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Activity of diclofenac used alone and in combination with streptomycin against Mycobacterium tuberculosis in mice

https://doi.org/10.1016/j.ijantimicag.2007.04.016Get rights and content

Abstract

The non-steroidal anti-inflammatory drug diclofenac (DCL) shows noteworthy in vitro and in vivo antimycobacterial activity. The aim of this study was to ascertain whether DCL used in combination with the first-line antitubercular antibiotic streptomycin (STM) synergistically augments its efficacy in vitro as well as in a murine tuberculosis infection model. In vitro minimum inhibitory concentrations (MICs) and synergistic activities of the drugs with respect to standard strains and clinical isolates of Mycobacterium tuberculosis were determined. Swiss albino male mice were intravenously infected with 2.3 × 107 M. tuberculosis H37Rv. Mice were treated with DCL or STM alone as well as in combination for 4 weeks to determine the survival rate, spleen weight and colony-forming unit (CFU) counts in the lungs and spleen. DCL was bactericidal at 40 μg/mL (4× MIC) against M. tuberculosis H37Rv and was synergistic with STM in vitro (fractional inhibitory concentration index 0.37). A dose of 10 μg/g/day DCL or 150 μg/g/day STM for 4 weeks, administered from 1 day post infection, significantly (P < 0.05) lowered bacterial counts and reduced mean spleen weight of mice compared with untreated animals. Simultaneous administration of both agents further decreased CFU counts (P < 0.05) in the lungs and spleen compared with mice receiving STM alone. Thus, the ability of extended antibiotic therapy may be improved with the help of this synergistic drug pair in murine tuberculosis, and further investigations may throw light on new directions to combat multidrug-resistant tuberculosis infections in humans.

Introduction

Multidrug resistance among Mycobacterium tuberculosis in particular and mycobacteria in general is a global problem requiring surveillance and control. Failure to cure effectively multidrug-resistant tuberculosis (MDR-TB) and latent infections using existing antibiotics and chemotherapeutics necessitates a search for newer and potent drugs, which also accelerate current treatment by shortening the total duration of therapy. Studies aimed at discovering antimycobacterial ‘non-antibiotics’ have demonstrated in vitro and in vivo enhancement of antibiotic activity against MDR-TB [1]. The anti-inflammatory drug diclofenac sodium (DCL) possesses powerful biocidal activity towards Gram-positive and Gram-negative bacteria [2], [3] and mycobacteria [4]. It was synergistic with streptomycin (STM) against Mycobacterium smegmatis [5] and strains of Escherichia coli and Staphylococcus aureus, and also with a non-antibiotic trifluoperazine against some Gram-positive and Gram-negative bacteria [6], [7].

Prolonged and widespread use of STM as monotherapy in tuberculosis has resulted in an average of 1 in 108 tubercle bacilli being resistant to STM at 10–100 μg/mL. Previously, STM was used at high doses, but its toxicity, coupled with development of resistance, limited its usefulness. As a result, short-course therapy with STM was started (lower doses given over long periods). This delayed the appearance of resistance and reduced toxicity. Subsequently, with the discovery of more drugs, combination therapy was started. However, successful treatment requires several months of therapy. Therefore, new options need to be explored to shorten the duration of treatment.

Patients treated for tuberculosis are often administered non-steroidal anti-inflammatory drugs such as DCL for inflammation. With this in mind, this study was undertaken to examine the possible effects of DCL on treatment of tuberculosis with STM in a murine model.

Section snippets

Drugs

DCL, rifampicin (RIF), STM, ethambutol (ETB) and isonicotinic acid hydrazide (INAH) were kindly donated by their respective manufacturers. Initial stock solutions (preserved at 4 °C) were made in distilled water or dimethyl sulphoxide (DMSO) at 10 μg/mL or 20 μg/mL, from which further dilutions were made in 7H9 broth (Difco Laboratories, Detroit, MI). The maximum concentration of DMSO was 0.5% to avoid interference.

Bacteria

Forty-five strains of mycobacteria were tested, of which 18 were Trudeau Memorial

MIC of DCL against mycobacteria

The MIC of DCL against the 45 strains of mycobacteria tested ranged from 10 μg/mL to 25 μg/mL (MIC for 50% of the organisms (MIC50) = 20 μg/mL). Of these, 18 were sensitive to conventional antimycobacterial drugs (STM, RIF, INAH and ETB), 8 were multidrug resistant and 19 were polydrug resistant. The MIC of DCL is five to six times higher than that of conventional drugs. Susceptible strains such as M. tuberculosis H37Rv 102 were inhibited at lower doses of the conventional agents (0.5–2 μg/mL); the

Discussion

Antimycobacterial non-antibiotics discovered so far mostly have in vitro MIC values ranging from 10 μg/mL to 25 μg/mL [1], [4], [5]. DCL is biocidal both to Gram-positive and Gram-negative bacteria [2], [3]. In the current study, the MIC (10 μg/mL) and MBC (40 μg/mL) of DCL against species of M. tuberculosis H37Rv 102 are in agreement with accounts from other workers. DCL has significant structural correlation with reported antimycobacterial non-antibiotics in the phenothiazine group.

This study

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These two authors contributed equally to this work.

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