Gatifloxacin in combination with rifampicin in a murine tuberculosis model

Abstract

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 ∼10⁶ 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.

Introduction

The development of shorter course treatment regimens for tuberculosis would likely facilitate improved tuberculosis control programmes. Gatifloxacin and moxifloxacin, two newer quinolones, were previously shown to have good in vitro and in vivo activities.^1–5 Recent studies in mice by Nuermberger et al.⁶ demonstrated that moxifloxacin plus rifampicin and pyrazinamide shortened the time required to achieve negative lung cultures by up to 2 months as compared with isoniazid combined with rifampicin and pyrazinamide. Subsequently Nuermberger et al. evaluated the proportion of mice with culture-positive relapse following various durations of treatment with moxifloxacin, rifampicin and pyrazinamide compared with isoniazid, rifampicin and pyrazinamide.⁷ No relapses were observed in mice treated for 4 months with the moxifloxacin regimen, however, the isoniazid-based regimen required 6 months of therapy to achieve similar results. Pyrazinamide was used during the initial 2 months in each of these regimens.

Moxifloxacin and gatifloxacin are currently being evaluated in multicentre trials for the treatment of tuberculosis. Moxifloxacin is being studied in combination with rifampicin plus pyrazinamide in comparison with isoniazid plus rifampicin and pyrazinamide. Gatifloxacin is being evaluated as an addition to the ‘standard regimen’ (isoniazid, rifampicin and pyrazinamide) compared with the standard regimen. The focus of both trials is to determine whether regimens containing moxifloxacin or gatifloxacin can shorten the length of therapy.

The aim of the present study was to evaluate the activities of gatifloxacin in combination with rifampicin using a ‘standard’ and high dose of each agent in comparison with isoniazid plus rifampicin.

Materials and methods

Drugs

Gatifloxacin was provided by Bristol-Myers Squibb Co., Princeton, NJ, USA. Isoniazid and rifampicin were purchased from Sigma Chemical Co., Saint Louis, MO, USA. Isoniazid was dissolved in distilled water. Gatifloxacin and rifampicin were dissolved in 20% dimethyl sulphoxide (DMSO) with subsequent dilution in distilled water (final concentration of DMSO was 4%). The solutions were prepared, aliquotted, frozen at −20°C and then thawed before use. Isoniazid and gatifloxacin were dosed in the mornings and rifampicin was given in the afternoons.

Isolate

Mycobacterium tuberculosis ATCC 35801 (strain Erdman) was obtained from the ATCC, Manassas, VA, USA. The MICs of the antimicrobial agents were determined using a microtitre methodology with 7H10 broth (pH 6.6; 7H10 agar formulation with agar and Malachite green omitted) supplemented with Middlebrook oleic acid-albumin-dextrose-catalase (OADC) enrichment (BBL, Sparks, MD, USA) and 0.05% Tween 80; the MICs were as follows: isoniazid, 0.03 mg/L; rifampicin, 0.06 mg/L; and gatifloxacin, 0.125 mg/L.

Inoculum preparation

The organism was grown in 7H10 broth with 10% OADC enrichment and 0.05% Tween 80 on a rotary shaker for 7 days at 37°C. The cells were diluted in 7H10 broth to yield 100 Klett units per mL (photoelectric colorimeter; Manostat Corp., New York, NY, USA) or ∼5 × 10⁷cfu/mL. Aliquots of the cell suspension were stored at −70°C prior to use.

On the day of infection the culture was thawed and sonicated. The inoculum size was determined by plating serial dilutions of the bacterial suspension in triplicate on 7H10 agar plates.

Infection study

Six-week-old female C57BL/6J mice (The Jackson Laboratory, Bar Harbor, ME, USA) were anaesthetized by intramuscular delivery of telazol (45 mg/kg)/xylazine (7.5 mg/kg) cocktail (Lederle Parenterals, Carolina, Puerto Rico and Bayer Corp., Shawnee Mission, KS, USA, respectively) and subsequently infected intranasally with ∼1 × 10⁶ viable M. tuberculosis in a 20 µL volume. There were six mice per group.

Treatment was started 1 week post-infection. An untreated group of mice was euthanized at the start of treatment (early controls). To ensure bacterial virulence a second group of untreated mice (late controls) was observed until the mice were moribund, and then euthanized. Therapy was given by oral gavage in a 0.2 mL volume 5 days per week for 4 or 12 weeks. In the initial study gatifloxacin 100 mg/kg plus rifampicin 10 mg/kg was compared with each agent alone for 4 weeks.

The subsequent 6 and 12 week treatment study consisted of the following treatment groups: isoniazid 25 mg/kg plus rifampicin 10 mg/kg; gatifloxacin 100 mg/kg plus rifampicin 10 mg/kg; and gatifloxacin 300 mg/kg plus rifampicin 20 mg/kg. Parallel groups of mice were treated for 12 weeks and then observed without therapy for an additional 8 weeks.

All mice were euthanized by CO₂ inhalation. The right lungs from the early control group and from each time point were aseptically removed and ground in a tissue homogenizer (IdeaWorks Laboratory Devices, Syracuse, NY, USA) containing 1 mL of 0.9% NaCl with 0.05% Tween 80. The number of viable organisms was determined by serial 10-fold dilution and subsequent inoculation onto 7H10 agar plates.

The right lungs from the 12 week and observation time points were homogenized in 0.5 mL of saline with Tween 80. Whole lung homogenates were plated in five equal portions.

The cultures were incubated at 37°C in ambient air for 4 weeks prior to counting colonies.

The animal protocol was approved by the Subcommittee for Animal Studies (SAS), Veterans Affairs Medical Center, Syracuse, NY, ACORP #010 “Mouse Model of Nasally Induced Mycobacterium Tuberculosis Infection”.

Statistical evaluation

Viable cell counts were converted to logarithms, which were then evaluated by one way analysis of variance. Statistically significant effects from the analyses of variance were further evaluated by the Tukey's multiple comparisons test to make pair-wise comparisons among means.⁸

Results

Four week treatment study

Each treatment group had significantly lower cfu/lung (P < 0.001) than was present at the initiation of therapy (early controls) (Table 1). The gatifloxacin plus rifampicin group had the lowest lung counts. There was no significant difference between this group and the gatifloxacin monotherapy group. There was a significant difference (P < 0.05) compared with rifampicin alone.

Six and twelve week treatment study

The mycobacterial load at the initiation of therapy (early control group) was 7.97 ± 0.43 log₁₀ cfu/lung. Mice from the late control group were moribund 14 days post-infection and were euthanized. Each of the 6 week treatment regimens reduced the mycobacterial counts in the lungs (P < 0.001) compared with those in the lungs of the early control group at the initiation of therapy (Table 2). The gatifloxacin 300 mg/kg plus rifampicin 20 mg/kg group was more active than the isoniazid plus rifampicin group or the low dose gatifloxacin plus rifampicin group (P < 0.001).

Each of the 12 week treatment regimens reduced the mycobacterial counts in the lungs (P < 0.001) compared with the early control group (Table 2). The mean log₁₀ cfu/lung for the isoniazid plus rifampicin, gatifloxacin 100 mg/kg plus rifampicin 10 mg/kg and gatifloxacin 300 mg/kg plus rifampicin 20 mg/kg groups were as follows: 0.20, 1.10 and 0, respectively. The number of mice in each group that exhibited no organism growth at this time point was 3, 1 and 6, respectively.

Observation phase

None of the regimens tested yielded a durable cure (no regrowth after the observation period). Five of 5 mice in the isoniazid plus rifampicin group had regrowth following the observation phase, while 6 of 6 and 3 of 5 mice in the gatifloxacin 100 mg/kg plus rifampicin 10 mg/kg and the gatifloxacin 300 mg/kg plus rifampicin 20 mg/kg groups had regrowth. The mean log cfu/lung for isoniazid plus rifampicin, gatifloxacin 100 mg/kg plus rifampicin 10 mg/kg and gatifloxacin 300 mg/kg plus rifampicin 20 mg/kg group was 2.86, 2.74 and 0.22, respectively. The high dose gatifloxacin plus rifampicin group was significantly different (P < 0.001) than either of the other treatment groups. There was no difference (P > 0.05) between isoniazid plus rifampicin, and gatifloxacin 100 mg/kg plus rifampicin 10 mg/kg.

Discussion

The high dose gatifloxacin plus rifampicin regimen nearly achieved a durable cure, whereas the low dose of gatifloxacin plus rifampicin failed to achieve a non-cultivatable state after 12 weeks of treatment. It would be of interest to evaluate the impact pyrazinamide might have during the initial 8 weeks of therapy with a gatifloxacin plus rifampicin regimen. Nuermberger et al.⁶ demonstrated that pyrazinamide in combination with moxifloxacin plus rifampicin significantly reduced the treatment time required to eliminate mycobacteria in the lungs of mice compared with the standard drug regimen. Similarly our laboratory showed that pyrazinamide enhanced the efficacy of gatifloxacin plus ethionamide in a previous study.² In an earlier study from this laboratory, isoniazid plus rifampicin in an intravenous infection model required ∼24 weeks of therapy to achieve a durable cure.⁹

We have demonstrated that gatifloxacin has impressive in vivo activity against M. tuberculosis in a murine tuberculosis model. It is likely that this agent would be more active against clinical tuberculosis than several of the older quinolones (ciprofloxacin, ofloxacin and levofloxacin) that are currently being used for the therapy of patients infected with multidrug-resistant tuberculosis. Both gatifloxacin and moxifloxacin appear to have relatively similar activities in vitro and in vivo (in mice). Moxifloxacin has recently been shown to be more bactericidal with chloramphenicol-treated M. tuberculosis than was gatifloxacin.¹⁰ This finding could explain in part the superior activity of moxifloxacin plus rifampicin observed by Nuermberger (using a BALB/c mouse model)^6,7 when compared with the results found with gatifloxacin plus rifampicin in this study. A study evaluating the in vitro activities of several fluoroquinolones against a rifampicin-tolerant population of M. tuberculosis found that moxifloxacin and gatifloxacin had similar sterilizing activities.⁴ A direct comparison of the activities of moxifloxacin and gatifloxacin was not made in the in vitro model that measured the elimination of rifampicin-tolerant persisters in the presence of rifampicin, which the authors suggested most closely resembled the clinical setting of first-line treatment.⁴

The rationale for the high dose group in this study was to determine if more intensive therapy would potentially impact the length of therapy required to achieve a durable cure (culture negative status after a post-treatment observation phase). It is not clear whether rifampicin or gatifloxacin can be administered to humans at levels that would provide exposure comparable to that given the mice, however, it might be possible to dose newer quinolones or other new chemical entities at higher levels during therapy to achieve the exposure that was modelled with the high dose regimen in this study. Rifampicin has been used in human prosthetic-joint infection studies at doses of 450 mg orally every 12 h for up to 6 months.¹¹

Evaluation of clinical tuberculosis isolates (wild-type and drug-resistant) in vitro and in vivo should be done to confirm our observations. Clinical studies could subsequently be undertaken to define the appropriate role of these drugs in tuberculosis treatment regimens.

Acknowledgements

Michelle DeStefano provided helpful editorial comments. The study was supported in part by the Department of Veterans Affairs.

Transparency declarations

None to declare.

References