Tuberculosis 90 (2010) 301e305

Contents lists available at ScienceDirect

Tuberculosis
journal homepage: http://intl.elsevierhealth.com/journals/tube

MODEL SYSTEMS

Bactericidal activity of the diarylquinoline TMC207 against Mycobacterium tuberculosis outside and within cells
Jasvir Dhillon a, Koen Andries b, Patrick P.J. Phillips c, Denis A. Mitchison a, *
a

St Georges University of London, UK Dept. of Antimicrobial Research, Tibotec BVBA, Johnson and Johnson, Beerse, Belgium c Medical Research Council Clinical Trials Unit, London, UK
b

a r t i c l e i n f o
Article history: Received 29 April 2010 Received in revised form 29 July 2010 Accepted 29 July 2010 Keywords: Diarylquinoline TMC207 Intra-cellular activity Peritoneal macrophages J774 cell line

s u m m a r y
The bactericidal activities of the diarylquinoline TMC207 in a liquid culture medium started with a bacteriostatic phase lasting about 7 days and then continued with a dose-related bactericidal phase. In comparison, its intra-cellular activity in primary mouse peritoneal macrophages (PM) and in the J774 macrophage-like cell line had little or no static phase so that the bactericidal kill was evident by 5e7 days presumably due to low bacterial ATP levels. Bactericidal activities in the three systems were compared by estimating [1] the rate of bacterial killing (K) during exposure to 0.12e1.0 mg/ml TMC207 which were similar at, 0.35 in the J774 cells and 0.27 in mouse PM (p 0.6) with each lower than 0.11 in extracellular cultures (p < 0.001) and [2] the TMC207 concentration at the intersection between the curve relating cfu count to TMC207 concentration and the cfu count at day-0, dened as the static concentration. Static concentrations were 0.22 mg/ml for extra-cellular cultures, 0.17 mg/ml for mouse PM and 0.06 mg/ml for J774 cells, signicantly lower than the extra-cellular value (p < 0.001). Thus, the intracellular activity of TMC207 is clearly greater than its extra-cellular activity mainly because the preliminary static phase was a shorter or absent. 2010 Elsevier Ltd. All rights reserved.

1. Introduction The diarylquinoline, TMC207 (formerly R207190), is a new antituberculosis drug1 under assessment in a phase II clinical trial in which it was added to a background regimen for the treatment of multi-drug resistant (MDR) pulmonary tuberculosis.2 In this trial, sputum conversion to negativity has occurred after 8 weeks of treatment in 48% of 23 patients on TMC207 background drugs compared to 9% of 24 on placebo background drugs. Peak plasma concentrations of 3.2 mg/ml and 1.7 mg/ml and trough concentrations of 1.0 and 0.7 mg/ml were found during dosage with 400 mg daily or 200 mg thrice weekly, respectively. These encouraging results were obtained essentially as a result of its action on the extra-cellular bacilli that comprise the majority of the bacterial population in cavitary pulmonary tuberculosis.3,4 Evidence of its intra-cellular activity is also necessary to assess its effect on the intra-cellular component of the bacterial population particularly because the bacilli that best survive chemotherapy and cause

relapse may well be those resident in macrophages.5 Since TMC207 acts by selective inhibition of mycobacterial ATP synthase,1 the factors that will affect its antibacterial activity in comparing intracellular and extra-cellular activity are (1) the concentration of ATP and the energy pool present in each of the bacterial populations at the start of its activity, (2) the MIC and MBC against each population and (3) the available concentration of TMC207. We therefore compared the extra-cellular activity of TMC207 with its activity on bacilli within mouse peritoneal macrophages and the J774 macrophage-like cell line, using dual criteria [1] the rate (K(c)) at which the concentration c of TMC207 kills, which will be affected by all of these factors and [2] the approach of earlier studies,6,7 in which the concentration that just inhibits growth is taken as the static concentration (SC) which is likely to reect the MIC but would not be affected by the available bacterial ATP.

2. Materials and methods 2.1. Culture media and drugs

* Corresponding author. Present address: Division of Cellular & Molecular Medicine, St Georges University of London, Cranmer Terrace, London SW17 0RE, UK. Tel.: 44 208 725 5704; fax: 44 208 672 0234. E-mail address: dmitchis@sgul.ac.uk (D.A. Mitchison). 1472-9792/$ e see front matter 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.tube.2010.07.004

All tissue culture media and supplements were obtained from Invitrogen Ltd (Paisley, UK). The broth used in extra-cellular studies was Middlebrook 7H9 plus ADC supplement (Becton Dickinson UK

302

J. Dhillon et al. / Tuberculosis 90 (2010) 301e305

Ltd, Oxford UK). Colony counts were done on Middlebrook 7H11 oleic acid-agar medium (Becton Dickinson). Mycobacterium tuberculosis, strain H37Rv, was kept as stock strain in liquid nitrogen and serially transferred at weekly intervals in 7H9 broth ADC. Pure powder TMC207, a gift from Johnson & Johnson, was dissolved and diluted in dimethyl sulphoxide (VWR International Ltd, Lutterworth, UK) at a concentration of 10 mg/ml and stored at 20  C. Final dilutions in the range 0.006e1.0 mg/ml or 0.25e32 mg/ml, as well as a control without drug were made in the culture media. 2.2. Extra-cellular activity A 6 ml volume of a 7-day culture of M. tuberculosis, H37Rv in 7H9 broth was added to 200 ml 7H9 broth. After incubation for 4 days, the inoculated medium was dispensed into 30-ml screw capped universal containers and TMC207 added Immediately (day-0). After 7, 15 and 22 days of incubation at 37 C, samples were ultrasonicated briey and viable counts set up. 2.3. Primary mouse peritoneal macrophages (mouse PM) For the preparation of macrophage monolayers, the peritoneal cavities of recently killed specic pathogen free BALB/c mice (Harland UK Ltd, Bichecster, UK) weighing 18e20 g were washed out with RPMI 1640 medium containing heparin (5 U/ml; SigmaeAldrich). Pooled washings were centrifuged at 150 g for 10 min and the cells were resuspended in RPMI 1640 10% fetal calf serum (FCS). From this suspension, 5 105 cells were put into 24 well plates (16 mm diameter VWR International) and these were incubated at 37  C with 5% CO2 for 2 h to allow adherence. Nonadherent cells were removed by washing three times with RPMI 1640 10% FCS and the resulting monolayer was cultured in RPMI 1640 10% FCS. For infection and addition of drug, a 7-day culture of M. tuberculosis in 7H9 broth was washed twice by centrifugation at 1000 g for 15 min and resuspended in RPMI 1640 10% FCS and then ultrasonicated for 21 s (Rinco Ultrasonics UK Ltd, London, UK). The infective inoculum was prepared to give a bacteria to cells ratio of 1:1. After a phagocytosis period of 2.5 h, monolayers were washed four times to remove unattached mycobacteria and overlaid with RPMI 1640 10% FCS TMC207 (day-0). The medium and drug over the monolayers were replaced daily for 2 days left unchanged for 2 days and changed again on day-5. Cells were removed by adding 0.25% SDS (SigmaeAldrich) into the 24 well plates and aspirating the cell suspensions into tissue culture tubes. A nal volume of 1 ml per cell was made up with RPMI 1640 10% FCS to abrogate the toxic effect of SDS. These were briey ultrasonicated to disrupt cell clumps and used for counting colonies at day-5. 2.4. J774 cell line The J774A.1 cell line derived from mouse macrophages was obtained from Dr Barry Walker (National Institute and Biological Safety Standards, Potters Bars UK). This was kept at stock concentrations in liquid nitrogen, thawed at 37 C and aliquoted into ltered 25 cm asks (Fisher Scientic UK, Loughborough, UK) with warmed RPMI 1640 10% FCS medium. Cells were allowed to adhere until conuent and the asks split twice a week to maintain the cell line. Splitting was carried out once the cells were conuent by discarding the medium and adding 4 ml of RPMI 1640 10% FCS. The cells were gently taken off the ask by pipetting up and down till the medium becomes cloudy and the back of the ask became clear. From this suspension, 0.5 ml was added into a new ask with medium for maintenance. A cell count was carried out on this suspension and 5 105 cells were added into 24 well plates and

allowed to adhere for 48 h with daily medium changes before infecting with M. tuberculosis. Drugs were added (day-0) after phagocytosis for 2.5 h. The culture medium and drugs were changed as for the mouse PM. Colony counts were done after incubation for 5 and 7 days. 2.5. Experimental design There were three experiments in each of which a comparison was made between the activities of TMC207 in the range of 0.006e1.0 mg/ml on M. tuberculosis in each of the three conditions: extra-cellular, in the J774 cell line and in primary mouse PM. In further single experiments in each condition, the range of TMC207 was increased to 0.25e32 mg/ml. In each experiment, test cultures were set up in duplicate for extra-cellular cultures and in triplicate with the J774 cells and peritoneal macrophages. Serial 10-fold dilutions were made from each culture, and an inoculum of 0.1 ml from each was added to a one-third segment of a 7H11 plate in duplicate. Plates were packed into polyethylene bags and incubated for 3e4 weeks at 37  C before colony forming units (cfu) were counted. Colony counts were recorded on an Excel sheet and converted to log10 cfu/ml using a standard formula. 2.6. Statistical methods Rates of kill (K) were calculated by tting a mixed effects linear regression model to the mean log ctu/ml counts over time. Counts on extra-cellular preparations at day-7 were omitted since with some concentrations bacteriostasis occurred during the 0e7 day period. Static concentrations (SC) were calculated for each replicate (two replicates extra-cellular and three replicates in the J774 cells and in peritoneal macrophages) and for each of three experiments using trigonometric methods with both colony count and drug concentration log transformed. Given the highest concentration, x on the log scale, with a cfu count, cfux, greater than the cfu count at day-0, cfu0, and the lowest concentration, y, with a cfu count, cfuy, smaller than the cfu0, the formula for the SC used was: Log (SC) R ((cfuy cfu0) y/R), where R (y x)/ (cfuy cfux). Using these estimates of SC, mixed effect models were used to estimate the mean SC for each type of culture condition. 3. Results 3.1. Bactericidal activities of TMC207 against bacteria in extracellular and intra-cellular locations The bactericidal activity of extra-cellularTMC207 was slow in onset with stasis or slight growth at low concentrations during the rst 7e14 days (Figure 1). Only by 21 days was there a clear distinction between concentrations of 0.006e0.12 mg/ml that allowed growth and concentrations of 0.25e32 mg/ml that were bactericidal in a dose-related manner. Reduction of the cfu count below the limit of detection (LOD) occurred with concentrations of 16e32 mg/ml The curves showed acceleration of killing during the 21-day exposure period. The corresponding results with J774 cells containing M. tuberculosis (Figure 2) showed earlier starts to bactericidal action with no initial static phase. The distinction in allowing growth became clear by 7 days with growth occurring at 0.006e0.025 mg/ml and inhibition in a concentration dependent manner at 0.12e32 mg/ml. The LOD was reached with 8e32 mg/ml by 5 days, more rapidly than in extra-cellular cultures. Again, the rate of kill accelerated between 5 and 7 days. The results with infected primary mouse peritoneal macrophages were similar to those obtained with the J774 macrophage cell line, except that counts were obtained only after incubation for 5 days since the

J. Dhillon et al. / Tuberculosis 90 (2010) 301e305

303

9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 0 7
TMC 0.006 TMC 0.012 TMC 0.025 TMC 0.12 TMC 0.25 TMC 0.5 TMC 1.0 TMC 0

9.0 8.0 7.0 6.0

TMC 0 TMC 0.006 TMC 0.012 TMC 0.025 TMC 0.12 TMC 0.25 TMC 0.5 TMC 1.0

Log cfu / ml

Log cfu /ml

5.0 4.0 3.0 2.0 1.0 0.0

14

21

28

9.0 8.0 7.0

B
TMC 0 TMC 0.25 TMC 0.5 TMC 1 TMC 2 TMC 4 TMC 8 TMC 16 TMC 32

9.0 8.0 7.0 6.0

TMC 0 TMC 0.25 TMC 0.5 TMC 1 TMC 2 TMC 4 TMC 8-32

Log cfu / ml

Log cfu /ml

6.0 5.0 4.0 3.0 2.0 1.0 0.0 0 7

LOD

5.0 4.0 3.0 2.0 1.0 0.0 0

LOD

14

21

28

3 4 5 Days of incubatiion

Days of incubation
Figure 1. The bactericidal action of TMC207 on extra-cellular cultures of M. tuberculosis. A. Effects on cfu counts over the range of 0.006e1 mg/ml TMC207. B Effects on cfu counts over the range of 0.25e32 mg/ml TMC207. LOD limit of detection.

Figure 2. The bactericidal action of TMC207 on cultures of M. tuberculosis in J774 cells. A. Effects on cfu counts over the range of 0.006e1 mg/ml TMC207. B Effects on cfu counts over the range of 0.25e32 mg/ml of detection TMC207. LOD limit of detection.

macrophages deteriorated if incubated longer (Figure 3). The distinction between growth and no growth at 5 days was at 0.12 mg/ml. The LOD was reached at 5 days by cultures containing 16e32 mg. These results indicate that there were only small differences in the static MICs between the three preparations, so that the MIC and the intra-cellular concentrations of TMC207 were fairly similar in all three. However the speed of onset of bactericidal activity was much slower in the extra-cellular cultures than in either of the two intra-cellular cultures. 3.2. Calculation of the speed of bactericidal action (K) The calculation of the rates of kill (K) under the three conditions is shown in Table 1. The differences between the extra-cellular rates and those obtained in the J774 cell line or in mouse PM were both highly signicant (p < 0.001), whereas no signicant difference was found between the rates for J774 cells and mouse PM. 3.3. Calculation of the static concentration The method for calculating the static concentration (SC) is exemplied in Figure 4 where the cfu count is plotted against log concentration of TMC207 using the data at 21 days for the extracellular cultures, at 7 days for the J774 cell line cultures and 5 days for the mouse PM. In each case the SC was calculated by interpolation between the mean counts in the low TMC207 concentrations. SCs were lower for the mouse PM and J774 cultures than for the extra-cellular cultures though only the difference between the

mean SC of 0.06 mg/ml for J774 cells and 0.22 mg/ml for the extracellular cultures attained signicance (p < 0.001) (Table 2). 4. Discussion A number of different factors could affect the actions of TMC207 on extra-cellular and intra-cellular M. tuberculosis. Concentrations in various mouse tissues have been found to be 6e20 fold higher than in plasma after a single dose of TMC207,1 but this does not necessarily imply that increased concentrations are available for inhibiting intra-cellular bacilli since the drug could be concentrated in vesicles separate from the bacilli. We hypothesise that the inhibition of the bacterial ATP synthase by TMC207 reduces the internal energy pool until it reaches a specic lethal level, perhaps causing membrane instability. The speed with which death occurs therefore depends on two processes (1) the speed with which the ATP synthase is inhibited presumably best measured by the static concentration, which is a type of MIC, and (2) the size of the energy pool at the start of the TMC207 exposure. As we have found that the static concentration is a little decreased in J744 cells and possibly decreased slightly in mouse PM, there is therefore evidence of slightly greater ATP synthase inhibition within cells possibly as a result of slightly higher effective intracellular concentrations of TMC207 but not to the extent indicated by the direct measurement of the intra-cellular concentration. We then have to account for the greater speed of onset of the bactericidal action in the J774 cells and the mouse PM. This cannot be a function of differences in intra-cellular drug concentration because no t was found between the intra-cellular and the extracellular kill curves by equating curves for high TMC207

304

J. Dhillon et al. / Tuberculosis 90 (2010) 301e305

9.0 8.0 7.0
TMC 0 TMC 0.006 TMC 0.012 TMC 0.025 TMC 0.12 TMC 0.25 TMC 0.5 TMC 1

10.0

9.0

Day 7 Day 15 Day 22

Log cfu / ml

6.0 5.0 4.0 3.0 2.0 1.0 0.0 0 1 2 3 4 5

8.0

Log cfu / ml

7.0
Day-0 zero

6.0

5.0
(0.01) (0.1)

Static MIC
(1.0)

9.0 8.0 7.0 6.0

4.0 -2.5

-2

-1.5

-1

-0.5

0.5

Log TMC207 concentration ( g/ml)

Figure 4. Calculation of the static concentration. Shown are the survival curves joining cfu counts of M tuberculosis after exposure to TMC207 for 21 days in extra-cellular cultures, for 7 days in J774 macrophage-like cells or for 5 days in mouse peritoneal macrophages. A vertical dotted line, indicating the value of the static concentration, is drawn where the cfu count at day-0, shown by a horizontal arrow line, cuts the survival curve.

Log cfu / ml

TMC 0

5.0 4.0 3.0 2.0 1.0 0.0

LOD

TMC 0.25 TMC 0.5 TMC 1 TMC 2 TMC 4 TMC 8-32

Days of incubatiion
Figure 3. The bactericidal action of TMC207 on cultures of M. tuberculosis in mouse peritoneal macrophages. A. Effects on cfu counts over the range of 0.006e1 mg/ml TMC207. B Effects on cfu counts over the range of 0.25e32 mg/ml of detection TMC207. LOD limit of detection.

concentrations in extra-cellular cultures with low concentrations in intra-cellular concentrations. The curves differ not just in their dose dependent phase but in the existence of the static early phase during exposure that characterizes extra-cellular experiments but not those on intra-cellular bacilli. A possible explanation for the differences seems to be a higher initial ability to generate ATP in the extra-cellular bacilli. Microarray studies indicate that energy production in M. tuberculosis changes from oxidative phosphorylation under conditions of logarithmic aerobic growth to beoxidation of fatty acids inside phagosomes probably because of the presence of NO.8 This change is accompanied by a reduction in energy production and a reduction in ATP synthase activity as well as induction of an additional NAD dehydrogenase. It is a reasonable hypothesis that initial contact of the ATP-rich bacilli with TMC207 causes immediate inhibition of ATP synthase and with it a signal that stops or reduces bacterial multiplication.

Table 1 Mixed effects analysis of the rate of the fall in cfu counts (K) of M. tuberculosis during contact with TMC207 in three conditions. TMC207 (mg/ml) K Extra-cellular 0.12 0.25 0.50 1.00 0.07 0.08 0.09 0.11 J774 0.13 0.21 0.28 0.35 Mouse PM 0.07 0.18 0.26 0.27

However, according to this hypothesis, redox potentials are maintained by pre-existing ATP from the energy pool. As the ATP is used up, bactericidal activity starts gradually at rst but then with increasing effect to kill the bacterial population. The accelerating bactericidal curves found in Figure 1 t this explanation but it can only be conrmed by further investigation of events during the early stages of contact between bacteria and TMC207. This explanation also accounts for the earlier observation that bactericidal action on extra-cellular bacilli is time but not dose dependent for at least the rst 12 days,1 on the assumption that the cultural conditions during these experiments allowed for a slightly greater accumulation of bacterial ATP and the energy pool than in our current extra-cellular experiments. While it seems that TMC207 kills extra-cellular bacilli in conventional culture very slowly, the nding that M. tuberculosis bacilli is sputum are fat and lazy with a microarray pattern that resembles but is not identical to that found in intra-phagosomal bacilli and, in particular, that energy is also derived from b eoxidation of fatty acids,9 suggests that reserves generating ATP may well be lower than in extra-cellular cultures. It seems likely that the speed of killing of TMC207 in tuberculous lesions is faster than in aerobic culture and also increases in speed as treatment continues. This contention is supported by the occurrence of a denite fall in the sputum cfu counts after treatment of patients with TMC207 alone for 7 days in the early bactericidal activity study,10 a period shorter than the 12 days of stasis found in log phase cultures1 As the stasis stage did not happen or happened very briey in our two intracellular preparations, TMC207 should be of value in the rapid eradication of the minority intra-cellular bacterial component in pulmonary tuberculosis and particularly those intra-cellular bacilli causing latent disease.

Table 2 Mixed effects statistical analysis of static concentrations. Experimental system Mean static concentration (mg/ml) 95% condence limits Extra-cellular Mouse PM J774 cells 0.22 0.17 0.06 0.13e0.37 0.09e0.31 0.04e0.10

Extra-cellular vs. mouse PM, p < 0.001. Extra-cellular vs. J774 cells, p < 0.001. Mouse PM vs. J774 cells, p 0.6.

Extra-cellular vs. mouse PM, p 0.5. Extra-cellular vs. J774 cells, p < 0.001.

J. Dhillon et al. / Tuberculosis 90 (2010) 301e305

305

Competing interests: None declared. Funding: This study was supported by a grant from Johnson & Johnson Pharmaceutical Research & Development. Ethical approval: Not required. References
1. Andries K, Verhasselt P, Guillemont J, Ghlmann HWH, Nefs J-M, Winkler J, et al. A diarylquinoline drug active on the ATP synthase of mycobacterium tuberculosis. Science 2005;307:223e7. 2. Diacon AH, Pym A, Grosbusch M, Patienta R, Rustomjee R, Page-Shipp L, et al. The diarylquinoline TMC207 for multidrug-resistant tuberculosis. N Engl J Med 2009;360:2397e405. 3. Canetti G. The tubercle bacillus in the tuberculous cavity. In: The tubercle bacillus in the pulmonary lesion of man: histobacteriology and its bearing on the therapy of pulmonary tuberculosis. New York: Springer; 1955. p. 62e8. 4. Grosset J. Mycobacterium tuberculosis in the extracellular compartment: an underestimated adversary. Antimicrob Agents Chemother 2003;47:833e6.

5. Mwandumba HC, Russell DG, Nyirenda MHJ, Anderson SA, White ME, Molyneux ME, et al. Mycobacterium tuberculosis resides in nonacidied vacuoles in endocytically competent alveolar macrophages from patients with tuberculosis and HIV infection. J Immunol 2004;172:4592e8. 6. Dhillon J, Mitchison DA. Activity and penetration of anti-tuberculosis drugs in mouse peritoneal macrophages infected with Mycobacterium microti OV254. Antimicrob Agents Chemother 1989;33:1255e9. 7. Dhillon J, Mitchison DA. Activity extra-cellular of rifabutin, FCE22807, rifapentine and rifampin against Mycobacterium microti and M. tuberculosis and their penetration into mouse peritoneal macrophages. Am Rev Respir Dis 1992;145:212e4. 8. Schnappinger D, Ehrt S, Voskuil MI, Liu Y, Mangan JA, Monahan IM, et al. Transcriptional adaptation of mycobacterium tuberculosis within macrophages: insights into the phagosomal environment. J Exp Med 2003;198: 693e704. 9. Garton NJ, Waddell SJ, Sherratt AL, Lee SM, Smith RJ, Senner C, et al. Cytological and transcript analyses reveal fat and lazy persister-like bacilli in tuberculous sputum. PLoS Med 2008 Apr 1;5(4):e75. 10. Rustomjee R, Diacon AH, Allen J, Venter A, Reddy C, Patientia RF, et al. Early bactericidal activity and pharacokinetics of the diarylquinoline TMC207 in treatment of pulmonary tuberculosis. Antimicrob Agents Chemother 2008;52:2831e5.