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1 Department of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD 21205, USA
2 Center for Tuberculosis Research, School of Medicine, Johns Hopkins University, Baltimore, MD 21231, USA
Correspondence
Ying Zhang
yzhang{at}jhsph.edu
Received 9 November 2006
Accepted 10 April 2007
Abbreviations: INH, isoniazid; KTC, ketoconazole; PZA, pyrazinamide; RIF, rifampicin; SIT, serum inhibitory titre; TB, tuberculosis.
These authors contributed equally to this work. 
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INTRODUCTION |
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 TOP INTRODUCTION METHODSRESULTS AND DISCUSSIONREFERENCES |
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Successful treatment of TB involves targeting the multiple populations of bacteria that reside in the host. Currently, TB is treated with a combination of drugs including isoniazid (INH), rifampicin (RIF), pyrazinamide (PZA) and ethambutol given for 2 months, followed by a 4 month treatment with INH and RIF (American Thoracic Society et al., 2003). The lengthy therapy is often associated with poor patient compliance and the emergence of drug resistance.
We have shown previously that members of the azole class of antifungal agents had significant anti-TB activity in vitro (Sun & Zhang, 1999). In addition, an interesting property of the azole drugs is that they are active against growing bacilli and, to some extent, stationary-phase bacilli. This latter property may be important for shortening the duration of TB therapy by killing a population of bacilli not readily killed by current TB drugs. Of the azole drugs, ketoconazole (KTC) was selected for examination, as it is a commonly used, systemic drug (unlike some of the other azoles that are used topically). This study evaluated KTC for its activity against M. tuberculosis in vitro and in the mouse model.
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METHODS |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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MIC and minimum bactericidal concentration (MBC) determination. Twofold dilutions of each drug were prepared in 7H9 broth with ADC. Each tube was inoculated 1 : 10 with a 10–1 dilution of a 10- to 14-day-old culture (final concentration of about 105 c.f.u.) of H37Rv or PAS- and streptomycin-resistant H37Rv ATCC 35824. After 14 days, tubes were inspected for visible growth and c.f.u. counts were determined for those dilutions with no visible growth by plating on 7H11 agar with ADC. The MIC value was the drug concentration that completely inhibited growth. The MBC value was the minimum concentration that killed 99 % of inoculated bacteria. Determination of MIC and MBC was performed under both neutral (pH=6.7–6.8) and acidic (pH=5.5–5.8) conditions.
Drug exposure assay.
Two-month-old cultures of strain H37Ra were washed and resuspended in an equal volume of 7H9 broth without ADC or glycerol supplementation and exposed for 3 days to KTC (1, 5, 10 or 50 µg ml–1), RIF (10 µg ml–1) or PZA (100 µg ml–1), or combinations of RIF, PZA and KTC (Table 1
). Drugs were dissolved in DMSO and diluted to a final concentration of 0.1 % DMSO. Prior to inoculation, the medium was adjusted to a pH of 5.5–5.8 with 1 M HCl (Fisher Scientific). Solvent alone was used as a control. Following exposure for the indicated time, cells were washed and resuspended to remove the drug(s) and plated on 7H11 agar with ADC and 0.5 % glycerol supplementation to enumerate the remaining live cells.
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Mouse infections. Infections were conducted using a method adapted from Nuermberger et al. (2004). Four- to six-week-old female BALB/c mice (NCI) were infected with high-dose, aerosolized M. tuberculosis. A frozen stock of mouse-passaged H37Rv was cultured in 7H9 broth with ADC and glycerol and incubated for 14 days at 37 °C before use in a Middlebrook Inhalation Exposure System (Glas-Col).
Treatments. Following infection, mice were randomized to a treatment group (four to five mice per group). To allow for established infections, therapy was initiated 16 days after aerosol infection and administered 5 days per week for a month. Drugs were suspended in 0.2 % agarose and administered by oral gavage in a total volume of 0.2 ml. Drugs in combination were administered simultaneously; one control group received carrier only.
Drugs were used in the experiments at the following concentrations: KTC, 75 mg kg–1; INH, 25 mg kg–1; RIF, 10 mg kg–1; and PZA, 150 mg kg–1 (compounds obtained from Sigma). Stock solutions of these drugs were stored at –20 °C between treatment sessions.
Determination of c.f.u. The day after the conclusion of treatment, mice were euthanized. Total lungs and spleens were homogenized in 2 ml PBS. Serial dilutions of 100, 10–2, 10–4 and 10–6 (250 µl) were plated on Middlebrook 7H11 agar (Becton Dickinson) supplemented with ADC (Becton Dickinson) and PANTA (Becton Dickinson) to enumerate the total c.f.u. of M. tuberculosis per organ per mouse. c.f.u. counts from one group of controls were determined at the start of treatment to establish a baseline of infection; c.f.u. counts were also obtained from control mice at the conclusion of the study.
Statistical analyses. c.f.u. counts were converted to log10 values and compared using Student's t-test.
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RESULTS AND DISCUSSION |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Synergy of KTC with PZA and RIF in vitro
In TB infection, it is thought that a subpopulation of bacteria, originally susceptible to antibiotics, is able to persist in tissues despite exposure to drugs (McCune et al., 1957). A compound that targets this subset of cells may be able to shorten the duration of therapy. The adoption of PZA, which has been shown to be active against non-replicating persisting bacilli in vitro (Zhang et al., 2002), into the regimen for TB treatment has partly been responsible for a shortening in the duration of therapy. Through a drug exposure assay that measured activity against non-growing bacteria, KTC was found to exhibit a synergistic effect with RIF and PZA after a 3 day exposure in 7H9 broth without ADC, pH 5.5 (Table 1
). Two-month-old M. tuberculosis H37Ra cultures were exposed for 3 days to KTC (1, 5, 10 or 50 µg ml–1), RIF (10 µg ml–1) or PZA (100 µg ml–1), or combinations of KTC with RIF and PZA under acidic conditions (pH 5.5). RIF was used as a positive control instead of INH because the culture used for drug exposure was 2 months old and it is known that old cultures are not susceptible to INH but are more susceptible to RIF. No synergy was detected following short-term exposure against exponential-phase M. tuberculosis (data not shown).
KTC at a concentration of 50 µg ml–1 exhibited significant anti-TB activity during a short-term exposure on non-growing cells, reducing viable bacterial counts from 5.88 log10 (c.f.u. ml–1) (control) to 4.00 log10 (c.f.u. ml–1) (Table 1). This decrease was greater than that seen with RIF or PZA alone or in combination. Furthermore, considerable anti-TB activity was observed when KTC (50 µg ml–1) was combined with RIF and PZA. After exposure to these three drugs for 3 days, no viable bacteria could be cultured [lower limit of detection was 2 log10 (c.f.u. ml–1)].
Lower concentrations of KTC (1, 5 and 10 µg ml–1) were found to have minimal effects on the non-growing population of TB after a 3 day exposure (reduction in c.f.u. values of less than tenfold). However, when combined with RIF and PZA, these lower concentrations of KTC showed greater anti-TB activity than RIF or PZA alone (Table 1).
The mode of action of KTC may be due to inhibition of M. tuberculosis cytochrome P450 mono-oxygenases (McLean et al., 2002). Azole antifungals have been shown to bind to the P450s CYP121 and CYP51 in M. tuberculosis and may exert their antimycobacterial properties by targeting these molecules (McLean et al., 2002). Azoles have also been found to inhibit the tricarboxylic acid cycle enzyme succinate dehydrogenase (Boshoff et al., 2004). The enhancement of PZA activity by KTC seen in this study could be related to the inhibition of succinate dehydrogenase by KTC such that it makes it easier for PZA, which depletes membrane energy, to kill tubercle bacilli. Further investigation into the mechanism of action of KTC on M. tuberculosis is needed.
Serum inhibitory testing
Before drug assessment in vivo, serum inhibitory testing was performed. Serum inhibitory testing is an in vitro assay that combines pharmacodynamic and pharmacokinetic principles to determine the antibacterial activity of a drug in the serum following administration (NCCLS, 1999). A single 75 mg kg–1 dose of KTC resulted in a serum concentration that was able to inhibit the growth of M. tuberculosis 2 h after oral administration. The SIT is the lowest dilution of serum that inhibits growth of M. tuberculosis in vitro. At the given dose, KTC was found to have an SIT of 1 : 2 to 1 : 4 against M. tuberculosis.
Dose optimization in unestablished murine infection
To optimize oral dosing of KTC in an in vivo murine model of unestablished infection, 4-week-old, female BALB/c mice were infected with an aerosolized, exponential-phase culture of a mouse-adapted strain of M. tuberculosis H37Rv. Treatment was initiated the day after infection and continued for 5 days per week for 4 weeks. By employing a dosing study, KTC at 75 mg kg–1 was found to be the lowest daily dose with activity against unestablished, murine TB infection, in a manner similar to treatment with INH alone (data not shown). Daily administration with this dose for 1 month was found to have no overt adverse effects in the infected animals.
Activity of KTC after short-duration treatment of established TB in the mouse model
The efficacy of KTC in combination with currently used TB drugs was assessed in a murine model of established pulmonary TB. KTC was co-administered daily for 1 month with various combinations of INH, PZA and RIF. Following treatment, the total number of c.f.u. in lung and spleen tissues was determined (Table 2).
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The group treated with the combination of INH, PZA and RIF had lung counts that were about 3 log10 c.f.u. less than those of the controls at the beginning of treatment and almost 4 log10 c.f.u. less than those of the controls at the end of the study (P <0.001). Mice receiving INH, PZA and RIF exhibited complete clearance of detectable bacilli from the spleen (Table 2).
The addition of KTC to INH, PZA and RIF resulted in a further decrease in the number of bacteria in the lungs compared with the INH, PZA and RIF group (P <0.05) and negative spleen counts.
Treatment with KTC, INH and RIF (replacement of PZA with KTC) led to a slight but insignificant decrease in the lung c.f.u. counts compared with the INH, PZA and RIF group (P >0.05). The effect of KTC, INH and RIF was essentially indistinguishable from the INH, PZA and RIF treatment group after 1 month of treatment in this model.
Mice given KTC, INH and PZA (replacement of RIF with KTC) had higher lung counts than the INH, PZA and RIF group, and one mouse in the group had a spleen positive for M. tuberculosis, whilst the rest were negative (Table 2).
The lowest numbers of bacilli in lungs were found in the group administered KTC, PZA and RIF. When KTC replaced INH in a 1 month combination therapy, there was a decrease of about 4 log10 c.f.u. per lung compared with the starting untreated controls and about 4.7 log10 c.f.u. per lung compared with the end count of untreated controls. The lung c.f.u. value for the KTC, PZA and RIF group was significantly less than that for the INH, PZA and RIF group (P <0.01) and also less than that for the KTC, INH, PZA and RIF group (P <0.05) (Table 2).
Treatment of established TB infection in animal models, as well as in patients, requires long-duration therapy. This study showed that the addition of KTC to the standard regimen of INH, PZA and RIF resulted in increased killing of bacilli in the first month of treatment. However, the observed enhancement of bacterial clearance after 1 month of therapy, whilst statistically significant, was not dramatic; it remains to be seen whether the addition of KTC would translate into a shorter time to cure in a longer treatment study. Interestingly, replacement of INH in a three-drug regimen with KTC led to an even greater reduction in the number of viable bacilli in infected animals. This phenomenon may be related to the well-known antagonism between INH and PZA (Grosset et al., 1992). As co-administration of KTC with RIF has been reported to lower the serum concentrations of RIF (Finch et al., 2002) and KTC (Engelhard et al., 1984) compared with administration of each drug separately, future studies may wish to examine the administration of higher doses of RIF or KTC to compensate for the pharmacokinetic interaction, even though a normal dose of RIF was shown to have an effect when combined with KTC in this study.
This account is the first report of the potential anti-TB activity of KTC in vivo. An enhancement of anti-TB activity was observed in vitro against non-growing M. tuberculosis when KTC was combined with PZA and RIF. The results from short-course treatment in this mouse model indicate that replacement of INH in the standard regimen with KTC may lead to increased killing of bacteria. These results are preliminary in nature and further investigations are underway to examine the effect of KTC in long-term treatment of M. tuberculosis infection in the mouse model.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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