Nutrient-starved incubation conditions enhance pyrazinamide activity against Mycobacterium tuberculosis

Pyrazinamide (PZA) is an unconventional frontline tuberculosis drug characterized by high in vivo sterilizing activity, but poor in vitro activity. The study on the mechanism of action of PZA has attracted significant attention because of the peculiarity of PZA and its ability to shorten the tuberculosis chemotherapy. In this study, we examined the effect of nutrient-starved conditions on PZA activity in vitroat acid pH. We also examined the effect of fatty acids, benzoic acid and salicylic acid on PZA activity. The results showed that nutrient-starved conditions lowered the membrane potential of Mycobacterium tuberculosis and enhanced the activity of PZA, with 5- and 10-day starvation conditions resulting in greater enhancement than 3-day starvation. Fatty acids, benzoic acid and salicylic acid enhanced PZA activity in both normal and starved bacilli, especially in starved bacilli. These findings provide further support for the recent model of PZA action and may have implications for developing new drugs that shorten therapy.     

Synergistic activity of R207910 combined with pyrazinamide against murine tuberculosis

In previous studies, the diarylquinoline R207910 (also known as TMC207) was demonstrated to have high bactericidal activity when combined with first- or second-line antituberculous drugs. Here we extend the evaluation of R207910 in the curative model of murine tuberculosis by assessing the activities of one-, two-, and three-drug combinations containing R207910 and isoniazid (INH), rifampin (RIF), pyrazinamide (PZA), or moxifloxacin (MXF) in the setting of a high initial bacillary load (7.2 log(10) CFU). Two months of treatment with the combinations R207910-PZA, R207910-PZA-INH, R207910-PZA-RIF, or R207910-PZA-MXF resulted in culture-negative lung homogenates in 70 to 100% of the mice, while mice treated with INH-RIF-PZA (the reference regimen) or RIF-MXF-PZA remained culture positive. Combinations including R207910 but not PZA (e.g., R207910-INH-RIF and R207910-MXF-RIF) were less active than R207910-PZA-containing regimens administered either alone or with the addition of INH, RIF, or MXF. These results reveal a synergistic interaction between R207910 and PZA. Three-drug combinations containing these two drugs and INH, RIF, or MXF have the potential to significantly shorten the treatment duration in patients, provided that these results can be confirmed in long-term experiments including periods of relapse.     

Reduced pyrazinamidase activity and the natural resistance of Mycobacterium kansasii to the antituberculosis drug pyrazinamide

Pyrazinamide (PZA), an analog of nicotinamide, is a prodrug that requires conversion to the bactericidal compound pyrazinoic acid (POA) by the bacterial pyrazinamidase (PZase) activity of nicotinamidase to show activity against Mycobacterium tuberculosis. Mutations leading to a loss of PZase activity cause PZA resistance in M. tuberculosis. M. kansasii is naturally resistant to PZA and has reduced PZase activity along with an apparently detectable nicotinamidase activity. The role of the reduction in PZase activity in the natural PZA resistance of M. kansasii is unknown. The MICs of PZA and POA for M. kansasii were determined to be 500 and 125 micrograms/ml, respectively. Using [14C]PZA and [14C]nicotinamide, we found that M. kansasii had about 5-fold-less PZase activity and about 25-fold-less nicotinamidase activity than M. tuberculosis. The M. kansasii pncA gene was cloned on a 1.8-kb BamHI DNA fragment, using M. avium pncA probe. Sequence analysis showed that the M. kansasii pncA gene encoded a protein with homology to its counterparts from M. tuberculosis (69.9%), M. avium (65.6%), and Escherichia coli (28.5%). Transformation of naturally PZA-resistant M. bovis BCG with M. kansasii pncA conferred partial PZA susceptibility. Transformation of M. kansasii with M. avium pncA caused functional expression of PZase and high-level susceptibility to PZA, indicating that the natural PZA resistance in M. kansasii results from a reduced PZase activity. Like M. tuberculosis, M. kansasii accumulated POA in the cells at an acidic pH; however, due to its highly active POA efflux pump, the naturally PZA-resistant species M. smegmatis did not. These findings suggest the existence of a weak POA efflux mechanism in M. kansasii.     

Dose-dependent activity of pyrazinamide in animal models of intracellular and extracellular tuberculosis infections

Recent in vitro pharmacokinetic data suggest that the currently recommended dose of pyrazinamide may be suboptimal for killing intracellular bacilli in humans. We evaluated a range of pyrazinamide doses against intracellular and extracellular Mycobacterium tuberculosis in chronically infected mice and guinea pigs, respectively. Antibiotics were given five times weekly for 4 weeks beginning 28 days after infection. Human-equivalent doses of isoniazid reduced lung bacterial counts 10-fold in each species. Pyrazinamide given at 1/4 and 1/2 the human-equivalent dose was minimally active, while human-equivalent doses reduced lung bacterial counts by ～1.0 log(10) in each species. Doubling the human-equivalent dose of pyrazinamide reduced the lung bacillary burden by 1.7 and 3.0 log(10) in mice and guinea pigs, respectively. As in humans and mice, pyrazinamide showed significant synergy with rifampin in guinea pigs. Clinical studies are warranted to investigate the sterilizing activity and tolerability of higher doses of pyrazinamide in combination tuberculosis regimens.     

Susceptibility of Mycobacterium tuberculosis to pyrazinamide and its relationship to pyrazinamidase activity.

Pyrazinamidase activity has been associated with pyrazinamide-susceptible Mycobacterium tuberculosis strains. The detection of pyrazinamidase activity by the Wayne method was found to be of limited value when compared with the results of standard pyrazinamide susceptibility tests, especially when a high level of pyrazinamide resistance was found. When resistance to pyrazinamide reached a level of 150 to 200 micrograms/ml, there was too much variability in Wayne test results to accurately define pyrazinamide susceptibility.     

Correlation between pyrazinamide activity and pncA mutations in Mycobacterium tuberculosis isolates in Taiwan

A total of 76 clinical Mycobacterium tuberculosis isolates from Taiwan were tested for pyrazinamidase activity, pyrazinamide susceptibility, and pncA mutations. Frequency of resistance to PZA rose with increases in resistance to first-line drugs. Of 17 pyrazinamide-resistant strains, 7 (3 of which had not been previously described) possessed mutations in the pncA gene.     

Does pyrazinoic acid as an active moiety of pyrazinamide have specific activity against Mycobacterium tuberculosis?

The commonly accepted hypothesis explaining the mechanism of action of pyrazinamide (PZA) is based on the assumption that PZA-susceptible Mycobacterium tuberculosis strains produce pyrazinamidase, which hydrolyzes PZA to the antibacterial moiety pyrazinoic acid (POA). It is not clear whether POA has specific antimicrobial activity or the inhibition of growth caused by POA is due to its ability to lower the pH of the environment below the limits of tolerance of M. tuberculosis growth. We confirmed in this study that POA, depending on the concentration, lowered the pH of 7H12 broth (pH 6.0), which ranged from 5.8 at 120.0 micrograms/ml to 4.6 at 960.0 micrograms/ml. Therefore, we tested the inhibitory effects of different concentrations of POA in broth in which the final pH was adjusted to 5.6 by adding appropriate amounts of phosphoric acid or dipotassium phosphate. Under these conditions, we found a clear dose-response correlation, proving that POA does have specific antimicrobial activity. The MIC of POA at pH 5.6 was 240 to 480 micrograms/ml, 8- to 16-fold higher than the MIC of PZA under the same conditions and much higher than the concentrations achievable in humans. This suggests that the action of POA in an acid environment is a combined effect of its specific activity and its ability to lower the pH below the limits of tolerance of the target organism.     

Lymphokine enhances oxygen-independent activity against intracellular pathogens

To determine if mechanisms other than the generation of toxic oxygen intermediates are active against intracellular pathogens, oxidatively deficient mouse L cells and monocyte-derived macrophages from patients with chronic granulomatous disease were stimulated with soluble lymphocyte products. Despite no enhancement in oxidative activity, these cells displayed effective microbistatic activity against both T. gondii and C. psittaci. These results suggest a potential role for nonoxidative mechanisms in the mononuclear phagocyte's activity against intracellular pathogens, and indicate that lymphokines can regulate both oxygen-dependent and oxygen-independent antimicrobial responses.     

Developmental status of new antituberculous drugs

Worldwide, there are nearly 9 million new cases of active tuberculosis (TB) and 1.8 million associated deaths every year. Especially, difficulty of the chemotherapy in TB/HIV co-infection and also prevalence of multi drug-resistant tuberculosis (MDR-TB) as well as extensively drug-resistant tuberculosis (XDR-TB) make serious clinical problems. Development of the new drugs is essential to overcome of such clinical difficulty as described above. The developmental status of the new drugs has at present advanced greatly by the leading contribution of the international organization such as Global Alliance for TB Drug Development and Stop TB Partnership in this decade. This article reviews the developing new compounds at presents as the key drugs for treatment of TB now.     

Tumor-targeting nanodelivery enhances the anticancer activity of a novel quinazolinone analogue

GMC-5-193 (GMC) is a novel anticancer small-molecule quinazolinone analogue with properties that include antimicrotubule activity and inherent fluorescence. The aim of this study was to produce and optimize a systemically administered liposomal formulation for tumor-targeting delivery of GMC to enhance the anticancer effect of this compound and evaluate its bioefficacy. GMC was encapsulated within a cationic liposome, which was decorated on the surface with an anti-transferrin receptor single-chain antibody fragment (TfRscFv) as the tumor-targeting moiety to form a nanoscale complex (scL/GMC). Confocal imaging of fluorescent GMC uptake in a human melanoma cell line, MDA-MB-435, showed higher cellular uptake of GMC when delivered via the liposome complex compared with free GMC. Delivery of GMC by the tumor-targeting liposome nanoimmunocomplex also resulted in a 3- to 4-fold decrease in IC(50) values in human cancer cells [DU145 (prostate) and MDA-MB-435] compared with the effects of GMC administered as free GMC. In addition, the GMC nanoimmunocomplex increased the sensitivity of cancer cells to doxorubicin, docetaxel, or mitoxantrone by approximately 3- to 30-fold. In the MDA435/LCC6 athymic nude mice xenograft lung metastases model, GMC was specifically delivered to tumors by the nanoimmunocomplex. These data show that incorporation of small-molecule therapeutic GMC within the tumor-targeting liposome nanocomplex enhances its anticancer effect.     

Use of pyrazinamidase activity on Mycobacterium tuberculosis as a rapid method for determination of pyrazinamide susceptibility.

Pyrazinamidase activity in clinical isolates of Mycobacterium tuberculosis has been previously found to correlate with susceptibility to the antituberculosis drug pyrazinamide. The Wayne method for determining pyrazinamidase activity, a technique also utilized as an aid in identification of mycobacteria, and thin-layer chromatography method were found to be useful screening methods for susceptibility testing, since resistant strains are pyrazinamidase negative. These simple methods overcome the difficulty in growing M. tuberculosis at pH 5.5, as is required in the conventional method of susceptibility testing.     

Iron blocks the accumulation and activity of tetracyclines in bacteria

The apparent sensitivities of several bacterial pathogens to tetracyclines varied by up to 128-fold with the medium content of Fe, but not of other metals. The effect of Fe was independent of superoxide dismutase activity and of intracellular Fe, but accumulation of tetracyclines was blocked in high-Fe medium. Thus, synergistic suppression of bacterial growth in the presence of a low Fe concentration and tetracyclines arises because of elevated antibiotic accumulation.     

Molecular detection of resistance to antituberculous therapy

Drug-resistant tuberculosis is becoming increasingly common and represents a worldwide threat. Therefore, new approaches for the rapid susceptibility testing of Mycobacterium tuberculosis are needed to replace traditional culture-based methods. This article presents the genetic background of drug resistance in tubercle bacillus, and the methods currently available for genotypic susceptibility testing.     

A rapid method for the evaluation of new antituberculous agents

Fifty-one new synthetic compounds belonging to four different series, namely (1a) substituted aryl-4-(substituted phenyl) succinimide; (1b) N-(substituted methyl)-4-(heterocyclic) succinimide; (2) heterocyclic 4-(5'-nitro-2-furyl) thiazoles; (3) substituted aryl-4-(3', 4'-dihydroxy phenyl) thiazoles, and (4) phenyl-N,N-1,2,3-bis-methoxy carbonyl guanidines were screened for antituberculous activity using a conventional broth dilution test (BDT) and a liquid scintillation radiometric method (LSRM). Eight compounds showed in vitro activity. LSRM showed 100% agreement with BDT. LSRM is completed within 60 h, while BDT requires 8-9 days. Unlike BDT, LSRM allowed one to measure the graded changes in the metabolism and the growth rate of Mycobacterium tuberculosis in response to various concentrations of the drug. It permits the measurement of the relative prolongation of the replication time by the drug or the test compound. With LSRM it was possible to detect the phase of growth during which the test compound shows or begins its antituberculous activity. It improves our understanding of the antituberculous activity of the test compound and hence is more advantageous. It is rapid, reliable, quantitative and more sensitive than BDT. LSRM is thus suitable for the evaluation of new drugs.     

Cholesterol enhances phospholipid-dependent activated protein C anticoagulant activity

The influence of cholesterol on activated protein C (APC) anticoagulant activity in plasma and on factor Va inactivation was investigated. Anticoagulant and procoagulant activities of phosphatidylcholine/phosphatidylserine (PC/PS) vesicles containing cholesterol were assessed in the presence and absence of APC using factor Xa-1-stage clotting and factor Va inactivation assays. Cholesterol at approximate physiological membrane levels (30%) in PC/PS (60%/10% w/w) vesicles prolonged the factor Xa-1-stage clotting time dose-dependently in the presence of APC but not in the absence of APC. APC-mediated cleavage of purified recombinant factor Va variants that were modified at specific APC cleavage sites (Q306/Q679-factor Va; Q506/Q679-factor Va) was studied to define the effects of cholesterol on APC cleavage at R506 and R306. When compared to control PC/PS vesicles, cholesterol in PC/PS vesicles enhanced factor Va inactivation and the rate of APC cleavage at both R506 and R306. Cholesterol also enhanced APC cleavage rates at R306 in the presence of the APC cofactor, protein S. In summary, APC anticoagulant activity in plasma and factor Va inactivation as a result of cleavages at R506 and R306 by APC is markedly enhanced by cholesterol in phospholipid vesicles. These results suggest that cholesterol in a membrane surface may selectively enhance APC activities.