/processing (all from Perkinlmer Instruments LLC, Shelton, CT). MOX was analysed bymploying a USP gradient program (USP 2000) with waterpH = 2.0, set with orthophosphoric acid)/acetonitrile (60/40)s the mobile phase (at 1.5 mL/min), 292 nm as the detectionavelength and reversed phase C18 column (COSMOSILC18-MS-II Waters type (Waters Corp., Milford, MA);50 × 4.6 mm; 5 �m particle size). ECZ and ATDs werenalysed by employing USP gradient/isocratic programs asescribed previously [8,9]. The sensitivity of the methodsas: MOX, 0.1 mg/L; ECZ, 0.2 mg/L; RIF, 0.4 mg/L; INH,.2 mg/L; PZA, 1.0 mg/L; and EMB, 0.5 mg/L.

.6. Preparation of drug doses for in vivo studies

As in the case of our previous reports, the drug doses usedhroughout the study were MOX 8 mg/kg, ECZ 3.3 mg/kg,IF 12 mg/kg, INH 10 mg/kg, PZA 25 mg/kg and EMB6 mg/kg body weight according to the standard adult humanoses [8,10]. As the dose was different for each drug, thenitial amount of drug taken to prepare the formulationsas calculated by the formula: (amount of drug requireder animal/mean drug encapsulation efficiency) × 100, asreviously described [11]. Once the total amount of drugequired was known, an equivalent amount of PLG was usedn the preparation process. The PLG nanoparticle formula-ion encapsulating drugs was suspended in distilled waterust before oral dosing in each experiment. Similarly, freerugs were also freshly dissolved in distilled water/methanol5:1 v/v) immediately before dosing.

.7. In vivo drug disposition studies

Mice were grouped as follows (12 mice per group):roup 1, free MOX; Group 2, free ECZ; Group 3, freeTDs; Group 4, MOX-loaded PLG nanoparticles; Group 5,CZ-loaded PLG nanoparticles; Group 6, ATD-loaded PLG

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44 Z. Ahmad et al. / International Journa

anoparticles; and Group 7, empty PLG nanoparticles (a pos-tive control to explore the influence of alginate nanoparticlesn drug estimations). The animals were bled at several timeoints. The plasma obtained from each mouse was depro-einised and analysed by HPLC for drugs to obtain the plasmarug concentration–time profile. The HPLC method for MOXas the same as described above for encapsulation stud-

es.The animals were sacrificed at different time points. Drug

evels were determined in 20% w/v of tissue homogenateslungs, liver and spleen) following the same analytical pro-edure as described for plasma.

.8. Experimental infection and chemotherapy

Mice were infected via the lateral tail vein with 1 × 107

acilli of M. tuberculosis H37Rv. Fifteen days later, estab-ishment of infection was confirmed by sacrificing five micend Ziehl–Neelsen staining of lung/spleen homogenates. Inddition, 100 �L of undiluted, 1:100 and 1:1000 dilutedomogenates were plated on Middlebrook 7H10 agar supple-ented with oleic acid–albumin–dextrose–catalase (OADC)

or the enumeration of basal colony-forming units (CFU).ubsequently, mice were grouped as follows (n = 6 perroup): Group 1, untreated control; Group 2, free ECZwice daily; Group 3, free MOX once daily; Group 4,ree ECZ + MOX; Group 5, free ECZ + MOX + RIF; Group, INH + RIF + PZA + EMB; Group 7, PLG ECZ weekly;roup 8, PLG MOX once daily; Group 9, PLG ECZ + MOX;roup 10, PLG ECZ + MOX + RIF; and Group 11, PLGIF + INH + PZA + EMB.

On Day 62 following the initiation of chemotherapy, allhe animals were sacrificed. The lungs and spleen wereemoved aseptically and homogenised in 3 mL of sterile iso-onic saline. Then, 100 �L of undiluted, 1:100 and 1:1000iluted homogenates were plated on Middlebrook media andFU were counted after 28 days of plating. CFU data werenalysed by one-way analysis of variance (ANOVA) followedy Student’s unpaired t-test to compare the untreated andreated groups.

. Results

.1. Physicochemical characterisation of PLGanoparticles

The average size of the PLG nanoparticles was 217 nm,ith a polydispersity index of 0.38. The drug encapsula-

ion efficiency of PLG nanoparticles for ECZ and MOX was

ound to be 52.27 ± 3.80% and 33.69 ± 3.88%, respectivelyresults are mean ± standard deviation of five replicates).he encapsulation efficiency of PLG nanoparticles with

espect to other ATDs was the same as described previously9,12].

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oxifloxacin or econazole to mice at a therapeutic dose. Values areean ± standard deviation of six animals. MIC, minimum inhibitory con-

entration.

.2. In vivo drug disposition studies

Oral free ECZ and MOX were detectable up to 3–4 h and2 h, respectively, in plasma. However, following a single oraldministration of drug-loaded PLG nanoparticles, therapeu-ic drug concentrations in plasma were maintained for up todays and 4 days in the case of ECZ and MOX, respectively

Fig. 1), whilst in the organs (lungs, liver and spleen) both

ig. 2. Tissue drug levels (�g/mL) on Day 6 in mice following a singleral administration of azole/moxifloxacin-loaded nanoparticles. Values areean ± standard deviation of six animals.

Z. Ahmad et al. / International Journal of Antimicrobial Agents 31 (2008) 142–146 145

Table 1Chemotherapeutic efficacy of oral free ECZ and MOX, and ECZ- and MOX-loaded poly-(dl-lactide-co-glycolide) (PLG) nanoparticles against murinetuberculosis

Type of drug/group Log10 CFUa

Lung Spleen

Untreated controls 6.88 ± 0.035 6.90 ± 0.025

FreeECZ twice daily (112 doses) 4.87 ± 0.040* 4.89 ± 0.020*

MOX once daily (56 doses) 4.96 ± 0.013* 4.96 ± 0.013*

ECZ + MOX (112 doses + 56 doses) 3.03 ± 0.070** 3.14 ± 0.130**

ECZ + MOX + RIF (112 doses + 56 doses + 56 doses) N.D. N.D.RIF + INH + PZA + EMB (56 doses) N.D. N.D.

PLG-encapsulatedECZ weekly (8 doses) 4.85 ± 0.050* 4.89 ± 0.040*

MOX weekly (8 doses) 4.94 ± 0.013* 4.96 ± 0.030*

ECZ + MOX (8 doses) 3.04 ± 0.120** 3.13 ± 0.140**

ECZ + MOX + RIF (8 doses + 4 doses) N.D. N.D.RIF + INH + PZA + EMB (6 doses) N.D. N.D.

ECZ, econazole; MOX, moxifloxacin; RFI, rifampicin; INH, isoniazid; PZA, pyrazinamide; EMB, ethambutol; CFU, colony-forming units; N.D., no detectablebacilli.

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.3. Chemotherapeutic efficacy

Based on tissue drug distribution, free ECZ and MOXere administered twice and once daily whilst the encap-

ulated drugs were administered weekly. All the freeTDs (INH, RIF, PZA and EMB) were administered onceaily and encapsulated drugs were administered every 10thay with the exception of EMB, which was adminis-ered weekly. Eight weeks of chemotherapy with ECZr MOX either in free form (112 or 56 doses admin-stered twice or once per day) or in encapsulated form8 doses administered weekly) resulted in approximatelylog10 CFU reduction from lungs and spleen of miceompared with untreated controls (P < 0.01) (Table 1). Fur-hermore, the combination of ECZ + MOX proved highlyignificant (P < 0.001) with respect to therapeutic poten-ial compared with either of these drugs individually, as itesulted in a reduction of 3.5 log10 CFU in free or encapsu-ated form (Table 1). Addition of RIF to this combinationesulted in the most potent regimen, which yielded total bac-erial clearance in mice within 8 weeks, comparable withour-ATDs conventional treatment (Table 1). No significantifference was observed between free versus encapsulatedounterparts.

. Discussion

In the present study, PLG nanoparticles encapsulating

CZ and MOX have been developed for evaluation of their

herapeutic potential against TB. Both these drugs havehown their immense potential against TB, especially in

DR and latent or persistent forms of the disease.

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A single oral administration of drug-loaded PLG nanopar-icles to mice maintained therapeutic drug levels in organs forp to 6 days; this formed the basis of the chemotherapeuticchedule, as TB infection is localised in different tissues.herefore, the PLG formulation was administered weekly,hereas free drugs were administered once or twice daily.Mice were infected with 1.5 × 107 bacilli of M. tuber-

ulosis H37Rv because a similar inoculum has been usedn other reports and is known to provide bacillary loadsimilar to those found in human TB cases [13]. Eight weeksf chemotherapy with ECZ or MOX reduced bacterialurden from organs by approximately 2 log CFU. Thesebservations are in accordance with previous studies [3].urthermore, PLG nanoparticles showed their potential toeduce the dosing frequency of azoles and MOX by 14-nd 7-fold, respectively, without compromising therapeuticfficacy. The combination of ECZ and MOX proved to beuch better as it reduced bacterial burden by approximately

.5 log CFU compared with 2 log CFU with individual drugs.hese results can be explained on the basis that the tworugs have different targets, therefore in combination theyight act synergistically. Indeed, recent studies have shown

hat ECZ inhibits the biosynthesis of glycopeptidolipids,hich in turn are responsible for maintaining the integrityf the mycobacterial cell envelope [14]. Thus, ECZ mightmprove the penetration of other drugs such as MOX and

ake their target (DNA gyrase in the case of MOX) moreccessible. The most interesting finding was the observationhat combination of ECZ, MOX and RIF resulted in total

acterial clearance compared with approximately 7 log CFUn untreated controls. This observation can be explainedn the basis of the fact that all three drugs of this com-ination are active against actively multiplying as well as

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on-dividing bacilli [6,15]. This combination is more benefi-ial than a conventional regimen on the basis of the fact thatCZ and MOX are highly active against drug-resistant andDR bacilli. In addition, all three drugs exhibit sterilising

ctivity owing to their activity against non-replicating bacilliompared with a conventional regimen where only RIF bearshis activity. This regimen is also expected to reduce theotal duration of TB chemotherapy owing to the sterilisingctivity of all its drugs compared with a conventionalegimen [5,15]. PLG nanoparticles have made this newegimen more acceptable, as dosing frequency was reducedy 14-, 10- and 7-fold for ECZ, MOX and RIF, respectively,s has been demonstrated earlier for all frontline ATDs.

This is the first report demonstrating the efficacy ofhree-drug combination (ECZ, MOX and RIF) in free orncapsulated form against murine TB. However, its evalua-ion against resistant and latent TB warrants further research.

cknowledgments

ZA gratefully acknowledges CSIR (New Delhi, India) forhe award of Senior Research Fellowship.

Funding: DST, New Delhi, India.Competing interests: None declared.Ethical approval: Animal Ethics Committee of PGIMER

Chandigarh, India).

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