BALB/c and Swiss mice are routinely used to validate the effectiveness of tuberculosis drug regimens, although these mouse strains fail to develop human-like pulmonary granulomas exhibiting caseous necrosis. Microenvironmental conditions within human granulomas may negatively impact drug efficacy, and this may not be reflected in non-necrotizing lesions found within conventional mouse models. The C3HeB/FeJ mouse model has been increasingly utilized as it develops hypoxic, caseous necrotic granulomas which may more closely mimic the pathophysiological conditions found within human pulmonary granulomas. Here, we examined the treatment response of BALB/c and C3HeB/FeJ mice to bedaquiline (BDQ) and pyrazinamide (PZA) administered singly and in combination. BALB/c mice consistently displayed a highly uniform treatment response to both drugs, while C3HeB/FeJ mice displayed a bimodal response composed of responsive and less-responsive mice. Plasma pharmacokinetic analysis of dissected lesions from BALB/c and C3HeB/FeJ mice revealed that PZA penetrated lesion types from both mouse strains with similar efficiency. However, the pH of the necrotic caseum of C3HeB/FeJ granulomas was determined to be 7.5, which is in the range where PZA is essentially ineffective under standard laboratory in vitro growth conditions. BDQ preferentially accumulated within the highly cellular regions in the lungs of both mouse strains, although it was present at reduced but still biologically relevant concentrations within the central caseum when dosed at 25 mg/kg. The differential treatment response which resulted from the heterogeneous pulmonary pathology in the C3HeB/FeJ mouse model revealed several factors which may impact treatment efficacy, and could be further evaluated in clinical trials.

New drugs and drugs with a novel mechanism of action are desperately needed to shorten the duration of tuberculosis treatment, to prevent the emergence of drug resistance, and to treat multiple-drug-resistant strains of Mycobacterium tuberculosis. Recently, there has been renewed interest in clofazimine (CFZ). In this study, we utilized the C3HeB/FeJ mouse model, possessing highly organized, hypoxic pulmonary granulomas with caseous necrosis, to evaluate CFZ monotherapy in comparison to results with BALB/c mice, which form only multifocal, coalescing cellular aggregates devoid of caseous necrosis. While CFZ treatment was highly effective in BALB/c mice, its activity was attenuated in the lungs of C3HeB/FeJ mice. This lack of efficacy was directly related to the pathological progression of disease in these mice, since administration of CFZ prior to the formation of hypoxic, necrotic granulomas reconstituted bactericidal activity in this mouse strain. These results support the continued use of mouse models of tuberculosis infection which exhibit a granulomatous response in the lungs that more closely resembles the pathology found in human disease.

Methodologies for preclinical animal model testing of drugs against Mycobacterium tuberculosis vary from laboratory to laboratory; however, it is unknown if these variations result in different outcomes. Thus, a series of head-to-head comparisons of drug regimens in three commonly used mouse models (intravenous, a low-dose aerosol, and a high-dose aerosol infection model) and in two strains of mice are reported here. Treatment with standard tuberculosis (TB) drugs resulted in similar efficacies in two mouse species after a low-dose aerosol infection. When comparing the three different infection models, the efficacies in mice of rifampin and pyrazinamide were similar when administered with either isoniazid or moxifloxacin. Relapse studies revealed that the standard drug regimen showed a significantly higher relapse rate than the moxifloxacin-containing regimen. In fact, 4 months of the moxifloxacin-containing combination regimen showed similar relapse rates as 6 months of the standard regimen. The intravenous model showed slower bactericidal killing kinetics with the combination regimens tested and a higher relapse of infection than either aerosol infection models. All three models showed similar outcomes for in vivo efficacy and relapse of infection for the drug combinations tested, regardless of the mouse infection model used. Efficacy data for the drug combinations used also showed similar results, regardless of the formulation used for rifampin or timing of the drugs administered in combination. In all three infection models, the dual combination of rifampin and pyrazinamide was less sterilizing than the standard three-drug regimen, and therefore the results do not support the previously reported antagonism between standard TB agents.For the first time in many years, there is a portfolio of promising new compounds at every level of tuberculosis (TB) drug discovery and development (4; www.tballiance.org). However, careful selection of new drug candidates is imperative, and efficient screening models for new drugs, including pertinent animal models, need to be further developed and studied. Preclinical testing in animals of newly discovered agents alone, and in combination with new and old agents, prior to being tested in humans is a crucial but lengthy process. These drug regimens include agents that provide bactericidal activities against rapidly growing bacilli, but they especially aim to include those that possess potent sterilizing activities and hence prevent relapse (8). The most commonly used animal species in TB drug development is the mouse, mainly because of economical and practical reasons, but also because of the limited requirement of compound (26).Various mouse Mycobacterium tuberculosis infection models are utilized by both industry and academia, and they differ in the route of infection with M. tuberculosis, inoculum and strain of M. tuberculosis, strain of mice, timing of the start of treatment after infection, the length of treatment, etc. A model where therapy is started immediately after in...

Intrapulmonary aerosol infection of GM-CSF KO mice with M. abscessus is a useful animal model for studying pathogenesis as well as pre-clinical testing of new compounds against M. abscessus in acute or chronic phases of infection.

Metronidazole, which is used for the treatment of infections caused by anaerobic organisms, was evaluated in Mycobacterium tuberculosis-infected guinea pigs. M. tuberculosis can adapt to hypoxia, which is present in the primary lesions of infected guinea pigs. Metronidazole treatment (for 6 weeks at 100 mg/kg of body weight) resulted in no reduction in the bacillary burden and significantly worsened lesion inflammation.The excellent distribution of metronidazole (MET) in all organs and its good bactericidal activity, including its activity against quiescent bacteria, make it the compound of choice for the treatment of many infections caused by anaerobic organisms. Mycobacterium tuberculosis also has the ability to adapt to gradual oxygen depletion or stationary growth. This phenomenon has been studied extensively by Wayne and Hayes and has been described as a sequential progression through two stages, defined as nonreplicating phase 1 (NRP-1) and NRP-2 (15). MET has no effect on exponentially growing bacilli or on NRP-1 bacilli, but it is bactericidal for bacilli in stationary phase and NRP-2 (6, 16).Nonprogressive M. tuberculosis lesions in the lungs often have limited vascularization (due to necrosis and dystrophic mineralization), causing a limited O 2 supply. Bacterial persistence in these hypoxic lesions has been thought to be accompanied by susceptibility to MET. In mouse models of tuberculosis (TB), MET has failed to show consistent activity (5, 6, 10). This is not surprising, as hypoxia was found to be completely absent in the M. tuberculosis lesions of infected mice (1, 11), and the progression of disease rarely reaches the stages of extensive necrosis (2,3,8,12). Lung lesions in guinea pigs infected with M. tuberculosis, on the other hand, show caseous necrosis, mineralization, and hypoxia, which are also seen in natural infections in humans (13,14). Guinea pigs develop necrotic primary lesions that differ in their morphology compared to those of the secondary lesions that result after the activation of adaptive immunity (8). In an earlier paper, we described that the persisting, acid-fast bacilli are primarily found extracellularly in a hypoxic microenvironment of primary lesion necrosis and that only few a bacilli are localized within the secondary granulomas (8). The same study also showed evidence of hypoxia in these primary lesions of guinea pigs when pimonidazole was used (8), and therefore, we chose to explore the potential bactericidal activity of MET in the guinea pig model.To establish appropriate drug doses in guinea pigs for the anti-M. tuberculosis drugs used in this study, pharmacokinetic analysis was performed with a single dose each of isoniazid (INH), rifampin (RIF), pyrazinamide (PZA), and MET. All drugs were obtained from Sigma Chemical Co. (St. Louis, MO). INH, PZA, and MET were dissolved in distilled water, while RIF was dissolved in dimethyl sulfoxide (final concentration, 0.5%) with sucrose (40%, wt/vol) to increase its palatability. The pharmacokinetic data obtained by validated...