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Bacterial peptide deformylase (PDF) belongs to a subfamily of metalloproteases catalyzing the removal of the N-terminal formyl group from newly synthesized proteins. We report the synthesis and biological activity of highly potent inhibitors of Mycobacterium tuberculosis (Mtb) PDF enzyme as well as the first X-ray crystal structure of Mtb PDF. Structure-activity relationship and crystallographic data clarified the structural requirements for high enzyme potency and cell based potency. Activities against single and multi-drug-resistant Mtb strains are also reported.

Objectives: Gatifloxacin previously demonstrated good in vitro and in vivo activities against Mycobacterium tuberculosis. Several regimens of gatifloxacin in combination with rifampicin were compared with isoniazid plus rifampicin in a mouse tuberculosis model., Methods: C57BL/6 mice were infected intranasally with approximately 10(6) viable M. tuberculosis organisms. Treatment with various regimens of gatifloxacin plus rifampicin was started 1 week post-infection and was administered for 4-12 weeks. Mice were euthanized at the end of therapy and their right lungs were removed and cell counts were determined., Results: Gatifloxacin 100 mg/kg plus rifampicin 10 mg/kg has activity similar to that of isoniazid plus rifampicin in the 12 week treatment model. Gatifloxacin 300 mg/kg plus rifampicin 20 mg/kg yields a non-cultivatable state after 12 weeks of therapy and approaches but does not achieve a durable cure., Conclusions: Gatifloxacin in combination with rifampicin is a promising combination for potential evaluation in human clinical trials. Gatifloxacin plus rifampicin regimens had activities similar to or better than isoniazid plus rifampicin. A quinolone plus rifampicin combination may provide the foundation for shorter course regimens than the current isoniazid plus rifampicin-based regimen.

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.

Previous experiments with rifalazil (RLZ) (also known as KRM-1648) in combination with isoniazid (INH) demonstrated its potential for short-course treatment of Mycobacterium tuberculosis infection. In this study we investigated the minimum RLZ-INH treatment time required to eradicate M. tuberculosis in a murine model. RLZ-INH treatment for 6 weeks or longer led to a nonculturable state. Groups of mice treated in parallel were killed following an observation period to evaluate regrowth. RLZ-INH treatment for a minimum of 10 weeks was necessary to maintain a nonculturable state through the observation period. Pyrazinamide (PZA) was added to this regimen to determine whether the treatment duration could be further reduced. In this model, the addition of PZA did not shorten the duration of RLZ-INH treatment required to eradicate M. tuberculosis from mice. The addition of PZA reduced the number of mice in which regrowth occurred, although the reduction was not statistically significant.

The use of isoniazid (INH) for the treatment of INH-resistant Mycobacterium tuberculosis infection has been controversial. The purpose of the present studies was to determine if there is a dose response with INH for INH-susceptible M. tuberculosis Erdman (ATCC 35801), and whether high-dose INH (100 mg/kg of body weight) was more effective than standard-dose INH (25 mg/kg) for therapy of tuberculosis infections caused by INH-resistant mutants of M. tuberculosis Erdman. Six-week-old CD-1 mice were infected with approximately 10(7) viable mycobacteria. Early control groups of infected but untreated mice were euthanized by CO(2) inhalation 1 week later when treatment was initiated. INH (25, 50, 75, and 100 mg/kg) was given by gavage 5 days/week for 4 weeks. Late control groups of untreated mice and treated mice were sacrificed 2 days after the last dose of drug. Spleens and right lungs were removed aseptically and homogenized, and viable cell counts were determined by titration on 7H10 agar plates. In the next study, INH at 100 mg/kg was compared to INH at 25 mg/kg against an isogenic mutant of M. tuberculosis Erdman (INH MIC, 2 microg/ml) and the parent strain. This mutant was found to have a mutation in the KatG protein (Phe to Leu at position 183). In the first study, there was no dose response with increasing doses of INH. In the second study, there was no significant difference between the reduction of viable cell counts for mice treated with INH at 100 mg/kg and that for mice treated with INH at 25 mg/kg (parent or INH-resistant mutant). These preliminary results suggest that INH may be useful in combination therapy of M. tuberculosis infections caused by low-level INH-resistant organisms (INH MICs, 0.2 to 5 microg/ml) and that higher doses of INH are unlikely to be more efficacious than the standard 300-mg/day dose.

Besides direct bactericidal activity, long-term effectiveness is one of the most important features to consider when developing new drugs for chemotherapy. In this study, we evaluated the ability of rifapentine (RFP), in monotherapy and combination therapy, to completely eradicate a Mycobacterium tuberculosis infection and to prevent relapse posttreatment in a Swiss mouse model. The combination of RFP, isoniazid (INH), and pyrazinamide (PZA) administered daily resulted in an apparent clearance of M. tuberculosis organisms in the lungs and spleens of infected mice after 10 weeks of treatment. However, 3 months after the cessation of therapy, bacterial regrowth occurred in mice treated for a 12-week period, indicating a relapse of infection. In intermittent treatment regimens of RFP in combination with INH and PZA, sterilization was achieved when mice were treated two to five times per week for 9 weeks. Bacterial growth was still observed in the once-weekly treatment group. Our results show that mouse models can predict important parameters for new drugs. We stress the necessity for long-term posttreatment observation in animal models for the routine evaluation of new drugs for antituberculosis chemotherapy.

The activities of linezolid, eperezolid, and PNU-100480 were evaluated in a murine model of tuberculosis. Approximately 10(7) viable Mycobacterium tuberculosis ATCC 35801 organisms were given intravenously to 4-week-old outbred CD-1 mice. In the first study, treatment was started 1 day postinfection and was given by gavage for 4 weeks. Viable cell counts were determined from homogenates of spleens and lungs. PNU-100480 was as active as isoniazid. Linezolid was somewhat less active than PNU-100480 and isoniazid. Eperezolid had little activity in this model. In the next two studies, treatment was started 1 week postinfection. A dose-response study was performed with PNU-100480 and linezolid (both at 25, 50, and 100 mg/kg of body weight). PNU-100480 was more active than linezolid, and its efficacy increased with an escalation of the dose. Subsequently, the activity of PNU-100480 alone and in combination with rifampin or isoniazid was evaluated and was compared to that of isoniazid-rifampin. The activity of PNU-100480 was similar to that of isoniazid and/or rifampin in the various combinations tested. Further evaluation of these oxazolidinones in the murine test system would be useful prior to the development of clinical studies with humans.

The activity of KRM-1648, alone and in combination with isoniazid, was compared with those of isoniazid, rifampin, and the combination of rifampin plus isoniazid in a murine model of tuberculosis. Four-week-old female CD-1 mice were infected intravenously with approximately 10(7) viable Mycobacterium tuberculosis ATCC 35801 organisms. Treatment was started 1 week postinfection and was given by gavage 5 days per week. The duration of the treatment phase was 12 weeks, with groups of mice sacrificed at 2, 4, 6, 8, and 12 weeks. For the observation phase, additional groups of treated mice were sacrificed at 4, 8, 16, and 24 weeks after the cessation of treatment. Viable cell counts were determined from homogenates of the spleens and the right lungs. KRM-1648 was the most active single agent evaluated and resulted in no detectable CFUs in the spleens and lungs by the end of 6 weeks of treatment. Neither rifampin nor isoniazid reduced cell counts to undetectable levels, even after 12 weeks of treatment. The combination of KRM-1648 plus isoniazid was much more active than rifampin plus isoniazid. KRM-1648 plus isoniazid resulted in the apparent sterilization of organs at 6 months following the cessation of treatment. The promising activity of KRM-1648 may allow for ultrashort-course therapy of tuberculosis, i.e., treatment regimens of 4 months or less.

The activity of pyrazinamide (PZA) against eight isolates of Mycobacterium tuberculosis in a murine infection model was evaluated. M. tuberculosis isolates with various degrees of in vitro susceptibility to PZA (MIC range, 32 to > 2,048 micrograms/ml) were used. Four-week-old female mice were infected intravenously with approximately 10(7) viable M. tuberculosis organisms. PZA at 150 mg/kg of body weight was started 1 day postinfection and given 5 days/week for 4 weeks. Infected but untreated mice were compared with PZA-treated mice. Mice were sacrificed at the completion of the treatment period, and viable cell counts were determined from homogenates of spleens and right lungs. PZA had activity in the murine test system against M. tuberculosis isolates for which the MICs were < or = 256 micrograms/ml. However, there was an inconsistent correlation between the absolute MICs and the reductions in organ viable cell counts. Studies with drug-resistant M. tuberculosis isolates with an isogenic background would improve evaluation of drug efficacy in the murine test system. Further evaluation of antimycobacterial agents against monodrug-resistant isolates will provide data that will be useful for development of algorithms for treatment of infection with drug-resistant organisms.

The activity of KRM-1648 was evaluated in a murine model of tuberculosis. Approximately 10(7) viable Mycobacterium tuberculosis ATCC 35801 organisms were given intravenously to 4-week-old female outbred mice. Treatment was started 1 week postinfection and given by gavage for 4 weeks. Viable-cell counts were determined from homogenates of spleen and lung tissues. The activity of KRM-1648 was compared with those of rifampin and rifabutin at 20 mg/kg of body weight. KRM-1648 was more active than either rifampin or rifabutin against organisms in spleens and lungs. KRM-1648 alone and in combination with either isoniazid, ethambutol, pyrazinamide, or levofloxacin was evaluated. Other treatment groups received isoniazid, ethambutol, pyrazinamide, or levofloxacin as single agents. KRM-1648 was the most active single agent evaluated. KRM-1648-pyrazinamide and KRM-1648-isoniazid were the most active combinations. These combinations were more active than KRM-1648 alone. The promising activity of KRM-1648 in M. tuberculosis-infected mice suggests that it is a good candidate for clinical development as a new antituberculosis agent.

The activity of azithromycin (AZI) was evaluated in the beige mouse model of disseminated Mycobacterium avium infection. Mice were infected intravenously with approximately 10(7) viable avium ATCC 49601. AZI at 50, 100, or 200 mg/kg of body weight or clarithromycin (CLA) at 200 mg/kg was given by gavage 5 days per week for 4 weeks. Groups of treated mice were compared with untreated control animals. A dose-related reduction in cell counts in organs was observed with AZI treatment. AZI at 200 mg/kg was more active than CLA at 200 mg/kg against organisms in spleens. The activities of these two agents at 200 mg/kg were comparable against organisms in lungs. In a second study, AZI at 200 mg/kg was given daily for 5 days; this was followed by intermittent AZI treatment for the next 3 weeks. The activities of AZI given on a three-times- and five-times-per-week basis in the continuation phase were comparable. AZI given on a once-weekly basis was less active. The regimen of AZI given in combination with rifapentine on a once-weekly basis for 8 weeks showed promising activity. Clinical evaluation of AZI and rifapentine will help to define the roles of these agents in the treatment of disseminated M. avium complex infection.

The dose-response activity of rifabutin and the comparative activities of rifabutin and rifapentine were evaluated in the beige mouse model of disseminated Mycobacterium avium complex (MAC) infection. In the dose-response study, mice were infected intravenously with approximately 10(7) viable M. avium ATCC 49601. Treatment with rifabutin at 10, 20, or 40 mg/kg of body weight was started 7 days postinfection and was administered daily for 10 days. The mice were sacrificed 3 to 5 days after the last dose. Spleens, livers, and lungs were homogenized, and viable cell counts were determined by serial dilution and plating onto Middlebrook 7H10 agar. A dose-related reduction in MAC cell counts in the organs was noted for this MAC isolate. The comparative activities of rifabutin and rifapentine were determined against a total of five MAC isolates in the beige mouse model. Rifabutin or rifapentine (20 mg/kg each) was administered to infected mice for 10 days. Groups of treated mice were compared with untreated control animals. Despite favorable in vitro susceptibility results, rifabutin and rifapentine had activities in the spleens against only two of the five MAC isolates. For these two MAC isolates, rifabutin was more active than rifapentine. These agents had activities in the lungs against three of five isolates. Further study of rifabutin or rifapentine against a broader range of clinical isolates in a murine infection model may be useful as part of the continuing development of newer rifamycins as anti-MAC agents.

The activity of clarithromycin alone and in combination with other antimycobacterial agents was evaluated in the beige (C57BL/6J bgj/bgj) mouse model of disseminated Mycobacterium avium complex (MAC) infection. A dose-response experiment was performed with clarithromycin at 50, 100, 200, or 300 mg/kg of body weight administered daily by gavage to mice infected with approximately 10(7) viable MAC. A dose-related reduction in spleen and liver cell counts was noted with treatment at 50, 100, and 200 mg/kg. The difference in cell counts between treatment at 200 and 300 mg/kg was not significant. Clarithromycin at 200 mg/kg of body weight was found to have activity against three additional MAC isolates (MICs for the isolates ranged from 1 to 4 micrograms/ml by broth dilution). Clarithromycin at 200 mg/kg in combination with amikacin, ethambutol, temafloxacin, or rifampin did not result in increased activity beyond that seen with clarithromycin alone. Clarithromycin in combination with clofazimine or rifabutin resulted in an increase in activity beyond that seen with clarithromycin alone. The combination of clarithromycin with clofazimine or rifabutin should be considered for evaluation in the treatment of human MAC infections.

This guideline addresses the evaluation of new antimycobacterial drugs in the treatment and prevention (secondary prophylaxis) of infection by M. tuberculosis. Patients may be enrolled in clinical trials on the basis of clinical and/or microbiological criteria. A therapeutic regimen will likely include a combination of drugs; a randomized, active-control, comparative clinical trial is recommended. If appropriate samples can be obtained for culture during follow-up without placing the patient at unwarranted risk, the assessment of microbiological outcome is paramount. Prophylaxis will probably require a single drug, and a similar study design is preferred.

The comparative activities of azithromycin and clarithromycin and the activities of azithromycin alone and in combination with other antimycobacterial agents were evaluated in the beige mouse model of disseminated Mycobacterium avium complex infection. Azithromycin was similar in activity to clarithromycin. Azithromycin plus clofazimine plus ethambutol reduced the number of splenic organisms more than azithromycin alone, while the combination was less active than azithromycin alone for bacteria in lungs. Rifabutin had activity similar to that of azithromycin for organisms in spleens and lungs. Rifabutin plus azithromycin was more active than either agent alone for organisms in spleens, but the combination's activity was not significantly different from that of rifabutin for organisms in lungs. The activity of azithromycin against several M. avium complex isolates was evaluated. The reduction of viable cell counts in spleens ranged from 1.7 to 0.8 log units. For the three isolates studied, there was little correlation between the in vitro MIC and the in vivo activity.

The activity of TLC G-65 (a liposomal gentamicin preparation), alone and in combination with rifapentine, clarithromycin, clofazimine and ethambutol, was evaluated in the beige mouse (C57BL/6J--bgj/bgj) model of disseminated Mycobacterium avium infection. TLC G-65 was found to be more active than amikacin. The combination of rifapentine and TLC G-65 was more active than either agent alone. The activity of clarithromycin in combination with TLC G-65 was similar to that of either agent alone. Clofazimine improved the activity of TLC G-65 with respect to the spleen, while ethambutol improved the activity with respect to the liver. Clofazimine and ethambutol enhanced the activity of TLC G-65 against bacteria in the lungs. TLC G-65 in combination with rifapentine appears to be an attractive regimen for the treatment of infections caused by bacteria in the M. avium complex.

The activity of rifapentine (MDL 473) was evaluated in the beige (C57BL/6J-bgj/bgj) mouse model of disseminated Mycobacterium avium infection. Approximately 10(7) cfu of M. avium, serotype 1, were given iv. Seven days later treatment was started with intraperitoneal rifapentine at 20 mg/kg of body weight. Treatment was given daily for five days followed by twice weekly for three weeks. The mice were killed two days after the last dose. Spleens, livers and lungs were homogenized and cfu/organ determined. Analysis of variance and Tukey honestly significant difference tests indicated that rifapentine reduced cfu in each of the organs compared with untreated controls. A dose-response experiment was performed with a daily rifapentine dose of 10, 20 or 40 mg/kg administered intraperitoneally. Dose-related reductions in cfu counts were observed in each of the organs. The activity of oral rifapentine at 20 mg/kg was demonstrated in a comparative experiment with rifampicin at 20, 40 or 60 mg/kg. Rifapentine significantly reduced cfu counts in organs compared with rifampicin. Rifapentine should be considered for further evaluation in the treatment of M. avium complex infection in humans.