Dose-Dependent Activity of Pyrazinamide in Animal Models of Intracellular and Extracellular Tuberculosis Infections

Although the antituberculous activity of pyrazinamide (Z) was recognized in the early 1950s, its mechanism of action remains poorly understood (25). After passive diffusion and active transport (35), Z accumulates intracellularly (52) and is converted to pyrazinoic acid by Mycobacterium tuberculosis pyrazinamidase (24, 39). Originally thought to act on mycobacterial fatty acid synthase I (54), pyrazinoic acid likely acts by disrupting the proton motive force required for essential cellular functions at acidic pH (53). Although Z is poorly active against multiplying bacilli (22), the synergy of Z with rifampin (R) (27) has permitted the shortening of tuberculosis (TB) chemotherapy from 9 months to 6 months (41). Because of the strict requirement for acidic pH, Z is believed to target dormant bacilli residing within an acidic environment (30). Although Z-susceptible bacilli have been postulated to persist in the macrophage phagolysosome (37), recent studies suggest that the interior pH of these organelles may be only slightly acidic (11, 43), and Z lacks even bacteriostatic activity against intracellular M. tuberculosis in human monocyte-derived macrophages (20). However, activation of macrophages by gamma interferon (IFN-γ) leads to phagosome acidification to a pH range where Z is highly active (45). An alternative hypothesis is that Z kills bacilli residing in acidified lung compartments present during the early inflammatory stages of infection (30), which is consistent with clinical observations that Z activity is primarily limited to the first 2 months of therapy (5-7). In addition, hypoxia enhances the antituberculous activity of Z (48). A recent study using an in vitro pharmacokinetic (PK) system found that that the area under the serum concentration-time curve (AUC)/MIC is the PK parameter most closely linked with M. tuberculosis killing by Z (18). Using Monte Carlo simulations of 10,000 patients, these investigators reasoned that the persistent bacilli targeted by Z are likely to be extracellular based on the unlikelihood of achieving bactericidal drug levels within pulmonary macrophages of most patients receiving standard Z doses, while effective exposures were achieved in the epithelial lining fluid of 80 to 90% of simulated patients treated with equal Z doses. Based on in vitro PK data and mathematical modeling, they suggested that increasing the currently recommended Z dose may improve treatment outcomes in patients with TB (18). In order to determine whether Z is active against intracellular and/or extracellular bacilli in the mammalian host, we evaluated the bactericidal activity of a range of Z doses against chronic TB infection in mice and guinea pigs. Unlike mice, guinea pigs infected with M. tuberculosis form necrotic granulomas histologically resembling their human counterparts (29). Such lesions in humans (46) and guinea pigs (28) likely harbor persistent bacilli, which may encounter microenvironmental stress conditions including hypoxia (19, 28, 47) and acidic pH (16). On the other hand, TB infection in mice appears to be confined to the intraphagosomal compartment within cellular lesions lacking necrosis and associated microenvironmental conditions (14).