Azoleantifungals asnovel chemotherapeutic agents againstmurine tuberculosisZahoor Ahmad, Sadhna Sharma & G.K. Khuller

Department of Biochemistry, Postgraduate Institute of Medical Education & Research, Chandigarh, India

Correspondence: G.K. Khuller, Department

of Biochemistry, Postgraduate Institute of

Medical Education & Research, Chandigarh

160 012, India. Tel.:191 0172 2755 175;

fax:191 0172 2744 401; e-mail:

gkkhuller@yahoo.co.in

Received 19 March 2006; revised 22 May 2006;

accepted 30 May 2006.

First published online 4 July 2006.

DOI:10.1111/j.1574-6968.2006.00350.x

Editor: Roger Buxton

Keywords

tuberculosis; chemotherapy; econazole;

antitubercular drugs.

Abstract

The present study was designed to evaluate the in vivo antimycobacterial potential

of econazole alone and in combination with antitubercular drugs against tubercu-

losis in mice. Econazole was found to reduce bacterial burden by 90% in the lungs

and spleen of mice infected with 1� 107 cells of Mycobacterium tuberculosis and

was found to be equipotent to rifampicin. Further, our results indicate that

econazole can replace rifampicin/isoniazid as well as both rifampicin and isoniazid

in chemotherapy of murine tuberculosis. Econazole alone or in combination

with antitubercular drugs did not produce any hepatotoxicity in normal or

M. tuberculosis-infected mice.

Introduction

Despite the introduction of directly observed treatment,

short-course (DOTS) in 1995, one-third of the world’s

population is still infected with Mycobacterium tuberculosis

(Cardona & Ruiz-Manzano, 2004). Therefore, novel drug

strategies are desperately needed to combat the rising

incidence of tuberculosis (TB), especially the multidrug

resistant form (MDR-TB), and to shorten the duration of

tuberculosis chemotherapy (Zhang, 2005). Bioinformatic

analysis of the genome of M. tuberculosis has offered new

insights and this has revealed a large number of ORFs that

are similar to known sterol biosynthetic enzymes, including

a homologue of a CYP P450, fungal gene encoding 14a-

demethylase, an important fungal protein required for sterol

biosynthesis (McLean et al., 2002). This fungal enzyme

(14a-demethylase) is inhibited by azole compounds or

their derivatives, which explains their antifungal activity

(Hartman, 1997). Sterols have been isolated from mycobac-

teria (Garbe, 2004) and inhibitory activity of several azoles

has been documented against Mycobacterium smegmatis,

M. tuberculosis H37Ra and Streptomyces coelicotar (Sun &

Zhang, 1999; Guardiola-Diaz et al., 2001). These drugs have

been shown to have multiple targets in mycobacteria

(McLean et al., 2002) and recent studies have demonstrated

that azole drugs inhibit the biosynthesis of glycopeptido-

lipids (GPLs), which in turn are responsible for maintaining

the integrity of the mycobacterial cell envelope (Burguiere

et al., 2005). Recently, we have demonstrated the in vitro and

ex vivo potential of azole drugs (econazole and clotrimazole)

against M. tuberculosis H37Rv as well as the synergism of

azole drugs with conventional antitubercular drugs (Zahoor

et al., 2005). The antituberculosis potential of azole drugs

against latent or persistent tuberculosis has also been

demonstrated and econazole-like rifampicin has been

shown to prevent the formation of persistent or latent bacilli

in mice and is more effective than rifampicin against

persistent bacilli (Zahoor et al., 2006). These results

prompted us to evaluate the in vivo antimycobacterial

potential of econazole alone and in combination with

antitubercular drugs (ATDs) against murine tuberculosis.

Materials and methods

Chemicals and drugs

Econazole, isoniazid, rifampicin, pyrazinamide and etham-

butol were obtained from Sigma Chemical Co. (St Louis,

MO). Middlebrook 7H11 agar and OADC were obtained

from Beckton and Dickinson, USA. Standard kits for

estimations of serum alanine aminotransferase (ALT), alka-

line phosphatase (ALP) and total bilirubin were obtained

FEMS Microbiol Lett 261 (2006) 181–186 c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

from Transasia Bio-medicals Ltd (Daman, India). All other

reagents were of analytical grade obtained from standard

companies.

Animals

Laca mice (out-bred) of either sex weighing 20–25 g ob-

tained from the Central Animal House, Postgraduate In-

stitute of Medical Education and Research, Chandigarh

(India) were used in the study. Animals were housed in

biosafety cabinets (Nuaire Instruments, NU 605-600E,

Series 6, 2100 Fern brook Lane, Plymouth, MN) and were

given pellet diet and water ad libitum. The Institute’s Animal

Ethics Committee approved the study.

Culture

Mycobacterium tuberculosis H37Rv, originally obtained from

National Collection of Type Cultures (NCTC, London) was

maintained on modified Youman’s medium.

Organ drug distribution studies

The drug doses used throughout the study were: rifampicin

12 mg kg�1, isoniazid 10 mg kg�1, pyrazinamide 25 mg kg�1,

ethambutol 16 mg kg�1 and econazole 3.3 mg kg�1 body

weight according to the standard adult human doses de-

scribed previously (Pandey et al., 2003; Pandey et al., 2005).

For the single oral dose drug disposition studies, mice were

grouped as follows (6–8 animals per group): Group 1,

econazole; Group 2, ATDs (isoniazid, rifampicin, pyrazina-

mide and ethambutol); Group 3, econazole1ATDs (isonia-

zid, rifampicin, pyrazinamide and ethambutol). The

animals were bled at several time points and the plasma

obtained from each animal was divided into two parts.

The first part (100mL) was deproteinized with 100 mL of

acetonitrile, vortexed for 5 min and centrifuged at 5000 g

for 20 min at 4–8 1C. The supernatant was used for the

analysis of rifampicin and ethambutol. The second

portion of plasma (50 mL) was deproteinized with 50 mL of

10% w/v trichloroacetic acid, processed as above and

analyzed simultaneously for isoniazid and pyrazinamide.

For the estimation of econazole, the plasma samples

were deproteinized with methanol (1/1 v/v), vortexed and

centrifuged at 5000 g for 20 min at 4–8 1C. Animals were

also sacrificed at different time points, 20% w/v of tissue

homogenates (lungs, liver and spleen) were prepared and

analyzed for drug levels by following the same analytical

procedure as described for plasma. All the drugs were

analyzed by HPLC (Perkin Elmer Instruments LLC, Shelton,

CT) as described earlier (Pandey et al., 2005; Zahoor et al.,

2005).

Toxicity studies

The mice were grouped as follows (n = 8 per group): Group

1, control animals PBS daily; Group 2, Econazole twice

daily; Group 3, 4-antitubercular drug combination once

daily; Group 4, Econazole and 4-antitubercular drug com-

bination once daily (econazole and ethambutol twice daily).

Each group received the mentioned drugs/PBS orally for 28

days at therapeutic doses. Blood was collected by cardiac

puncture for the analysis of total bilirubin, alanine transa-

minase and alkaline phosphatase using standard kits. Simi-

lar estimations were done in infected animals on the 57th day

of chemotherapy to evaluate hepatotoxic effects, if any,

during chemotherapy.

Chemotherapeutic studies

Mice were infected via the lateral tail vein with 1� 105/

1� 107 bacilli of M. tuberculosis H37Rv as described earlier

(Zahoor et al., 2005; Pandey et al., 2006). The confirmation

of infection and basal bacterial load were determined as

described earlier (Zahoor et al., 2005). Subsequently, mice

were grouped as follows (eight animals per group): Group I,

untreated controls (received PBS); Group II, econazole;

Group III, rifampicin; Group IV, isoniazid, pyrazinamide,

ethambutol and rifampicin; Group V, econazole, isoniazid,

pyrazinamide and ethambutol; Group VI, isoniazid,

pyrazinamide and ethambutol; Group VII, econazole,

pyrazinamide, ethambutol and rifampicin; Group VIII,

pyrazinamide, ethambutol and rifampicin; Group IX, eco-

nazole, pyrazinamide and ethambutol. Antitubercular drugs

(isoniazid, pyrazinamide, ethambutol and rifampicin) were

administered once daily; however, econazole and ethambu-

tol (in the presence of econazole) were administered twice

daily. Animals were sacrificed on days 31, 46 and 58 of

chemotherapy; lungs and spleen were isolated under sterile

conditions and homogenized in 3 mL isotonic saline. One

hundred microlitres of undiluted, 1 : 10 and 1 : 1000 diluted

homogenates were plated on Middlebrook 7H11 agar plates

supplemented with oleic acid albumin dextrose catalase

(OADC) for CFU enumeration and colonies were counted

on day 28 post-inoculation.

Statistical analysis

The CFU data were analyzed by one-way analysis of variance

(ANOVA) followed by the Student’s unpaired t-test to com-

pare the control and treated groups.

Results and discussion

All the drugs alone or in combination were cleared from the

circulation within 24 h. However, plasma levels of rifampicin

were found to be higher, while levels of isoniazid and

ethambutol were lower in the presence of econazole (Fig. 1).

FEMS Microbiol Lett 261 (2006) 181–186c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

182 Z. Ahmad et al.

No significant differences were observed in plasma levels

of pyrazinamide or econazole between combination (ATD1

econazole) and ATD/econazole administered groups

(Fig. 1). The pharmacokinetic evaluation revealed that

rifampicin in the presence of econazole attained

significantly higher values of Cmax and (AUC)0�1 and low

values of Tmax and Kel (Table 1). The pharmacokinetic

parameters of isoniazid, pyrazinamide and ethambutol were

such that the (AUC)0�1 of each of these drugs decreased in

the presence of econazole (Table 1). The pharmacokinetics

of econazole were not significantly different in the presence

of ATDs.

Econazole Rifampicin

0

0.05

0.1

0.15

0.2

0.25

0.5 0.75 1 1.5 2 2.5 3 3.5Time (h) Time (h)

Free Econazole Free Econazole + 4ATDs

Isoniazid Pyrazinamide

Ethambutol

0

0.5

1

1.5

2

2.5

1 2 3 4 6 12 24

Time (h)1 2 4 6 12 24

Time (h)1 2 4 6 12 24

Time (h)1 2 4 6 12 24

Free 4ATDs Free Econazole + 4ATDs

00.20.40.60.8

11.21.41.61.8

2

Free oral ATDs Free oral ATDs+Econazole

0

5

10

15

20

25

30

Free oral ATDs Free oralATDs+Econazole

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

mcg

mL−1

mcg

mL−1

mcg

mL−1

mcg

mL−1

mcg

mL−1

Free oral ATDs Free oral ATDs+Econazole

(a) (b)

(d)(c)

(e)

Fig. 1. Plasma drug profile following oral administration of free econazole alone and in combination with ATDS to mice.

FEMS Microbiol Lett 261 (2006) 181–186 c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

183Azoles as antimycobacterial agents

All the ATDs were detected at or above the minimum

inhibitory concentration (MIC) in tissues (lungs, liver and

spleen) up to day 1 following the oral administration of

antitubercular drugs in the presence or absence of econazole

(Table 2), except ethambutol, which was detected only up to

12 h after co-administration with econazole (Table 2).

Econazole was detected in tissues up to 12 h after adminis-

tration alone or in combination with ATDs (Table 2) above

MIC, as reported earlier for mycobacteria (Zahoor et al.,

2005).

Based on the tissue drug distribution profile, all the four

ATDs (isoniazid, rifampicin, pyrazinamide and ethambutol)

were administered once daily. However econazole and

ethambutol (in presence of econazole) were administered

twice daily.

Eight weeks of chemotherapy with econazole alone re-

sulted in approximately 90% clearance of bacilli from lungs

and spleens of animals infected with 1� 107 cells of M.

tuberculosis as compared to untreated controls (Table 3).

This observation can be explained on the basis of multiple

targets of econazole in M. tuberculosis, as has been shown by

in vitro binding of econazole to various CYP 450s. To date,

econazole has been shown to bind three CYP 450s of M.

tuberculosis and, in fact, azoles bind more tightly to CYP 121

than CYP 151 (the initial suspected target) (McLean et al.,

2002). It has also been demonstrated that MIC of azoles

against M. smegmatis correlated with the Kd values of azoles

for CYP 121, thereby supporting the multiple targets of

azoles in mycobacteria (Leys et al., 2003). Further che-

motherapeutic potential of econazole was comparable to

that of rifampicin, as both these drugs decreased the

bacterial burden from 6.88–6.9 Log10 CFU to 4.87–4.89

Log10 CFU and 4.85–4.88 Log10 CFU, respectively, from the

lungs and spleens of infected mice (Table 3). In view of the

higher frequency of INH/RIF-or INH1RIF-resistant iso-

lates, the potential of econazole to replace these key frontline

antitubercular drugs during tuberculosis chemotherapy was

also evaluated. It was encouraging to observe that the

administration of each four-drug combination (isoniazid,

pyrazinamide, ethambutol and rifampicin or econazole,

isoniazid, ethambutol and pyrazinamide or econazole, ri-

fampicin, ethambutol and pyrazinamide) resulted in unde-

tectable CFU in lung and spleen homogenates as compared

to �4 log CFU in the untreated control group within 4

weeks (Table 4). The most significant observation was the

total bacterial clearance by the three-drug combination

(econazole, pyrazinamide and ethambutol) without RIF

and INH in 6 weeks (Table 4). These findings are in

agreement with the previous reports of 4ATD formulation,

which resulted in total clearance of CFU after 4 weeks of

administration (Pandey et al., 2006; Zahoor et al., 2006).

The present observations can be explained on the basis of

our earlier study, in which the combination of econazoleTab

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FEMS Microbiol Lett 261 (2006) 181–186c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

184 Z. Ahmad et al.

with either rifampicin or isoniazid has been demonstrated

to act synergistically against M. tuberculosis H37Rv (Zahoor

et al., 2005). Further, the role of econazole in reducing

bacterial burden from lungs and spleens of infected mice in

the presence of other ATDs (isoniazid, pyrazinamide and

ethambutol/rifampicin, pyrazinamide and ethambutol/pyr-

azinamide and ethambutol) is supported by the fact that all

of these combinations without econazole cleared half the

bacterial burden in comparison to untreated controls

(Table 4). These results are further supported by the pre-

vious studies, wherein the more potent antitubercular drugs

(isoniazid pyrazinamide1rifampicin) also failed to yield

undetectable CFUs in 4 weeks of chemotherapy (Pandey

et al., 2006). It is also emphasized here that pharmacokinetic

and tissue distribution data clearly demonstrate that econa-

zole mediates its antitubercular effect directly by its bacter-

icidal action and not by elevating the levels of other

antitubercular drugs. Moreover, econazole alone or in

combination was observed not to elevate levels of bilirubin,

ALT and ALP as compared to control animals in normal

(Table 5)/infected animals (data not shown), indicating no

evidence of hepatotoxicity on biochemical basis.

The observation that econazole shows comparable effi-

cacy to that of rifampicin and can replace INH/RIF/INH and

Table 2. Organ drug levels following oral administration of econazole with/without ATDs to mice

Drug Time (h)

Lungs Liver Spleen

ATDs/Econazole ATDs1Econazole ATDs/Econazole ATDs1Econazole ATDs/Econazole ATDs1Econazole

Econazole 12 0.45�0.01 0.15� 0.01 2.21� 0.04 0.16�0.01 0.47� 0.00 0.15� 0.01

Rifampicin 24 0.18�0.00 0.30� 0.01 0.25� 0.00 0.79�0.09 0.25� 0.00 0.30� 0.02

Isoniazid 24 0.11�0.03 0.31� 0.01 0.20� 0.03 0.31�0.01 0.15� 0.03 0.28� 0.02

Pyrazinamide 24 7.00�2.10 10.07� 0.13 7.20� 1.26 11.53�0.09 7.90� 0.94 11.53� 0.09

Ethambutol 24 1.20�0.20 – 0.80� 0.11 – 1.30� 0.23 –

ATDs = INH, PZA, EMB and RIF.

Table 3. Chemotherapeutic efficacy of econazole/rifampicin against

tuberculosis in mice infected with high dose (1x 107) of Mycobacterium

tuberculosis H37Rv

Groups

Log10 CFU

Lung Spleen

Untreated controls 6.88�0.035 6.9� 0.025

RIF 4.85�0.07� 4.88� 0.04�

Econazole 4.87�0.04� 4.89� 0.02�

Values are mean� SD of eight animals.�Po0.01 as compared to untreated controls.

Table 4. Chemotherapeutic efficacy of azoles with or without antitubercular against tuberculosis in mice infected with low dose (1� 105) of

Mycobacterium tuberculosis H37Rv

Groups

4 weeks

chemotherapy

6 weeks

chemotherapy

Log10 CFU Log10 CFU

Lung Spleen Lung Spleen

Untreated controls 4.02� 0.03 4.1�0.04 4.71� 0.04 4.73� 0.03

INH, PZA, EMB and RIF o1.0 o1.0 o1.0 o1.0

Econazole, INH, EMB and PZA o1.0 o1.0 o1.0 o1.0

INH, EMB and PZA 2.5� 0.6�� 2.65�0.5�� o1.0 o1.0

Econazole, RIF, EMB and PZA o1.0 o1.0 o1.0 o1.0

RIF, EMB and PZA 2.37� 0.38�� 2.72�0.42�� o1.0 o1.0

Econazole, EMB and PZA 2.3� 0.03�� 2.32�0.05�� o1.0 o1.0

EMB and PZA N.D. N.D. 2.1�0.62� 2.35� 0.45�

Values are mean� SD of eight animals.�Po0.01, ��Po0.001 as compared to untreated controls. Ao1.0 value in the figure indicates no detectable bacilli.

Table 5. Toxicity studies of econazole and antitubercular drugs in mice

Groups

Serum bilirubin

(mg 100�1 mL)

Serum ALT

(U L�1)

Serum ALP

(U L�1)

Untreated

controls

0.10–0.42 9–37 77–212

Econazole 0.28–0.37 13–37 51–98

ATDs 0.26–0.42 20–42 43–55

Econazole1

ATDs

0.23–0.35 6–13 105–293

ATDs = INH, PZA, EMB and RIF, ALT =alanine aminotransferase and

ALP = alkaline phosphatase.

FEMS Microbiol Lett 261 (2006) 181–186 c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

185Azoles as antimycobacterial agents

RIF during chemotherapy will have a great impact in

curbing multidrug-resistant tuberculosis. This finding is of

immense value because the commonest and most dangerous

forms of resistance in tubercle bacilli are INH/RIF/INH and

RIF. In addition, econazole has been shown to have strong

antimycobacterial potential against persistent/latent tuber-

cle bacilli (Zahoor et al., 2006).Thus, it appears that

econazole will be the real substitute for these two frontline

antitubercular drugs.

To the best of our knowledge, this is the first report

demonstrating the in vivo potential of econazole alone

as well as in combination with ATDs against murine

tuberculosis.

Acknowledgements

Z.A.P. thanks CSIR, New Delhi, India, for the award of

Senior Research Fellowship.

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