Activity of 7-methyljuglone in combination with antituberculous drugs against Mycobacterium tuberculosis.

Abstract

The recent increase in the incidence of tuberculosis with the emergence of multidrug-resistant (MDR) cases has lead to the search for new drugs that are effective against MDR strains of Mycobacterium tuberculosis and can augment the potential of existing drugs against tuberculosis. In the present study, we investigated the activities of a naphthoquinone, 7-methyljuglone, isolated from the roots of Euclea natalensis alone and in combination with other antituberculous drugs against extracellular and intracellular M. tuberculosis. Combinations of 7-methyljuglone with isoniazid or rifampicin resulted in a four to six-fold reduction in the minimum inhibitory concentration of each compound. Fractional inhibitory concentration (FIC) indexes obtained were 0.2 and 0.5, respectively, for rifampicin and isoniazid, suggesting a synergistic interaction between 7-methyljuglone and these anti-TB drugs. The ability of 7-methyljuglone to enhance the activity of isoniazid and rifampicin against both extracellular and intracellular organisms suggests that 7-methyljuglone may serve as a promising compound for development as an anti-tuberculous agent.

[c] 2006 Elsevier GmbH. All rights reserved.

Keywords: Antituberculous drug; 7-methyljuglone; Synergy; Isoniazid; Rifampicin

Introduction

The evaluation of the effectiveness of newly developed antituberculous agents against isolates of M. tuberculosis has become more important in the past few years, predominantly due to the appearance of multidrug-resistant tuberculosis (Bergmann and Woods, 1998). Such testing may also provide useful information for the treatment of patients who have active disease and cannot tolerate one or more of the primary antituberculous agents. Active tuberculosis is treated with more than one drug, therefore, in vitro evaluation of the activity of existing drug combination with novel bioactive anti-TB compounds is imperative.

An important consideration in the treatment of tuberculosis is the fact that the ethiological agent, M. tuberculosis, has the ability to persist intracellularly in the host macrophage for long periods of time (Quenelle et al., 2001). Optimum therapy, therefore, must depend upon the ability of the antituberculous agent to eradicate the bacteria within the macrophage. This becomes even more important when one considers the ability of M. tuberculosis to persist in a dormant state, thus giving rise to a large group of infected individuals who carry the organism in a sub-clinical state without having active disease (Collins, 1998). Novel bioactive compounds which could act synergistically with the classic tuberculosis drugs within the host macrophages might have direct access to dormant organisms that presumably would be within macrophages or in the surrounding lymphatic area (Quenelle et al., 2001).

First-line drugs used for the treatment of infections caused by M. tuberculosis include isoniazid (INH), rifampicin (RMP), ethambutol (EMB), streptomycin (SM) and pyrazinamide (PZA) (Snider and La Mont-agne, 1994). Most in vitro studies evaluate the efficacy of new drugs as single agents but since, in clinical practice, a combination of various drugs is necessary to prevent the development of drug resistance during therapy, studies of drug combinations are therefore necessary (Bergmann and Woods, 1998).

Euclea natalensis is a tree belonging to the family Ebeneceae that comprises about 500 species is widely distributed in tropical, subtropical Africa and is common on the East coast of South Africa. The phytochemical and biological studies of E. natalensis have been initiated due to continuous use of this plant in traditional African medicine. The Zulu and Shangaans (tribes of South Africa) use roots of the plant for curing different forms of chest complaints such as bronchitis, pleurisy and chronic asthma. The customs of cleaning teeth and gums with the charred roots of E. natalensis, in the belief that it benefits oral health, is practiced in South Africa by married women of the Zanzibari community. Charred and powdered root are used as a dye, applied to the skin lesions in leprosy and for ancylostomasis (Bryant, 1966; Watt and Breyer-Brand-wijk, 1962). The powdered roots are also used to relieve headache and toothache by local inhabitants of South Africa (Palgrave and Drummond, 1977).

The large subtropical genus Euclea is well known as a source of naphthoquinones. Naphthoquinones have a wide range of activities, including antispasmodic, antibacterial and antipruritic effects (Bruneton, 1995). Monomers, complex dimers and trimers such as 7-methyljuglone (7-MJ), diospyrin, and mamegakinone isolated from Euclea have been reported to possess antibacterial activity against Neisseria gonorrhoea, Shigella dysenteriae and Shigella flexnerri (Khan et al., 1978; Hazra and Sur, 1984).

In a previous paper, the activity of 7-methyljuglone, a naphthoquinone isolated from the roots of Euclea natalensis, was determined both in axenic medium and in a J774A.1 macrophage cell line (Lall et al., 2005). In this study, we investigated the effects of 7-MJ alone and in combination with RMP, INH, EMB and SM, against M tuberculosis H37Rv and two clinical strains of M. tuberculosis.

Materials and methods

Compounds used

RMP, INH, EMB and SM were purchased from Sigma (Sigma Chemical Company, St. Louis, MO). A naphthoquinone, 7-MJ was isolated from the roots of E. natalensis as described previously (Lall, 2002) and dissolved in dimethyl sulfoxide (DMSO) to a final stock solution of 10 mg/ml. The highest concentration of DMSO used (1%) was found to be non-toxic to all M. tuberculosis strains tested.

Mycobacterial strains

The H37Rv ATCC 27294 strain of M. tuberculosis was obtained from the American Type Culture Collection (Rockville, MD). Two clinical strains of M. tuberculosis (WH51/02 and WC64/02), isolated from sputum samples of patients suffering from tuberculosis, were obtained from the Tuberculosis research Institute of the South African Medical Research Council (Pretoria, South Africa).

Antimicrobial activity

All the cultures of M. tuberculosis were plated onto Lowenstein-Jensen medium and allowed to grow for 3-4 weeks at 37 [degrees]C before use. Once there was satisfactory growth of bacteria on a Lowenstein-Jensen slant, bacteria were carefully scraped and transferred into a sterilized glass tube containing a few glass beads (2 mm in diameter). Five milliliters of 0.04% Tween 80/saline was added to the culture. The suspension was homogenized by using a vortex mixer (Heidolph, Germany) and then left undisturbed for about 15-30min to allow clumped bacteria to settle to the bottom of the tube. The supernatant was carefully aspirated with a pipette, transferred to an empty sterile tube, and turbidity adjusted to approximate that of a McFarland no.1 standard by adding 0.04% Tween 80/saline.

Minimum inhibitory concentrations (MIC) were determined radiometrically using the BACTEC 460 apparatus (Becton Dickinson Diagnostic Instrument Systems, Sparks, MD) as reported previously (Lall et al., 2003). A volume of 0.1 ml of a bacterial suspension equivalent to McFarland no.1 was injected into a BACTEC 12B vial. The vial was incubated until a Growth Index (GI) of 300-500 was obtained. A 0.1 ml volume from the inoculum was then added to 2 drug-free control vials (medium + 1% DMSO). An additional 1:100 dilution of the inoculum was made and added to 2 drug-free control vials. Final concentrations of the test compounds ranged in a twofold dilution from 0.031 to 1 [micro]g/ml for 7-MJ, from 0.007 to 0.25 [micro]g/ml for RMP, from 0.007 to 0.25 [micro]g/ml for INH, from 0.016 to 5 [micro]g/ml for EMB and 0.078 to 2.5 [micro]g/ml for SM. Vials inoculated with compounds were incubated at 37[degrees]C. Growth indices on the BACTEC 460 instrument were monitored daily. When the 1:100 drug-free controls reached a GI of 30 or more, the experiment was stopped and the MIC determined. The MIC was defined as the minimum concentration of the compounds for which GI was below that of the 1:100 drug-free control vials.

Cytotoxic activity

7-MJ was dissolved initially in DMSO to make a stock solution of 10mg/ml; then serial two-fold dilutions were made, so that the final concentration of DMSO in each well was 1%. Cytotoxicity was determined by exposing different concentrations of 7-MJ (0.78-50 [micro]g/ml) to MRC-5, a primary fibroblast cell line (ATCC CCL-171), human lymphocytes, human monocyte-derived macrophages and THP-1, a human myelomo-nocytic cell line displaying macrophage-like activity (ATCC TIB 2.2). MRC-5 was maintained in DMEM (Sigma Diagnostics, St. Louis, MO, USA), supplemented with 10% fecal calf serum (heat inactivated at 37[degrees]C), 2mM glutamine, and 1% penicillin/streptomycin solution. Blood obtained from healthy volunteers was used to prepare lymphocytes and monocyte-derived macrophages using Histopaque-1077 (Sigma Diagnostics) as described by van Rensburg et al. (2000). Lymphocytes and monocyte-derived macrophages were stimulated with Phytohaemagglutinine (PHA) (BIOWEB Pty Ltd, Eden Glen, Johannesburg, South Africa). Lymphocytes, human monocyte-derived macrophages and THP-1 cells were maintained in RPMI-1640 supplemented with 10% autologous serum/fetal calf serum (heat inactivated at 37 [degrees]C), 2mM glutamine, and 1% penicillin/streptomycin solution. Cytotoxicity was determined in 96-well tissue culture plates (Becton Dickinson Labware, Lincoln Park, NJ) at an initial cell concentration of 6 x [10.sup.4] cells/well and incubated with 7-MJ at 37[degrees]C in an atmosphere of 5% C[O.sub.2] in air. After 4 days, cell viability was determined by the MTT assay (Mosman, 1983) with modifications as described by Van Rensburg et al. (2000). The 50% inhibitory concentration (I[C.sub.50]) value represents the concentration of the compounds which inhibits 50% of cell growth.

Intracellular activity

THP-1 cells were maintained at 37[degrees]C in 5% C[O.sub.2] in RPMI-1640 medium supplemented with 10% fetal calf serum, 2 mM glutamine, and 1% penicillin/streptomycin solution. For the experimental procedure 200 [micro]l of the cell suspension was added to each well of a 96 well plates at a concentration of 5 x [10.sup.5] cells/well. These cells were induced to differentiate into adherent cells by a 24-h exposure to 5g/ml phytohemagglutinin (PHA, Difco laboratories, Detroit, MI).

After incubation, the RPMI-1640 medium was removed, replaced with fresh RPMI-1640 medium and the cells incubated for another hour. Macrophage monolayers were infected with bacteria at a ratio of 50:1 (bacteria: macrophages), incubated for 4 h at 37 [degrees]C in humidified 5% C[O.sub.2] atmosphere and washed three times with RPMI-1640 medium (without antibiotic) to remove extracellular bacteria. After the third wash, 180 [micro]l RPMI-1640 (without antibiotics) was added to the wells and 20 [micro]l of the test compound was added to all the wells except the control wells. Final concentrations of the test compounds were between 0.312 and 10 [micro]g/ml for 7-MJ, between 0.031 and 1 [micro]g/ml for RMP and between 0.003 and 0.120 for INH. The cultures were then incubated for 5 days at 37 [degrees]C in a humidified 5% C[O.sub.2] atmosphere.

After the incubation period, the medium was removed and the intracellular bacteria released by lyses with 0.5% sodium dodecyl-sulphate (SDS). MICs were determined by the radiometric BACTEC 460 method (Siddiqi et al., 1981). BACTEC vials were inoculated with the bacterial suspension as explained before (2.3) and incubated at 37 [degrees]C overnight. The change in GI was recorded daily until the vials containing the 1:100 dilution of the untreated control suspension reached a GI of 30 or more. The MIC was defined as the minimum concentration of the test compound for which change in GI was below that of the 1:100 untreated control vials.

Combined drug action

The activity of two-drug combinations was evaluated in axenic medium and in a macrophage cell line (THP-1) at sub-MIC levels (below original MIC values) so that each compound was present at con centrations corresponding to 1/2, 1/4, 1/8, 1/16 and 1/32 of the MIC. Analysis of the drug combination data was achieved by calculating the fractional inhibitory concentration (FIC) index (Berenbaum, 1978; De Logu et al., 2002) as follows: FIC = (MI[C.sub.a combination]/MI[C.sub.a alone]) + (MI[C.sub.b combination]/MI[C.sub.b alone]) where a = irst compound and b = second compound used in the synergistic study. The FIC was interpreted as: FIC [less than or equal to]0.5, synergistic activity; FIC = 1, indifference/additive activity; FIC [greater than or equal to] 2, antagonistic activity.

Results

Combined drug activity determined by the radiometric method

The MICs obtained by 7-MJ, RMP, INH, EMB and SM as well as the MIC and FICs obtained with combinations of 7-MJ with the standard antituberculosis drugs against the M. tuberculosis strains tested are listed in Table 1. The MICs and FICs for each two-drug combination tested were similar for all three strains tested. Combination of 7-MJ with RMP reduced the MIC four-fold and produced an FIC of 0.5 suggesting a borderline synergistic effect. The most pronounced effect of the two-drug combination was demonstrated by the combination of 7-MJ with INH. This combination reduced the MICs of both compounds by eight-fold. The FIC for this combination was 0.25 indicating a synergistic combination. Combination of 7-MJ with either EMB or SM showed an FIC of 1.0 indicating an indifferent or additive effect of these drugs for the M. tuberculosis strains tested.

Cytotoxic activity

Sensitivity of various cell cultures for 7-MJ is shown in Table 2. 7-MJ is cytotoxic for resting and stimulated lymphocytes with I[C.sub.50] values of 3.97 and 3.46 [micro]g/ml. MRC-5, THP-1 and monocyte derived macrophages were not sensitive to 7-MJ up to a concentration of 50 [micro]g/ml.

Combined intracellular drug activity

The MICs and FICs obtained by the combination of 7-MJ with either RMP or INH against intracellular M. tuberculosis (H37Rv) are listed in Table 3. Combination of 7-MJ with RMP reduced the MIC of both compounds by four-fold. The FIC was 0.5 suggesting a borderline synergistic effect. Combination of 7-MJ with INH reduced the MIC of both compounds six-fold. The FIC index for this combination was 0.24 indicating a more pronounced synergistic action between 7-MJ and INH versus 7-MJ and RMP.

Discussions

The combination of 7-MJ with either RMP or INH showed both extracellular and intracellular synergistic activity against the tested strains of M. tuberculosis. Interestingly, sub-inhibitory concentrations of 7-MJ combined with INH showed a more pronounced synergistic activity. INH is a first-line drug used for direct treatment programmes because its activity against both extracellular and intracellular bacilli inhibits the development of resistant M. tuberculosis. Bulatovic et al. (2002) showed that M. tuberculosis, exhibited increased susceptibility to INH when combined with plumbagin (2-methyljuglone), another naphthoquinone with similar structure, indicating synergistic activity. Plumbagin is known to increase the intracellular superoxide concentration (Bulatovic et al., 2002) whereas the KatG enzyme of M. tuberculosis also appears to act as a peroxidase, utilizing hydrogen peroxide to oxidize INH to its active form (Mo et al., 2004), possibly explaining the synergistic activity that exist between these two compounds. This might also be the case with 7-MJ together with INH.

Cushion et al. (2000) showed that Atavaquone, a naphthoquinone, inhibits the electron transport chain in Pneumocytis carinii leading to the inhibition of oxygen consumption by the organism. 7-MJ may have similar mechanism of action which might inhibit the oxygen consumption of M. tuberculosis and its subsequent inhibition. Atovaquone has also been used in combination with proquanil hydrochloride for the treatment of acute Plasmodium falciparum malaria in children (Ana-bwani et al., 1999). The mechanism of action in malarial parasites has been shown to be inhibition of mitochondrial transport at the cytochrome [bc.sub.1] complex and subsequent breakdown of the mitochondrial membrane potential and ATP synthesis. This blockade results in the repression of pyrimidine biosynthesis by the inhibition of dihydroorotate dehydrogenase (DHOD). Since Plasmodium spp. cannot utilize host pyrimidines, blockage of DHOD function and de novo pyrimidine synthesis is lethal for the organism (Srivastava et al., 1997). DHOD is essential for M. tuberculosis growth and its collapse leads to the killing of the bacterium. Since we have shown that 7-MJ is toxic to cells it might be that it is also toxic to the mycobacterial mitochondria by blocking of DHOD function and subsequent killing of the bacterium.

Lall et al. (2005) found that 7-MJ has superior extracellular and intracellular activity against M. tuberculosis (H37Rv) relative to SM and EMB. In this study, 7-MJ was found to have synergistic activity when combined with either INH or RMP. The lack of sensitivity of monocytic cells to 7-MJ and the moderate cytotoxicity of 7-MJ for human lymphocytes suggests that it needs further evaluation as lymphocytes are critically important in tuberculosis and HIV.

Although the mechanism of synergy between 7-MJ with either RMP or INH in drug susceptible strains of M. tuberculosis is unknown, this finding is new and potentially significant. It would be of interest to determine if this synergistic effect could be demonstrated in an animal model of infection. If proven effective and relatively non-toxic in humans, addition of 7-MJ to INH or RMP may provide an alternative regimen for prophylactic or preventive treatment of persons likely to be infected with single drug-resistant strains of M. tuberculosis.

Acknowledgements

We thank Mr B Mooketsi and Dr T Muthivhi for their assistance on extracellular experiments. Mr F van der Kooy, Dr GK Joone and Ms M Nell for their technical support on the macrophage and cytotoxicity work. This work was supported by Grants from the South African Medical Research Council and the National Research Foundation.

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N.B. Bapela (a), N. Lall (b), P.B. Fourie (c), S.G. Franzblau (d), C.E.J. Van Rensburg (a,*)

(a) Department of Pharmacology, University of Pretoria, PO Box 2034, Pretoria, 0001, South Africa

(b) Department of Botany, University of Pretoria, Pretoria, South Africa

(c) Medical Research Council of South Africa, TB Research Lead Programme, Pretoria, South Africa

(d) Institute for Tuberculosis Research, College of Pharmacy, University of Illinois, Chicago, USA

*Corresponding author. Tel.: [phone]; fax: [phone].

E-mail address: [e-mail] (C.E.J. Van Rensburg).

Table 1. Synergistic activity of 7-MJ with existing antituberculous
drugs against drug-sensitive M. tuberculosis strains using the BACTEC
method

               MIC (a) ([micro]g/ml) and FIC (b) of compounds and
               compound combinations against drug-sensitive M.
               tuberculosis strains
Compounds and  H37Rv              WC 64/02           WH 51/02
combinations   MIC          FIC   MIC          FIC   MIC          FIC

7-MJ           0.5          --    0.5          --    0.5          --
RMP            0.125        --    0.125        --    0.125        --
INH            0.062        --    0.062        --    0.062        --
EMB            1.25         --    1.25         --    1.25         --
SM             0.625        --    0.625        --    0.625        --
7-MJ + RMP     0.125/0.031  0.5   0.125/0.031  0.5   0.125/0.031  0.5
7-MJ + INH     0.062/0.007  0.25  0.062/0.007  0.25  0.062/0.007  0.25
7-MJ + EMB     0.250/0.625  1     0.250/0.625  1     0.250/0.625  1
7-MJ + SM      0.250/0.312  1     0.250/0.312  1     0.250/0.312  1

(a) Minimum inhibitory concentration.
(b) Fractional inhibitory concentration.

Table 2. Cytotoxicity of 7-MJ for different cells

                        I[C.sub.50]        Therapeutic index
Cells                   ([micro]g/ml) (a)  (I[C.sub.50]/MIC)

MRC-5 fibroblasts       > 50               > 100
Resting lymphocytes        3.46                6.92
Stimulated lymphocytes     3.97                7.94
Monocyte-derived        > 50                > 50
macrophages
THP-1 macrophages       > 50                > 50

(a) 50% Inhibitory concentration.

Table 3. Intracellular MICs and FICs of combination of drugs acting
against M. tuberculosis by the radiometric BACTEC method

             MIC (a) ([micro]g/ml) and FIC (b) of compound
             and compound combinations against M.
             tuberculosis H37Rv strain
                   H37Rv
Combination  MIC         FIC

7-MJ         5           --
RMP          0.5         --
INH          0.06        --
7-MJ + RMP   1.25/0.125  0.5
7-MJ + INH   0.62/0.007  0.24

(a) Minimum inhibitory concentration.
(b) Fractional inhibitory concentration.