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Antimicrobial Agents and Chemotherapy, November 2000, p. 3167-3168, Vol. 44, No. 11
Departments of Medicine, Veterans Affairs Medical
Center and State University of New York Upstate Medical University,
Syracuse, New York 13210
Received 22 June 2000/Returned for modification 6 July
2000/Accepted 2 August 2000
The newer rifamycin, rifalazil (RLZ) (previously known as
KRM-1648), has been shown in prior experiments to be a highly potent drug against Mycobacterium tuberculosis. In this report, we
studied the efficacy of RLZ in combination with pyrazinamide (PZA) and ethambutol (EMB) in a long-term in vivo experiment and compared their
activity with the isoniazid (INH)-rifampin (RIF) combination which is
presently used in the clinic. Combinations of RLZ with PZA alone or
with both PZA and EMB were both found to have sterilizing activities
comparable to that of the INH-RIF combination but significantly better
activity with respect to relapse of infection. These results suggest
that RLZ, or other agents with similar activity, could be combined with
available agents to act as a potential alternative drug regimen to the
currently used INH-RIF combination.
Therapy for tuberculosis is arduous
due to the long duration of therapy and multidrug regimens. The current
standard regimen of isoniazid (INH), rifampin (RIF), and pyrazinamide
(PZA) requires 6 to 9 months of daily treatment. Approaches to improve
patient compliance include instituting an intermittent treatment
regimen or shortening the duration of therapy. With the discovery of
the newer rifamycins, rifapentine and rifalazil (RLZ) (previously known
as KRM-1648), the potential was created for shortening existing treatment regimens.
In the last decade, several groups extensively compared the efficacy of
the newer rifamycins in Mycobacterium tuberculosis in vitro
(6, 14, 15) and in vivo mouse models (1, 2, 5, 7, 8, 9,
16). RLZ is the most active single drug available against
M. tuberculosis today. In previous experiments in our
laboratory, we compared the activity of RLZ to that of RIF when given
as a single drug or in a combination regimen with INH. RLZ was
significantly more active than RIF as a single drug (11).
The combination of RLZ and INH was more effective than that of RIF and
INH, reducing the treatment period to apparent sterilization by half (6 versus 12 weeks). Six months following cessation of a 12-week RLZ-INH
treatment regimen, the organs still appeared to be sterilized, whereas
the RIF-INH regimen yields regrowth as early as 4 weeks after
completion of therapy (10).
In this report, we studied the efficacy of RLZ in combination with PZA
and ethambutol (EMB) in long-term in vivo experiments in order to test
their ability in potential alternative regimens to the currently used
INH, RIF, and PZA combination.
Six-week-old outbred female Swiss mice (Charles River, Wilmington,
Mass.) were infected intravenously through a caudal vein with M. tuberculosis ATCC 35801 (strain Erdman) as described previously (13). Each mouse received 3.6 × 107 viable
organisms suspended in 0.2 ml of modified 7H10 broth. The inoculum size
was verified by plating serial dilutions of the bacterial suspension in
triplicate on 7H10 agar plates (BBL Microbiology Systems, Cockeysville,
Md.) supplemented
with 10% Middlebrook oleic acid-albumin-dextrose-catalase (OADC)
enrichment. Plates were incubated at 37°C in ambient air for 4 weeks
prior to counting viable M. tuberculosis colonies (CFU).
Every group consisted of eight mice at each time point unless stated
otherwise. Treatment was started 1 week postinfection. A control group
of infected mice was sacrificed at the start of treatment (early
control group). A second group of infected but untreated mice was
sacrificed 4 weeks after therapy was initiated (late control group).
Drugs were administered orally by gavage, 5 days per week for 12 weeks.
RLZ (20 mg/kg) was given alone or in combination with PZA (150 mg/kg)
and/or EMB (150 mg/kg) and was compared to RIF (20 mg/kg) combined with
INH (25 mg/kg). RLZ was provided by Kaneka Corporation, Osaka, Japan.
PZA, RIF, EMB, and INH were purchased from Sigma Chemical Co., St.
Louis, Mo. RLZ and RIF were dissolved in dimethyl sulfoxide (DMSO),
with subsequent dilution in distilled water prior to administration. The final concentration of DMSO in the drug preparations was 5%. INH,
EMB, and PZA were dissolved in water. The water-soluble drugs were
administered in the morning; the DMSO-soluble drugs were given in the
afternoon. The MICs of RLZ, RIF, and INH for ATCC strain 35801 are
0.00047, 0.06, and 0.03 µg/ml, respectively. MICs were determined in
modified 7H10 broth (pH 6.6) as described previously (3).
Mice were sacrificed after completion of the 12-week treatment period,
and viable cell counts were determined from the homogenates of spleens
and lungs. Parallel treatment groups were sacrificed after a 12-week
observation period. Sacrifice was performed by CO2
inhalation. Spleens and right lungs were aseptically removed and ground
in a tissue homogenizer (IdeaWorks! Laboratory Devices, Syracuse,
N.Y.). The number of viable organisms was determined as described
above. The entire volume of organ homogenates was plated to determine
the number of culturable mycobacteria per organ. Statistical analysis
to evaluate the number of mice showing regrowth of infection after
cessation of treatment was evaluated by the Fisher exact test for a
2-by-2 contingency (4).
The results of the study are displayed in Tables
1 and 2.
After 12 weeks of treatment, in every treatment group a significant reduction in the number of M. tuberculosis organisms was
achieved, with an apparent clearance of the organs in the INH-RIF and
RLZ-PZA-EMB groups (Table 1). The numbers of culture-positive mice
between the treatment groups were not significantly different. At 12 weeks after the cessation of therapy, regrowth was detected in all
groups. Of note, however, only one mouse in the RLZ-PZA-EMB group
demonstrated regrowth in the lungs (one colony). Also, in the spleens
of mice from this group, regrowth was observed in only half of the mice compared to almost all mice in the other groups (Table 2). In spleens,
no significant difference was observed in the number of mice showing
relapse of infection between all treatment groups. In contrast, in the
lungs of the RLZ-PZA and RLZ-PZA-EMB groups the number of
culture-positive mice was significantly lower than in the RLZ and
RLZ-EMB groups. The addition of EMB to RLZ-PZA did not significantly
improve the treatment outcome.
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Copyright © 2000, American Society for Microbiology. All rights reserved.
Evaluation of Rifalazil in a Combination Treatment
Regimen as an Alternative to Isoniazid-Rifampin Therapy in a
Mouse Tuberculosis Model
ABSTRACT
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TABLE 1.
M. tuberculosis CFU counts in spleens and
lungs after 12 weeks of therapy
TABLE 2.
M. tuberculosis CFU counts in spleens and
lungs after 12 weeks of therapy and 12 weeks of observation
We conclude from this long-term drug study that the combinations RLZ-PZA and RLZ-PZA-EMB were the most active regimens evaluated. The addition of EMB did not significantly improve the treatment outcome in our experimental setting. However, it is possible the three-drug therapy might be superior to the two-drug regimen with regard to decreasing the emergence of drug resistance. Future experiments studying the frequency of drug resistance in long-term experiments might reveal a beneficial effect of adding a third drug. In conclusion, a sufficiently strong agent such as RLZ in combination with agents with modest activities, e.g., PZA and EMB, might be a valuable alternative to the presently used INH combination with RIF and PZA and may allow for a significantly shorter course regimen.
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ACKNOWLEDGMENTS |
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We acknowledge the technical assistance provided by M. S. DeStefano.
This study was supported in part by the NCDDG-OI program, a cooperative agreement U19-AI40972 with NIAID, and grants from the Kaneka Corporation, Osaka, Japan, and PathoGenesis, Seattle, Wash.
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FOOTNOTES |
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* Corresponding author. Mailing address: VAMC, 800 Irving Ave., Syracuse, NY 13210. Phone: (315) 476-7461, ext. 3324. Fax: (315) 476-5348. E-mail: Michael.Cynamon{at}med.va.gov.
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Antimicrobial Agents and Chemotherapy, November 2000, p. 3167-3168, Vol. 44, No. 11
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Copyright © 2000, American Society for Microbiology. All rights reserved.
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