Published ahead of print on August 11, 2004, doi:10.1164/rccm.200407-885OC
© 2004 American Thoracic Society doi: 10.1164/rccm.200407-885OC
Moxifloxacin-containing Regimens of Reduced Duration Produce a Stable Cure in Murine TuberculosisCenter for Tuberculosis Research, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland; Foundation for Innovative New Diagnostics, Geneva, Switzerland; and Division of Tuberculosis Elimination, Centers for Disease Control and Prevention, Atlanta, Georgia Correspondence and requests for reprints should be addressed to Eric L. Nuermberger, M.D., 1503 East Jefferson Street, Baltimore, MD 21231–1002. E-mail: enuermb{at}jhmi.edu
In a recent experimental study using the mouse model of tuberculosis, treatment with a combination of rifampin, moxifloxacin, and pyrazinamide was able to shorten the time to negative lung cultures by up to 2 months compared with the standard regimen of rifampin, isoniazid, and pyrazinamide. To confirm that this substitution of moxifloxacin for isoniazid permits a shorter duration of treatment, a second study was performed in which mice were assessed for relapse after treatment with combination therapy for 3, 4, 5, or 6 months. Although no relapse was observed among mice treated for at least 4 months with rifampin, moxifloxacin, and pyrazinamide, mice treated with rifampin, isoniazid, and pyrazinamide required 6 months of treatment before no relapse could be detected. For mice treated with rifampin, moxifloxacin, and pyrazinamide, similar efficacy was noted whether pyrazinamide was administered for 1 month, 2 months, or the entire duration of therapy. These results suggest that the use of rifampin, moxifloxacin, and pyrazinamide may substantially shorten the duration of therapy needed to cure human tuberculosis and that the full benefit of pyrazinamide in this regimen may be realized after just 1 month of treatment.
Key Words: fluoroquinolone • mouse • moxifloxacin • pyrazinamide • tuberculosis The 8-methoxyfluoroquinolone moxifloxacin has potent activity against Mycobacterium tuberculosis in vitro in the mouse model and in humans as measured by early bactericidal activity (1–7). Up to now, it has been used as a second-line drug for the treatment of patients with multidrug-resistant tuberculosis or patients intolerant of first-line agents. In a recent study, we assessed its potential as a first-line drug in the mouse model of tuberculosis. We found that the treatment of infected mice with a combination of rifampin (R), moxifloxacin (M), and pyrazinamide (Z) for 2 months, followed by rifampin and moxifloxacin for 4 months (regimen abbreviated as 2RMZ/4RM) resulted in a shorter time to lung culture conversion than did treatment with the standard regimen of rifampin, isoniazid (H), and pyrazinamide for 2 months followed by rifampin and isoniazid for 4 months (regimen abbreviated as 2RHZ/4RH) (8). Mice receiving 2RMZ/4RM were nearly all culture negative after just 3 months of therapy, whereas no mouse receiving 2RHZ/4RH was culture negative before receiving 5 months of therapy. Although these data suggest that the sterilizing activity of the standard isoniazid-containing regimen could be greatly improved by replacing isoniazid with moxifloxacin, the study was not designed to determine whether the duration of treatment might be shortened by the use of moxifloxacin-containing regimens because all mice received 6 months of therapy before being assessed for relapse. Because the frequency of relapse is the most stringent measure of sterilizing activity, the primary objective of this study was to characterize better the potential of a RMZ-based regimen to permit shortening the duration of therapy by assessing mice for relapse after treatment with abbreviated treatment regimens. A second issue raised by the previous study regards the optimal duration of treatment with pyrazinamide. The addition of pyrazinamide to regimens containing rifampin and isoniazid permits shortening the duration of modern, short-course therapy from 9 to 6 months (9). It has been well demonstrated that the maximum benefit of pyrazinamide is achieved in the first 2 months of therapy and that continued administration together with rifampin and isoniazid beyond the first 2 months results in no beneficial effect (10–12). However, the minimum duration of therapy necessary to realize the benefit of pyrazinamide has not been determined. Moreover, it remains unclear whether longer treatment with pyrazinamide would be beneficial in the absence of isoniazid (i.e., with the RMZ regimen). On one hand, the use of pyrazinamide for longer than 2 months has the potential to increase further the sterilizing activity of the RMZ regimen. On the other hand, shorter durations of pyrazinamide may be easier to administer and better tolerated in humans. To address the issue of the optimal duration of pyrazinamide administration, we studied additional treatment regimens in which pyrazinamide was combined with RM for only the first month or for the entire duration of treatment.
Antimicrobials Drugs were obtained and stock solutions prepared as previously described (8).
M. tuberculosis Strain
Aerosol Infection
Treatment Treatment began 19 days after infection (D0), when the bacillary burden is typically 107–108 cfu, like that in a human tuberculous pulmonary cavity (14, 15). Drugs were administered daily by gavage in the following dosages (mg/kg): rifampin, 10; isoniazid, 25; pyrazinamide, 150; and moxifloxacin, 100, as previously described (8).
Assessment of Treatment Efficacy
Statistical Analysis Cfu counts were log-transformed as log10(x + 1), where x equals the total organ cfu count. Multiple pairwise comparisons of group means were performed by one-way analysis of variance with Bonferroni's post-test (GraphPad InStat version 3.05, San Diego, CA).
Body Weight, Spleen Weight, and Gross Appearance of the Lung Body weight, spleen weight, and gross appearance of the lung were assessed for each group at each time point. There were no significant differences in body weight between groups at any time point. At D0, the mean spleen weight was 105 ± 18 mg. After 2 weeks of treatment, the mean spleen weights were 124 ± 38 and 81 ± 30 mg in the groups treated with RHZ and RMZ, respectively. Neither value was significantly different from the D0 control, but the difference between the RHZ and RMZ groups was significant by unpaired t test (p < 0.05). After 1 month of treatment, the mean spleen weights were 192 ± 15, 93 ± 10, and 89 ± 9 mg for mice receiving no treatment, RHZ, or RMZ, respectively. The differences between treated and untreated animals were highly significant (p < 0.0001), but the mean spleen weights were not different between mice receiving RHZ- and RMZ-based regimens at this or any later time point. The gross examination of the lung surface at D0 revealed multiple tubercles in both lungs, consistent with heavy infection. After 1 month, the lungs of untreated mice had increased in size, and the lesions had begun to coalesce. In contrast, the lungs of treated mice had 50–100 discrete visible lesions that were smaller than those seen at the onset of treatment. These lesions were progressively fewer in number after 2 and 3 months of treatment and were no longer visible after 4 months of treatment. There were no apparent differences in gross lung appearance between RHZ- and RMZ-treated mice or between mice that were later found to relapse and those that did not.
Organ cfu Counts during Treatment
Relapse after Treatment Completion The proportion of mice with culture-positive relapse after the end of treatment was determined for each regimen (Table 3). There were significant differences noted in the frequency of relapse between RMZ- and RHZ-treated mice after 3 months of treatment. The group treated with 2RMZ/1RM had significantly fewer relapses than the group treated with 2RHZ/1RH (p < 0.001) even when the analysis was corrected for multiple comparisons. Both the 1RMZ/2RM and 3RMZ groups also had fewer relapses compared with the group treated with the standard regimen (p < 0.01), but the statistical significance of the difference was lost in the corrected analysis. After 4 months of therapy, there were no relapses among mice treated with any one of the three RMZ-based regimens, whereas 5 of 12 mice treated with the standard regimen relapsed. Again, this difference was statistically significant before, but not after, correction for multiple comparisons (p < 0.05). When analyzed together, mice receiving any RMZ-based regimen for 3 or 4 months were less likely to relapse than were mice receiving the standard regimen for the same duration (p < 0.001). In other words, mice receiving any of the three RMZ-based regimens achieved a stable cure more rapidly than those receiving the standard regimen. Although stable cure was achieved in all mice receiving any one of the three RMZ-based regimens for 4 months, treatment with the standard regimen required 6 months of therapy for stable cure of all mice. No differences could be discerned between any of the RMZ-based regimens at any time point.
In this study, we directly measured the proportion of mice with culture-positive relapse after various durations of treatment with RMZ- and RHZ-based regimens. This design allowed us to determine better the extent to which use of an RMZ-based regimen would permit shortening the duration of therapy. Although we observed no relapse among mice treated for at least 4 months with 2RMZ/2RM, mice treated with 2RHZ/4RH required 6 months of treatment before no relapse could be detected. In other words, stable cure was achieved 2 months earlier in mice treated with an RMZ-based regimen rather than the standard RHZ-based regimen. The magnitude of this shortening effect is similar to that achieved with the introduction of pyrazinamide into first-line use. It is also worth noting that the moxifloxacin dose used in this experiment (100 mg/kg) is more likely to have underestimated, rather than overestimated, the activity of moxifloxacin expected in humans receiving the standard 400-mg dose (8, 16). Therefore the findings in mice are likely to be reproducible in humans, for reasons discussed in our previous article (8). We also found similar efficacy for all RMZ-based regimens whether pyrazinamide was administered for 1 month, 2 months, or the entire duration of therapy. Although inclusion of pyrazinamide during the intensive phase clearly adds to the sterilizing activity of rifampin and isoniazid in the mouse model (15) and in the treatment of human disease, the duration of therapy necessary to derive its maximum benefit has never been determined with precision. Randomized clinical trials have failed to demonstrate any benefit to extending the use of pyrazinamide beyond the first 2 months of therapy when the continuation regimen included rifampin and isoniazid (10–12), but the minimum duration of therapy necessary to realize the benefits of pyrazinamide remains unknown. Two clinical trials have compared a 1-month daily intensive phase regimen of streptomycin (S), isoniazid, rifampin, and pyrazinamide to a 2-month intensive phase regimen with the same drugs before switching to a three times weekly regimen of rifampin and isoniazid (17–19). The two regimens had similar relapse rates in both studies. Although the proportion of subjects with conversion to negative sputum cultures was lower at 2 months for subjects receiving the 1-month intensive phase (85% vs. 99%) in one of the two studies, 97% of subjects receiving the 1-month intensive phase had negative sputum cultures after 3 months of therapy (17). The delayed sputum conversion in patients receiving the 1-month intensive phase regimen could have been related to the receipt of only 1 month of pyrazinamide, only 1 month of streptomycin, and/or only 1 month of daily rifampin and isoniazid before the intermittent continuation phase regimen. However, the cure rates were not negatively affected (18). The results of this trial should not discourage the evaluation of shorter durations of pyrazinamide administration in future clinical trials of RMZ-based regimens. If our results with the use of only 1 month of pyrazinamide in the intensive phase of therapy can be confirmed in clinical trials, the result would be regimens that would be easier to deliver and would likely have fewer adverse effects. This may be critical if the recent finding that the combination of rifampin and pyrazinamide appears to be more hepatotoxic than RHZ also applies when RMZ is compared with RHZ (20). There are two caveats to consider in the interpretation of these results. First, despite initial passage in the mouse, the strain of M. tuberculosis used for infection appeared to be of somewhat reduced virulence because the bacillary burden in the lungs increased by only three logs, rather than the expected four logs, during the period before treatment. The lower bacillary burden at the onset of treatment resulted in more rapid attainment of negative lung cultures compared with our previous study. Although we cannot exclude the possibility that the diminished replication capacity of the organism differentially favored one regimen over another, we believe it is unlikely. Second, the relatively small numbers of mice used to determine the frequency of relapse limited the power to detect small differences between treatment groups. These two limitations are being addressed by a study that involves larger numbers of mice for relapse determinations and infection with a mouse-passaged isolate of the standard strain, M. tuberculosis H37Rv.
The development of an efficacious ultrashort course (i.e.,
Supported by the Global Alliance for TB Drug Development, the National Institutes of Health (supplement to grant AI43846), and Bayer (moxifloxacin). Conflict of Interest Statement: E.L.N. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; T.Y. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; S.T. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; K.W. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; I.R. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; R.J.O. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; A.A.V. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; R.E.C. has served as a consultant for Bayer on therapeutic uses of recombinant interleukins in human immunodeficiency virus–infected patients receiving a compensation of $800 in 2002 and 2003; W.R.B. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; J.H.G. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. Received in original form July 8, 2004; accepted in final form August 10, 2004
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ABSTRACT
METHODS
4 months) regimen for the treatment of TB in humans is expected to promote treatment completion rates and facilitate the worldwide implementation of directly observed treatment programs (