Should Moxifloxacin Be Used for the Treatment of Extensively Drug-Resistant Tuberculosis? An Answer from a Murine Model

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1 ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Nov. 2010, p Vol. 54, No /10/$12.00 doi: /aac Copyright 2010, American Society for Microbiology. All Rights Reserved. Should Moxifloxacin Be Used for the Treatment of Extensively Drug-Resistant Tuberculosis? An Answer from a Murine Model Julien Poissy, 1,4 Alexandra Aubry, 1,2,3 Christine Fernandez, 5 Marie-Catherine Lott, 5 Aurelie Chauffour, 1 Vincent Jarlier, 1,2,3 Robert Farinotti, 5 and Nicolas Veziris 1,2,3 * UPMC Université Paris 06, ER5, EA 1541, Laboratoire de Bactériologie-Hygiène, F Paris, France 1 ; AP-HP, Hôpital Pitié-Salpêtrière, Laboratoire de Bactériologie-Hygiène, F Paris, France 2 ; Centre National de Référence des Mycobactéries et de la Résistance des Mycobactéries aux Antituberculeux, F Paris, France 3 ; Service Universitaire de Maladies Infectieuses et Tropicales, Centre Hospitalier Gustave Dron, Tourcoing, France 4 ; and AP-HP, Hôpital Pitié-Salpêtrière, Service Pharmacie-Pharmacocinétique, F Paris, France 5 Received 15 July 2010/Returned for modification 10 August 2010/Accepted 24 August 2010 The prevalence of extensively drug-resistant tuberculosis (XDR-TB), defined as TB that is resistant to isoniazid, rifampin, fluoroquinolones, and aminoglycosides, is rising worldwide. The extent of Mycobacterium tuberculosis resistance to fluoroquinolones depends on the mutation in the DNA gyrase, the only target of fluoroquinolones. The MIC of moxifloxacin, the most active fluoroquinolone against M. tuberculosis, may be lower than its peak serum level for some ofloxacin-resistant strains of Mycobacterium tuberculosis. Therefore, if the MIC of moxifloxacin is lower than its peak serum level, it may be effective against XDR-TB. Our objective was to determine the efficacy of moxifloxacin in treating ofloxacin-resistant TB. We selected isogenic fluoroquinolone-resistant mutants of M. tuberculosis H37Rv in vivo. We infected Swiss mice with either wild-type H37Rv or one of three mutant strains with different MICs that are commonly seen in clinical practice. The MICs of the mutant strains ranged from below to above the peak moxifloxacin level seen in humans (3 g/ml). Each mouse was treated with one of four moxifloxacin doses for 1 month. Moxifloxacin was effective against mutant strain GyrB D500N, with the lowest MIC (0.5 g/ml), when the standard dose was doubled. Moxifloxacin reduced mortality in mice infected with mutant strain GyrA A90V with an intermediate MIC (2 g/ml). However, it had no impact on the mutant strain GyrA D94G with the highest MIC (4 g/ml). Our study underscores current WHO recommendations to use moxifloxacin when there is resistance to early-generation fluoroquinolones such as ofloxacin, restricting this recommendation to strains with moxifloxacin MICs of less than or equal to 2 g/ml. * Corresponding author. Mailing address: Laboratoire de Bactériologie, Faculté de Médecine Pierre et Marie Curie, 91 Boulevard de l hôpital, Paris Cedex 13, France. Phone: (33) Fax: (33) nicolas.veziris@upmc.fr. Published ahead of print on 30 August As the leading cause of death from curable infectious diseases worldwide, tuberculosis (TB) is a serious global health issue (15). The high rates of TB incidence and prevalence in developing countries have a considerable impact on population-level morbidity and mortality, particularly in settings where HIV incidence rates are high (43). Inappropriate administration of standard anti-tb drugs can lead to the emergence of bacilli that are resistant to one or more of these drugs. These bacilli may spread within populations, thus posing an additional obstacle to the control of TB (16). In 2004, the World Health Organization (WHO) reported more than 400,000 new cases of multidrug-resistant TB (MDR-TB), defined as TB resistant to isoniazid and rifampin, the two most common anti-tb drugs (48). Successful treatment of MDR-TB involves a second-line regimen, which may consist of fluoroquinolones and aminoglycosides, the second most effective drugs against TB. Although fluoroquinolones have been shown to improve prognoses in clinical studies (8, 9, 38), their extensive use has led to the emergence of extensively drug-resistant (XDR) bacilli, which are resistant to isoniazid, rifampin, fluoroquinolones, and either amikacin, kanamycin, or capreomycin (45). XDR-TB represents 4 to 19% of MDR-TB cases (6, 7, 28), and its prognosis is poor, with mortality rates of as high as 65 to 100%, especially in HIV-positive patients (5, 7, 17, 23). Several recent studies have shown that some treatment regimens may reduce XDR-TB mortality, especially if they contain newer fluoroquinolones such as moxifloxacin (22, 29). Fluoroquinolones target the Mycobacterium tuberculosis DNA gyrase (GyrA 2 GyrB 2 ), a catalytically active complex (1, 11, 25). Mutations in the gyra and/or gyrb subunit of DNA gyrase lead to fluoroquinolone resistance, defined by the WHO as resistance to at least 2 g/ml of ofloxacin (46). Moxifloxacin is an 8-methoxy fluoroquinolone with enhanced activity against M. tuberculosis; its MIC against wild-type M. tuberculosis is half of that of ofloxacin (2, 41). Although resistance is crossed within the fluoroquinolone group, MICs differ between molecules and may still be lower in the drug-resistant M. tuberculosis strains. The MIC of moxifloxacin, for example, may be lower than its maximum serum drug concentration, depending on the mutation in the resistant strain s DNA gyrase (2, 21). This led WHO to recommend including moxifloxacin in the treatment of XDR-TB patients despite in vitro resistance to ofloxacin (47). The aim of this study was to determine the efficacy of moxifloxacin against ofloxacin-resistant M. tuberculosis, using a murine model. 4765

2 4766 POISSY ET AL. ANTIMICROB. AGENTS CHEMOTHER. MATERIALS AND METHODS We followed the animal experiment guidelines of the Faculté de Médecine Pitié-Salpêtrière. Antimicrobial agents. We used the following anti-tb drugs: enoxacin (Sigma), ofloxacin, levofloxacin (Aventis), moxifloxacin (Bayer Pharma), and gatifloxacin (Grünenthal). These drugs were provided by their respective manufacturers. M. tuberculosis strains. We grew the H37Rv strain of M. tuberculosis, from a mouse organ used in a previous experiment, on Lowenstein-Jensen medium. Colonies were subcultured in Dubos broth (Diagnostics Pasteur, Paris, France) for 7 days at 37 C. This process was repeated twice to homogenize the suspension. We adjusted the turbidity of the resulting suspension with saline to match that of a standard 1-mg/ml suspension of Mycobacterium bovis BCG. We used saline to further dilute the solution to a concentration of 0.2 g/ml, which could be used to inoculate the mice. We obtained mutant strains by treating mice infected with M. tuberculosis H37Rv with fluoroquinolone monotherapy (moxifloxacin at 100 mg/kg or levofloxacin at 200 mg/kg) for 6 months (41). Sequencing of QRDRs. We sequenced the quinolone resistance-determining regions (QRDRs) of the gyra and gyrb subunits of each strain selected in vivo. The QRDRs of the gyra subunits were amplified using PCR and a primer pair consisting of pri9 (5 -CGCCGGGTGCTCTA-3 ) and pri8 (5 -CCGGTGGGTC ATTGCCTGGCG-3 ). The QRDRs of the gyrb subunits were amplified using PCR and a primer pair consisting of GBA (5 -GAGTTGGTGCGGCGTAAG AGC-3 ) and GBE (5 -CGGCCATCAGCACGATCTTG-3 ). The PCR was performed as follows: one 7-min cycle at 94 C, 40 1-min cycles at 94 C, 1 min at the hybridization temperature (65 C for gyra and 55 C for gyrb), 1 min at 72 C, and a final 10-min extension step at 72 C. PCR fragments were purified in spin columns (Qiagen, Hilden, Germany) and sequenced using the method described by Sanger et al. (34). We used the primers described above, the Big Dye Terminator v3.1 cycle sequencing kit (Applied Biosystems), and an ABI PRISM 3100 automatic sequencer (Applied Biosystems, France). We read the resulting nucleotide sequences using SeqScape (Applied Biosystems). We identified mutations by comparing them to the reference sequence imported from GenBank ( Determination of MICs. MICs were determined using 7H11 agar supplemented with 10% oleic acid-albumin-dextrose-catalase (OADC) (19). The MIC was defined as the lowest concentration that inhibited 99% of bacterial growth. We evaluated the fluoroquinolones ofloxacin, moxifloxacin, gatifloxacin, levofloxacin, and enoxacin. Enoxacin is a quinolone that has less activity against M. tuberculosis than the other fluoroquinolones, thus facilitating the detection of low-level resistance to fluoroquinolones (2). Murine model of TB. We purchased 4-week-old outbred Swiss mice from the Janvier breeding center (Le Genest Saint-Isle, France). We inoculated each mouse in the tail vein with a 0.5-ml bacterial suspension that contained 7 to 7.6 log 10 CFU of each M. tuberculosis strain. We conducted two murine experiments. The objective of the first experiment was to compare the virulence of ofloxacin-resistant mutants and M. tuberculosis H37Rv wild-type strains. We inoculated 120 mice in the first experiment, including 20 mice for each strain (the wild-type strain H37Rv and five mutants harboring mutations A90V, D94G, D94N, and S91P in the GyrA subunit and D500N in the GyrB subunit). The second experiment aimed to measure the effect of various doses of moxifloxacin on ofloxacin-resistant mutants. We inoculated 240 mice. Treatment was initiated at 1 day after infection and was administered by oral gavage 5 days per week for 4 weeks (18). We prepared the appropriate concentrations of moxifloxacin in distilled water that contained 0.05% agar. We established four therapeutic schedules: 33 mg/kg three times a day (33 mg/kg 3/ day), 66 mg/kg 3/day, 100 mg/kg 1/day, and 200 mg/kg 1/day. For each M. tuberculosis strain (the H37Rv wild-type strain and mutants with A90V and D94G in the GyrA subunit and D500N in the GyrB subunit; see below), 60 mice were inoculated: 10 mice for the enumeration of the bacterial load in lungs, 10 mice for survival analysis, and 10 mice for each schedule (33 mg/kg 3/day, 66 mg/kg 3/day, 100 mg/kg 1/day, and 200 mg/kg 1/day). We used the 100-mg/ kg 1/day dose since it was established earlier in mouse models to be the safest and clinically relevant dose (30, 41). The 200-mg/kg 1/day dose was administered to evaluate the benefits of increasing doses in subjects infected with strains with poor drug susceptibility. Since the maximum concentration (C max ) of moxifloxacin is higher for mice than for humans (26, 30, 31), we assessed two other drug regimens, which consisted of administering the 100-mg/kg and 200-mg/kg doses via three gavages per day (33 mg/kg 3/day and 66 mg/kg 3/day every 8 h). These regimens more closely approximated human moxifloxacin pharmacokinetics. TABLE 1. MICs of fluoroquinolones for wild-type M. tuberculosis H37Rv and derivative strains with mutations in DNA gyrase M. tuberculosis strain a Assessment of efficacy. Treatment efficacy was measured in terms of survival rates and lung CFU counts measured on both right and left lungs. Ten mice from each treatment group were sacrificed at 1 day after infection. Surviving mice were sacrificed at the completion of their treatment regimens. Additional DNA gyrase mutations at treatment completion. At treatment completion, we checked all strains of M. tuberculosis for increased resistance to ofloxacin and additional DNA gyrase mutations compared to those at the start of the experiment. We determined the proportion of surviving bacilli growing in highly concentrated ofloxacin-containing agar plates, with 16 g/ml for the GyrB D500N mutant strain and 64 g/ml for the GyrA A90V and D94G mutant strains. We sequenced the QRDRs of the gyra and gyrb subunits of colonies growing on ofloxacin-containing medium. Pharmacokinetic analysis. We measured the pharmacokinetic parameters of each moxifloxacin dose after the first dose was administered. Once the drug was orally administered, we anesthetized the mice using halogenic gas (isoflurane). We collected blood by performing cardiac puncture in three to five mice (depending on the volume of blood collected). When the dose was 100 mg/kg or 200 mg/kg, blood was drawn at 0, 10, 20, 45, and 90 min and at 3, 4, and 12 h after gavage. When the dose was 33 mg/kg or 66 mg/kg, blood was drawn at 0, 10, 20, 45, and 90 min and at 3, 4, and 8 h. Plasma was separated using centrifugation and was then frozen at 20 C until it was analyzed by liquid chromatography. This methodology has been previously described by Siefert et al. (36). We determined the maximum concentration (C max ) and the time to maximum concentration (T max ) by visually inspecting the experimental data. We estimated the area under the concentration-time curve (AUC) using the trapezoidal rule for n 1 subintervals. The terminal half-lives (t 1/2 ) were assessed using WinNonLin software (Pharsight Corporation, Mountain View, CA) and were fitted using a noncompartmental model. Statistical analysis. We analyzed qualitative data using the nonparametric Mann-Whitney-Wilcoxon test, and we evaluated quantitative data using the Student t test. We considered differences to be statistically significant when the P value was RESULTS MIC ( g/ml) Gatifloxacin Moxifloxacin Levofloxacin Ofloxacin Enoxacin Wild-type H37Rv GyrB D500N GyrA A90V GyrA S91P GyrA D94G GyrA D94N a GyrA and GyrB are subunits of DNA gyrase Characteristics of fluoroquinolone-resistant M. tuberculosis. The QRDRs of the four M. tuberculosis H37Rv strains derived from the wild-type strain had the mutations A90V, S91P, D94N, and D94G in the GyrA subunit and D500N in the GyrB subunit (Escherichia coli numbering system; amino acids 83, 84, and 87 in the GyrA subunit and amino acid 426 in the GyrB subunit). We did not find any additional mutations when we sequenced the entire gyra and gyrb genes of mutant strains. Table 1 shows the moxifloxacin MICs for mutant strains and compares them to the MIC for the H37Rv wild-type strain. The moxifloxacin MICs were 0.25 g/ml for the wild-type strain, 0.5 g/ml for the strain with the GyrB D500N mutation, 2 g/ml for the strain with the GyrA A90V mutation, and 4 g/ml for the strains with the GyrA S91P, D94G, and D94N mutations. Virulence of fluoroquinolone-resistant M. tuberculosis. Mice were infected with inocula ranging from 7.0 to 7.6 log 10 CFU.

3 VOL. 54, 2010 MOXIFLOXACIN AGAINST XDR TUBERCULOSIS 4767 FIG. 1. Survival among mice infected with wild-type M. tuberculosis strain H37Rv and treated with one of four doses of moxifloxacin: 33 mg/kg 3/day, 100 mg/kg/day, 66 mg/kg 3/day, and 200 mg/kg/day (curves can be superimposed). When infected mice remained untreated, mortality was similar among those infected with the H37Rv wild-type strain and those infected with any of the five derivative mutant strains (data not shown). At the end of the first experiment assessing virulence, we sequenced the QRDRs of the H37Rv wild-type strain and mutant strains, which were harvested from mouse organs. All of the mutations described above were stable. Moxifloxacin activity against fluoroquinolone-resistant M. tuberculosis. We used three mutant strains to conduct the therapeutic experiment: GyrB D500N, GyrA A90V, and GyrA D94G (MICs, 0.5, 2, and 4 g/ml, respectively). This choice allowed us to assess a range of moxifloxacin MICs both above and below the C max s seen in humans, which are generally around 3 g/ml (26). Survival. Survival rates over time by dose and by strain are shown in Fig. 1 to 4. Survival differed by mutation. Moxifloxacin was equally effective against wild-type H37Rv and the GyrB D500N mutant strain, regardless of dosage. The efficacies of moxifloxacin against the GyrA A90V mutant strain were 10/10 when the dose was 200 mg/kg 1/day, 6/10 when the dose was 66 mg/kg 3/day, 4/10 when the dose was 100 mg/kg 1/day, and 3/10 when the dose was 33 mg/kg 3/day. Moxifloxacin was not effective against the GyrA D94G mutant strain. Mortalities in treated and untreated mice infected with this strain were similar, regardless of dosage. FIG. 3. Survival among mice infected with the M. tuberculosis GyrA A90V mutant strain and treated with one of four doses of moxifloxacin: 33 mg/kg 3/day, 100 mg/kg/day, 66 mg/kg 3/day, and 200 mg/kg/ day (curves can be superimposed). Lung CFU counts. Table 2 shows CFU counts at treatment initiation (day 0) and 4 weeks later among surviving mice, by strain and by moxifloxacin dosage. CFU counts at day 0 were similar in all four groups. Lung CFU counts for mice infected with wild-type M. tuberculosis H37Rv declined significantly 1 month after treatment initiation at all doses (P ). CFU counts were lower for mice treated with doses of 200 mg/kg/day and 66 mg/kg 3/day than for mice treated with doses of 100 mg/kg 1/day and 33 mg/kg 3/day. This difference did not reach statistical significance, however. Among mice infected with the GyrB D500N mutant strain, CFU count declines were significant when moxifloxacin doses were 100 mg/kg 1/day, 66 mg/kg 3/day, and 200 mg/kg 1/day (P 0.005, , and , respectively). CFU count declines were not significant when the moxifloxacin dose was 33 mg/kg 3/day. Among mice infected with the GyrA A90V and D94G mutant strains, lung CFU counts increased between treatment initiation and 1 month at all moxifloxacin doses ( 0.11 to 1.07 and 0.95 to 1.23 log 10 CFU, respectively), demonstrating that the disease continued to evolve despite treatment. Additional DNA gyrase mutations at treatment end. Five of the 40 mice infected with the GyrB D500N mutant strain and treated with moxifloxacin had additional mutations (GyrA A90V, 4 mice; GyrA D94A, 1 mouse). Of these mice, three received moxifloxacin doses of 66 mg/kg 3/day, one received a FIG. 2. Survival among mice infected with the M. tuberculosis GyrB D500N mutant strain and treated with one of four doses of moxifloxacin: 33 mg/kg 3/day, 100 mg/kg/day, 66 mg/kg 3/day, and 200 mg/kg/ day (curves can be superimposed). FIG. 4. Survival among mice infected with the M. tuberculosis GyrA D94G mutant strain and treated with one of four doses of moxifloxacin: 33 mg/kg 3/day, 100 mg/kg/day, 66 mg/kg 3/day, and 200 mg/kg/ day (curves can be superimposed).

4 4768 POISSY ET AL. ANTIMICROB. AGENTS CHEMOTHER. M. tuberculosis strain TABLE 2. CFU in the lungs of mice before treatment and 4 weeks after treatment initiation (number of surviving mice) Before treatment CFU in lungs, mean SD (no. of surviving mice) 4 wk after initiation of treatment (mg/kg gavages/day) Wild type H37Rv (10) (10) (10) (10) (10) GyrB D500N (10) (10) (9) (10) (10) GyrA A90V (10) (3) (4) (6) (10) GyrA D94G (10) (6) (8) (8) (5) dose of 33 mg/kg 3/day, and one received a dose of 100 mg/kg/day. The proportion of second-step resistant strains was almost 1% (0.5 to 2%) of the bacillary population for three out of four mice, demonstrating that second-step mutants were replacing after drug treatment. Four of the 40 mice infected with the GyrA A90V mutant strain and treated with moxifloxacin had additional mutations (GyrA D94G or D94N). Of these mice, two received moxifloxacin doses of 100 mg/kg/day, one received a dose of 66 mg/kg 3/day, and one received a dose of 33 mg/kg 3/day. The proportion of second-step resistant mutants remained very low (less than 1/10 5 ), suggesting that only a small proportion of the initial bacillary population was being replaced by second-step mutants. Among mice infected with the GyrA D94G mutant, colonies did not grow on media with high ofloxacin concentrations, demonstrating that no second-step mutants had emerged. Pharmacokinetic data. Pharmacokinetic data are shown in Table 3. The area under the concentration-time curve divided by the MIC (AUC/MIC ratio) and the C max /MIC ratio for each strain are presented in Table 4. The highest C max (10 g/ml, two to three times the usual human C max ) was measured in mice treated with 200-mg/kg dosing. The C max measured in mice treated with 100-mg/kg dosing (9 g/ml) was nearly equal to that of mice treated with 200-mg/kg dosing. The C max measured in mice treated with 33-mg/kg dosing (5 g/ml) was equivalent to the highest C max measured in humans. The C max measured in mice treated with 66-mg/kg dosing (7 g/ml) was 1.5 to 2 times higher than the usual human C max (Table 3). The AUCs measured in mice treated with 33 mg/kg 3/day (24 g h/ml), 100 mg kg 1/day (22 g h/ml), and 200 mg/ kg 1/day (27 g h/ml) were equivalent to the lowest AUC TABLE 3. Pharmacokinetic data for moxifloxacin in a murine model Parameter (unit) Value for: Mice given moxifloxacin dose (mg/kg gavages/day) of: Humans given 400 mg moxifloxacin/day a T max (min) C max ( g/ml) t 1/2 (h) AUC b ( g h/ml) a This is the standard dose administered to humans (26, 36, 37, 40, 44). b For doses of 33 mg/kg 3/day and 66 mg/kg 3/day, the AUC was obtained by multiplying the AUC measured after one dose by 3. measured in human. The AUC in mice treated with 66 mg/ kg 3/day (39 g h/ml) was equivalent to the highest AUC measured in humans. DISCUSSION The aim of this study was to determine the efficacy of moxifloxacin against ofloxacin-resistant M. tuberculosis, using a murine model. For this purpose we selected isogenic strains of M. tuberculosis H37Rv in vivo that contained mutations in the gyra and gyrb genes of DNA gyrase, the only target of fluoroquinolones in M. tuberculosis (1, 11). GyrA mutations have been described elsewhere (2, 24, 39). The mutation we found in GyrB has been observed in laboratory-selected fluoroquinolone-resistant strains of M. tuberculosis (24), one clinical strain of M. tuberculosis, and many clinical fluoroquinoloneresistant strains of other bacteria (14, 32). Although the GyrB mutation has been observed in only one clinical strain, we included it for two reasons. First, it acted as a control for common mutations that display high levels of resistance (GyrA A90V and D94G). Second, even if they are rare, strains with low levels of resistance have been described in clinical settings (42). The moxifloxacin MICs that we found for mutant strains are consistent with previously published data (2, 24, 39), which showed that strains with mutations at position 94 had higher moxifloxacin MICs than those with mutations at position 90 (Table 1) (2). The mutant strains of M. tuberculosis H37Rv had equal virulence in our study. This finding is consistent with previous descriptions of DNA gyrase mutant strains of other bacterial species, such as Streptococcus pneumoniae or Campylobacter jejuni (20, 27). We initiated treatment at 1 day after infection (preventive model) to ensure that the bacillary load would be low, thus reducing the probability of selecting second-step mutants. This allowed us to measure the precise moxifloxacin activity, whereas an experiment with a high bacillary load would have measured a mix of a decrease of the initial bacillary population and an increase of second-step mutants. Future studies evaluating the efficacy of moxifloxacin-containing drug combinations against fluoroquinolone-resistant strains should use a mouse model with a high initial bacillary load (curative model). This study shows that moxifloxacin activity decreases with increasing MICs. Moxifloxacin had the lowest MIC against wild-type strain H37Rv and the highest against the GyrA D94G mutant strain (Table 1). All of the mice infected with wild-type M. tuberculosis H37Rv and the GyrB D500N mutant, which had the lowest moxifloxacin MIC of the mutant strains

5 VOL. 54, 2010 MOXIFLOXACIN AGAINST XDR TUBERCULOSIS 4769 TABLE 4. AUC/MIC and C max /MIC ratios associated with four doses of moxifloxacin and four strains of M. tuberculosis H37Rv Ratio after moxifloxacin dose (mg/kg gavages/day) of a : Strain MIC ( g/ml) C max /MIC AUC/MIC C max /MIC AUC/MIC C max /MIC AUC/MIC C max /MIC AUC/MIC Wild type GyrB D500N GyrA A90V GyrA D94G a For doses of 33 mg/kg 3/day and 66 mg/kg 3/day, the AUC was obtained by multiplying the AUC measured after one dose by 3. (0.5 g/ml), survived (Fig. 2). Moxifloxacin exhibited bactericidal activity with the GyrB D500N mutant only when doses were higher (200 mg/kg/day and 66 mg/kg 3/day). Moxifloxacin activity was lower in mice infected with the GyrA A90V mutant strain, which had an intermediate moxifloxacin MIC (2 g/ml). Higher doses of moxifloxacin prevented mortality but did not have an impact on lung CFU counts (partial bacteriostatic activity). Mice infected with the GyrA D94G mutant strain, which had the highest moxifloxacin MIC (4 g/ml), had the worst outcomes: neither mortality nor lung CFU counts declined, even when moxifloxacin doses were doubled. GyrA A90V and GyrA D94G are the most commonly found mutations in clinical practice and are responsible for almost 80% of fluoroquinolone resistance in M. tuberculosis. To date, lowlevel resistance, such as that conferred by the GyrB D500N mutation, remains rare. Our results suggest that when testing ofloxacin-resistant patients for fluoroquinolone susceptibility, moxifloxacin should be tested at 2 g/ml to distinguish between high-level and low-level resistance. The present study also reveals the pharmacokinetic parameters that predict fluoroquinolone efficacy against sensitive and resistant M. tuberculosis strains. Fluoroquinolones are concentration-dependent antibiotics. The AUC/MIC ratio is generally considered to be the main predictor of clinical outcomes (40). A previous study with mice showed that an AUC/MIC ratio of 125 was associated with optimal outcomes for TB (35). Our study confirms that bactericidal activity against M. tuberculosis H37Rv in vivo was higher when AUC/MIC ratios were high, especially at levels above 100, as seen previously for fast-growing bacterial species (Table 4) (3). Our observations regarding pharmacokinetic drivers varied among the ofloxacin-resistant strains. All moxifloxacin doses prevented mortality in mice infected with GyrB D500N mutants (Fig. 2). The highest AUC/ MIC ratio (at 66 mg/kg, 78) reduced CFU counts very effectively. However, when AUC/MIC ratios were lower (44 to 54), the CFU counts declined proportionally with the C max /MIC ratio. The highest CFU count decline was observed with the highest C max /MIC ratio (at 200 mg/kg, 20), whereas the lowest CFU count decline was observed with the lowest C max /MIC ratio (at 33 mg/kg 3, 10). Moxifloxacin did not reduce CFU counts when mice were infected with GyrA A90V mutants, regardless of dosage. Mortality was lowest in mice that received doses of 200 mg/kg 1/day, leading to the highest C max / MIC ratio (5). Mortality was highest in mice that received doses of 33 mg/kg 3/day, leading to the lowest C max /MIC ratio (2.5). Intermediate mortality rates were associated with intermediate C max /MIC values (3.5 to 4.5). The 66-mg/kg 3/day dose was associated with a lower mortality rate than the 100- mg/kg 1/day dose, likely because the AUC/MIC ratio was nearly two times higher. Moxifloxacin doses that had the largest impact on strains with low and intermediate resistance levels were associated with either the highest C max (10 g/ml) or the highest AUC (39 g h/ml). These data suggest that fluoroquinolones should be administered once daily to patients infected with XDR-TB. In order to compare our results to those reported for humans, we measured AUC values for each dose. For doses of 33 mg/kg 3/day, 100 mg/kg/day, and 200 mg/kg/day, AUC values were close to those found in humans taking the recommended oral dose (400 mg/day) (26, 37). The AUC values observed for mice that received doses of 66 mg/kg 3/day were similar to the highest AUC values found in humans taking the recommended oral dose (Table 3). It is thus likely that a moxifloxacin dose of 400 mg/day is active in humans infected with strains that have MICs of 2 g/ml. Several studies to date have shown that the 400-mg/day dose is well tolerated in the long term. Though adverse effects do occur (including nausea, vomiting, muscle pain, tremors, insomnia, and dizziness), none are irreversible or fatal (4, 10, 12, 13, 33). It is probably not possible to achieve the same C max in humans as obtained in mice that received doses of 200 mg/kg 1/day (10 g/ml). Humans may be able to reach the C max obtained in mice that received doses of 66 mg/kg 3/day (7 g/ml), but only if their doses are doubled to 800 mg/day. Increasing moxifloxacin doses to 800 mg/day would increase AUC values and perhaps lead to a higher efficacy than that found in our study. Future studies should evaluate the effect of an 800-mg/day dose on isolates with MIC values of 2 g/ml. Moxifloxacin doses of 800 mg/day seem to be well tolerated (40), but little is known about their long-term tolerability in TB patients. Finally, the serum levels in patients who take moxifloxacin to treat fluoroquinolone-susceptible and -resistant TB should be measured, since human pharmacokinetics in the presence of moxifloxacin are heterogeneous. Our study has several limitations. First, our experiment was conducted with mice. We analyzed pharmacokinetic data using several doses of moxifloxacin to predict possible outcomes in humans taking standard and double doses of moxifloxacin. Future studies should confirm these data in humans. Second, the emergence of second-step mutants in the strains against which moxifloxacin was effective reveals that this drug must be administered in combination with other drugs. Moreover, the inocula used were lower than that usually seen in human tuberculosis and could have minimized the risk of emergence of second-step mutants. Third, we studied single-drug therapy, and a curative murine model should be used to determine the best multidrug regimen.

6 4770 POISSY ET AL. ANTIMICROB. AGENTS CHEMOTHER. In conclusion, we conducted a study on murine TB in vivo using isogenic ofloxacin-monoresistant strains of M. tuberculosis H37Rv that were representative of resistance levels seen in clinical practice. The results demonstrate that moxifloxacin is active against strains with low levels of resistance (MIC, 0.5 g/ml) and reduces the mortality associated with strains with intermediate resistance (MIC, 2 g/ml). Moxifloxacin is inactive, however, against strains with high levels of resistance (MIC, 2 g/ml). These results have important implications for moxifloxacin use and dosage in patients with XDR-TB. Our study provides data which support the current WHO recommendation to use moxifloxacin when there is resistance to early-generation fluoroquinolones such as ofloxacin. Human studies should be conducted to confirm these observations and to assess the long-term tolerability of higher doses of moxifloxacin in TB patients. ACKNOWLEDGMENTS We thank Chantal Truffot, Mélanie Leroy, and Murad Ibrahim for their technical assistance. We are indebted to Caroline Sloan for editing the paper. The laboratory is supported by an annual grant from Université Paris 6, Pierre et Marie Curie (EA1541), and a research grant from the French Ministry of Health. REFERENCES 1. Aubry, A., X. S. Pan, L. M. Fisher, V. Jarlier, and E. Cambau Mycobacterium tuberculosis DNA gyrase: interaction with quinolones and correlation with antimycobacterial drug activity. Antimicrob. Agents Chemother. 48: Aubry, A., N. Veziris, E. Cambau, C. Truffot-Pernot, V. Jarlier, and L. M. Fisher Novel gyrase mutations in quinolone-resistant and -hypersusceptible clinical isolates of Mycobacterium tuberculosis: functional analysis of mutant enzymes. Antimicrob. Agents Chemother. 50: Azoulay-Dupuis, E., J. P. Bedos, J. Mohler, P. Moine, C. Cherbuliez, G. Peytavin, B. Fantin, and T. 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