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Journal of Clinical Microbiology, July 2008, p. 2212-2215, Vol. 46, No. 7
0095-1137/08/$08.00+0     doi:10.1128/JCM.00177-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Prevention of Drug Carryover Effects in Studies Assessing Antimycobacterial Efficacy of TMC207

Nacer Lounis,^1* Tom Gevers,¹ Joke Van Den Berg,¹ Tom Verhaeghe,² Rolf van Heeswijk,¹ and Koen Andries¹

Tibotec BVBA, Johnson & Johnson, Turnhoutseweg 30, Beerse 2340, Belgium,¹ Pharmaceutical Research and Development, Johnson & Johnson, Turnhoutseweg 30, Beerse 2340, Belgium²

Received 29 January 2008/ Returned for modification 23 March 2008/ Accepted 3 May 2008

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The levels of TMC207 (R207910) that can be reached in mouse organs and the sputa of treated patients easily exceed the MIC of the compound and can therefore interfere with in vitro bacterial titrations. We studied the usefulness of protein-enriched media for the prevention of such drug carryover effects. The average MIC of Mycobacterium tuberculosis was determined on three different media: unsupplemented 7H11 agar (MIC = 0.03 µg/ml), 7H11 agar supplemented with 5% bovine serum albumin (BSA; MIC = 1 µg/ml), and Lowenstein-Jensen medium (MIC = 14.33 µg/ml). In a second stage of the study, the maximal noninhibitory concentrations (MNICs) of TMC207 were determined by adding TMC207 to the bacterial inoculum rather than to the culture medium. These MNICs were 0.97 µg/ml for 7H11 agar, 32.33 µg/ml for 7H11 agar with 5% BSA, and 96.33 µg/ml for Lowenstein-Jensen medium. Both protein-enriched media were able to prevent drug carryover effects, but the use of 7H11 medium supplemented with 5% BSA is preferred for practical reasons.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The drug carryover phenomenon can be defined as the inhibition of bacterial growth in vitro which is not due to the inhibition of growth in vivo but, rather, to the presence of high inhibitor concentrations in the tested samples. Failure to recognize the contribution of drug carryover may result in overestimation of a drug's in vivo efficacy. Antibiotics combining high levels of tissue penetration (high volumes of distribution) with low MICs, such as the diarylquinoline TMC207, the nitro-dihydro-imidazo-oxazole OPC-67683, the nitroimidazo-oxazine PA-824, and the pyrrole LL-3858 (8), are at risk of displaying the carryover phenomenon when sputum from treated patients or organs from treated animals are cultivated for the isolation of Mycobacterium tuberculosis.

TMC207 (also known as R207910) is being assessed in a phase IIb, placebo-controlled, double-blind, randomized trial for the evaluation of its antibacterial activity in subjects with smear-positive pulmonary infection caused by multidrug-resistant M. tuberculosis. Patients are treated for either 2 or 6 months with TMC207 in combination with a background regimen recommended for use for the treatment of multidrug-resistant M. tuberculosis infections (a combination of second-line drugs, such as kanamycin, pyrazinamide, ofloxacin, and ethionamide).

Following repeated oral administration of TMC207 to the mouse, rat, and dog, the tissue trough levels in all species are high in the lung, spleen, lymph nodes, and thymus, with an average tissue concentration/plasma concentration ratio above 30 (1). Theoretically, concentrations of 10 to 15 µg/ml or higher could be achieved in the sputum after several months of treatment with TMC207, and these concentrations exceed the MIC of TMC207 against M. tuberculosis by >100-fold.

When TMC207 was assessed in vitro by equilibrium dialysis, TMC207 was found to be extensively bound to plasma proteins, resulting in a free fraction in the buffer compartment below the quantification limit of the liquid chromatography-tandem mass spectrometry method used. At a concentration of 5 µg/ml, the level of plasma protein binding was found to exceed 99.9% in all animal species, including humans (unpublished data).

As TMC207 is highly protein bound, we attempted to prevent drug carryover by using culture media containing high protein concentrations, such as the Lowenstein-Jensen medium (4) and Middlebrook 7H11 agar containing 5% bovine serum albumin (BSA). Middlebrook 7H11 medium (with 10% oleic acid, albumin, dextrose, and catalase [OADC]) was used as a control.

In initial experiments, Mycobacterium smegmatis was used as a surrogate for M. tuberculosis. We cultivated M. smegmatis in the presence of different concentrations of TMC207 on different culture media in order to determine the maximum concentration of TMC207 in sputum or animal organs that did not result in drug carryover effects and therefore that did not interfere with the estimation of CFU counts. The results obtained with M. smegmatis were extrapolated to M. tuberculosis by taking into account the differences in the MICs of TMC207 between M. smegmatis and M. tuberculosis. In order to confirm these predictions, M. tuberculosis was cultivated on the three media in the presence of different concentrations of TMC207.

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Antimicrobial agent. TMC207 was synthesized by Johnson & Johnson (Beerse, Belgium).

Mycobacterial strains. Mycobacterium smegmatis ATCC 607 (from ATCC) and Mycobacterium tuberculosis H37Rv (from V. Jarlier, Paris, France) were used in this study.

Media. The following media were used to grow either M. smegmatis or M. tuberculosis in the presence of different concentrations of TMC207: Middlebrook 7H11 agar (with 10% OADC) alone, Middlebrook 7H11 agar (with 10% OADC) and 5% BSA, and Lowenstein-Jensen medium.

Preparation of media. The media were prepared as described in the following sections.

(i) Double-concentrated 10% OADC-enriched 7H11 agar. A total of 12.6 g of Middlebrook 7H11 agar (Becton Dickinson, Le Pont de Claix, France) was dissolved in 240 ml of distilled water containing 3 ml of glycerol (VWR, Fontenay sous Bois, France). The mixture was heated with continuous stirring and was then boiled for 1 min to dissolve all the powder. The solution was then autoclaved for 15 min at 121°C and then cooled to 55°C so that 60 ml of Middlebrook OADC (Becton Dickinson, Sparks, MD) could be added. The final solution is a double-concentrated 7H11 agar.

(ii) Middlebrook 7H11 agar enriched with 10% OADC. A total of 250 ml of the double-concentrated 7H11 agar was mixed with 250 ml of sterile distilled water in order to get regular 7H11 agar.

(iii) Middlebrook 7H11 agar enriched with 10% OADC and containing 5% BSA. A total of 200 ml of the double-concentrated 7H11 agar was mixed with 200 ml of 10% BSA (Sigma, Steinheim, Germany).

(iv) Lowenstein-Jensen medium. A total of 18.6 g of the Lowenstein base form (Becton Dickinson, Le Pont de Claix, France) was dissolved in 300 ml of distilled water containing 6 ml of glycerol (VWR). The mixture was heated and stirred continuously and then boiled for 1 min to dissolve all the powder. The solution was then autoclaved for 15 min at 121°C and then cooled to 55°C so that 200 ml of fresh, uniform egg suspension could be added aseptically to the cooled medium. The medium was distributed into plates and was allowed to coagulate for 45 min at 85°C.

Preparation of inocula. A stock solution of M. smegmatis which was estimated to contain 10⁶ CFU/ml was diluted 500 times in phosphate-buffered saline (PBS) in two steps (10-fold and 50-fold) and was used to inoculate the different media. A stock solution of M. tuberculosis which was estimated to contain 10⁹ CFU/ml was diluted 250,000 times in PBS in five steps (10-fold, 10-fold, 10-fold, 10-fold, and 25-fold) and was used to inoculate the different media.

Preparation of TMC207. TMC207 was first dissolved in dimethyl sulfoxide (DMSO), and further dilutions were made in PBS. In three experiments (see Table 2), PBS was replaced by sputum in order to mimic the clinical situation more closely. The sputum was pasteurized to prevent contamination of the media with fast-growing bacteria and was digested with dithiothreitol to improve its liquidity, as is done in clinical practice.

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TABLE 2. MIC/COs and MNICs of TMC207 for M. smegmatis and M. tuberculosis measured on different media by the carryover simulation method

Inoculation of plates. For both the MIC and the maximal noninhibitory concentration (MNIC) experiments, petri dishes 180 mm in diameter were divided into two compartments of 90 mm each. Each 90-mm compartment was filled with 10 ml of culture medium. For the conventional MIC experiments, 0.1 ml of the bacterial inoculum was plated on each 90-mm compartment. In the experiments carried out to determine the MIC by the carryover simulating method (MIC/CO) and the MNIC, 0.1 ml inoculum (which consisted of a mixture of 0.05 ml of bacteria and 0.05 ml of TMC207) was added to each 90-mm-diameter compartment immediately after the bacteria were mixed with TMC207. Plates inoculated with either M. smegmatis or M. tuberculosis were incubated at 37°C for 3 days and 4 weeks, respectively.

MIC determination by conventional method. For MIC determination by the conventional method, TMC207 was incorporated into the culture medium and not into the inoculum. The threefold dilution concentrations of TMC207 tested were 0.001, 0.004, 0.012, 0.04, 0.1, 0.3, 1, 3, and 9 µg/ml for 7H11 agar and 5% BSA-enriched 7H11 agar. For Lowenstein-Jensen medium, the concentrations tested were 1, 2.5, 5, 10, 12.5, 20, 25, and 50 µg/ml for both mycobacteria. These concentrations were selected on the basis of previous data for Lowenstein-Jensen medium. Control plates contained either PBS or DMSO, the solvent of TMC207. The highest concentration of DMSO used, 1%, did not interfere with the bacterial titrations. In all experiments, the MIC was defined as the concentration that killed 99% of the initial inoculum, resulting in the absence of colonies after 4 weeks of incubation.

MIC/CO and MNIC determinations. For determination of the MIC/CO and the MNIC by the carryover simulation method, TMC207 was not incorporated into the medium but was mixed with the inoculum (mycobacteria diluted in PBS or sputum) to mimic what happens when TMC207-treated patient sputum or TMC207-treated animal organs are plated into these media. Control plates contained either PBS or DMSO, the solvent of TMC207. The highest concentration of DMSO used, 1%, did not interfere with the bacterial titrations. The threefold dilution concentrations of TMC207 tested were 0.02, 0.06, 0.18, 0.54, 1.6, 4.9, 15, 44, and 131 µg/ml for M. smegmatis and either 0.11, 0.33, 1, 3, 9, 27, 81, and 243 µg/ml or 0.06, 0.18, 0.54, 1.62, 4.9, 15, 44, 131, and 394 µg/ml for M. tuberculosis for all media. The MIC/CO was defined as the concentration that killed 99% of the initial inoculum. The MNIC was defined as the highest concentration that resulted in the survival of >99% of the bacteria obtained on the different media. Both the MIC and the MNIC were determined in this study.

Statistical analysis. Comparisons of the mean MNICs were not performed since too few studies were done to have enough power to show significance. We based our conclusions on the observed increase in the MNICs.

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Determination of TMC207 MICs for M. smegmatis and M. tuberculosis by the conventional method. For M. smegmatis, the mean MIC of TMC207 on 7H11 agar medium was 0.009 µg/ml. When 5% BSA was added to the 7H11 agar medium, the mean MIC increased 21-fold to reach 0.19 µg/ml. When Lowenstein-Jensen medium was used, the mean MIC increased 1,667-fold to reach 15 µg/ml (Table 1). For M. tuberculosis, the mean MIC of TMC207 on 7H11 agar medium was 0.03 µg/ml. When 5% BSA was added, the mean MIC increased 33-fold to reach 1.0 µg/ml. When Lowenstein-Jensen medium was used, the mean MIC increased 478-fold to reach 14.33 µg/ml (Table 1).

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TABLE 1. MICs of TMC207 against M. smegmatis and M. tuberculosis measured on different media by the conventional method

Determination of TMC207 MIC/COs for M. smegmatis and M. tuberculosis. For M. smegmatis, the mean MIC/COs of TMC207 were 2.05 µg/ml, 26.4 µg/ml, and >131 µg/ml on 7H11 agar medium, 5% BSA-containing 7H11 agar medium, and Lowenstein-Jensen medium, respectively (Table 2). For M. tuberculosis, when TMC207 was present in the inoculum and not in the agar, the mean MIC/COs were 16.25 µg/ml, 290 µg/ml, and >344 µg/ml on 7H11 agar medium, 5% BSA-containing 7H11 agar medium, and Lowenstein-Jensen medium, respectively (Table 2).

Determination of TMC207 MNICs for M. smegmatis and M. tuberculosis by carryover simulation method. For M. smegmatis, the mean MNICs by the carryover simulation method were 0.23 µg/ml, 1.16 µg/ml, and 4.9 µg/ml on 7H11 agar medium, 5% BSA-containing 7H11 agar medium, and Lowenstein-Jensen medium, respectively (Table 2). The use of 5% BSA increased the mean MNIC by a factor of 5, while the use of Lowenstein-Jensen medium increased the mean MNIC by a factor of 21. For M. tuberculosis, the mean MNICs were 0.97, 32.33, and 96.33 µg/ml on 7H11 agar medium, 5% BSA-containing 7H11 agar medium, and Lowenstein-Jensen medium, respectively. When 5% BSA was included in the 7H11 agar medium, the mean MNIC increased by a factor of 33. When M. tuberculosis was grown on Lowenstein-Jensen medium, the mean MNIC increased by a factor of 99 (Table 2). The results showed a clear increase in the MNICs of TMC207 for M. tuberculosis when they were measured on 5% BSA-supplemented 7H11 agar or Lowenstein-Jensen medium.

Impact of using dithiothreitol. The mean MICs and the mean MNICs for M. smegmatis were similar irrespective of the use of TMC207 solutions in PBS or in pasteurized and dithiothreitol-digested sputum (Table 2).

Extrapolation of results obtained with M. smegmatis to M. tuberculosis. The mean MIC of TMC207 for M. tuberculosis by the conventional method was 3.33 times higher than the mean MIC of TMC207 for M. smegmatis (0.009 and 0.03 µg/ml, respectively). The mean MIC/COs of TMC207 for M. smegmatis determined on 7H11 agar medium, 5% BSA-containing 7H11 agar medium, and Lowenstein-Jensen medium were 2.05, 26.4, and >131 µg/ml, respectively. The expected MIC/COs of TMC207 for M. tuberculosis determined on the same culture media were 6.82 µg/ml (2.05 µg/ml x 3.33), 87.9 µg/ml (26.4 µg/ml x 3.33), and >436.2 µg/ml (>131 µg/ml x 3.33), respectively. The measured mean MIC/COs of TMC207 for M. tuberculosis were 16, 290, and >344 µg/ml, respectively.

The mean MNICs of TMC207 for M. smegmatis determined on the three media by the carryover simulation method were 0.2, 1.2, and 4.9 µg/ml, respectively. The expected MNICs of TMC207 for M. tuberculosis with the same culture media were 0.67 µg/ml (0.2 µg/ml x 3.33), 4 µg/ml (1.2 µg/ml x 3.33), and 16.3 µg/ml (4.9 µg/ml x 3.33), respectively. The measured mean MNICs were 1, 32.33, and 96.33 µg/ml, respectively.

The MICs or MNICs expected in the carryover simulation were predicted from the increase observed between M. smegmatis and M. tuberculosis by the conventional method (the M. tuberculosis MIC was 3.33 times higher than the M. smegmatis MIC).

The data obtained with M. smegmatis could indeed be extrapolated to M. tuberculosis: no significant discrepancies were found between the calculated and the measured values. The measured values were actually somewhat higher than the predicted values.

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The drug carryover phenomenon has been described for several antibiotics, e.g., β-lactams, fluoroquinolones, and clofazimine, and also for antiseptics, such as betadine, chlorhexidine, and niacinamide (2, 3, 4, 7, 9). Some methods that can be used to overcome this phenomenon are available, but none of them can be universally applied. Weakly active compounds are less problematic in this respect than highly active compounds, as they are less active both in vivo and in vitro. The low activity in vivo results in relatively high bacterial concentrations, which are counted in high dilutions of tissue samples. In these high dilutions, the concentration of a weakly active compound usually does not exceed its MIC or MNIC. Weakly active compounds that are highly concentrated in cells can be problematic. When samples from treated animals are cultivated on culture medium, the amount of compound carried over is high, and this concentration may inhibit the growth of the bacterium, although no activity is observed in vivo. This was observed when mice infected with Mycobacterium avium were treated with clofazimine. Undiluted, 10-fold-diluted, and 100-fold-diluted spleen tissue cultures were negative for bacterial growth, while colonies of M. avium did emerge in 1,000- and 10,000-fold dilutions of spleen tissue suspensions. The splenomegaly observed in clofazimine-treated mice was not different from that observed in untreated control mice, confirming that clofazimine was not active in vivo, and the inhibition of growth in vitro in the lowest dilutions was entirely due to a drug carryover effect (4).

Highly active compounds are much more problematic than weakly active compounds, as they are more active both in vivo and in vitro. The high level of activity in vivo results in low bacterial concentrations, which are counted in low dilutions of tissue samples in vitro. In these low dilutions, the concentration of a highly active compound can exceed the MIC and the MNIC. TMC207 is a diarylquinoline compound that exhibits very potent activity against M. tuberculosis both in vitro and in vivo (1). TMC207 in combination with antituberculosis drugs was able to render mice culture negative after only 2 months of treatment of infections caused by both a sensitive M. tuberculosis strain (1) and a multidrug-resistant M. tuberculosis strain (6). The activity of this compound against M. tuberculosis was also confirmed in a second animal model, the guinea pig (5). In a first phase IIa clinical trial with treatment-naive patients with pulmonary tuberculosis with smear-positive sputum, the average area under the concentration-time curve from 0 to 24 h for TMC207 was 64.8 ± 20.7 µg·h/ml and the minimum concentration in plasma of TMC207 given at 400 mg for 7 days was 1.45 ± 0.44 µg/ml (8a). The maximal concentration in the sputa of these patients was about 5 µg/ml. This concentration did not interfere with the growth of M. tuberculosis, despite the use of 7H11 agar medium, because the bacilli were counted in high dilutions of sputum (>1/10,000), which resulted in drug levels below the MNIC. The expected sputum TMC207 concentrations will be higher in future trials, in which TMC207 will be dosed in combination with other active drugs for several months. Due to the expected efficacy of these combinations, the bacilli will be quantified in low sputum dilutions. The use of 7H11 agar medium will be inappropriate, since the MNIC of 0.97 µg/ml will be exceeded in these dilutions.

Our study shows that protein-enriched media such as 5% BSA-containing 7H11 agar medium, Lowenstein-Jensen medium, and 50% fetal calf serum containing 7H11 agar medium (data not shown) can be used to increase MICs and MNICs. Lowenstein-Jensen medium, which contains a high concentration of avian albumin, was most effective in this respect. All animal efficacy studies performed so far used this medium to prevent overestimation of the drug's efficacy because of drug carryover effects. However, several issues are associated with the use of Lowenstein-Jensen medium in clinical laboratories. The preparation of this medium is cumbersome and requires a coagulator and the use of laminar airflow. Several clinical tuberculosis laboratories prefer to use agar media, such as 7H10 or 7H11 agar medium, which are much easier to prepare. An additional problem is the chemical instability of antibacterials and antifungals (which are added to the medium to prevent contamination) during the coagulation process. This necessitates the use of NaOH to decontaminate the sputa, and NaOH itself can kill an important number of M. tuberculosis bacilli.

The use of 7H11 agar supplemented with 5% BSA can be recommended for prevention of the carryover of TMC207. Use of this medium allows the use of other antibiotics to prevent the growth of contaminating organisms and avoids the need to use more drastic agents for sputum decontamination. The drug carryover phenomenon should be taken into account in any study with a highly active compound with a high level of tissue distribution.

 
* Corresponding author. Mailing address: BVBA, Johnson & Johnson, Turnhoutseweg 30, Beerse 2340, Belgium. Phone: 32 14 60 65 71. Fax: 32 14 60 54 03. E-mail: nlounis{at}tibbe.jnj.com

Published ahead of print on 14 May 2008.

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Journal of Clinical Microbiology, July 2008, p. 2212-2215, Vol. 46, No. 7
0095-1137/08/$08.00+0     doi:10.1128/JCM.00177-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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