Jansy Sarathy

The association between sterilizing activity and drug distribution into tuberculosis lesions.

Finding new treatment-shortening antibiotics to improve cure rates and curb the alarming emergence of drug resistance is the major objective of tuberculosis (TB) drug development. Using a matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging suite in a biosafety containment facility, we show that the key sterilizing drugs rifampicin and pyrazinamide efficiently penetrate the sites of TB infection in lung lesions. Rifampicin even accumulates in necrotic caseum, a critical lesion site where persisting tubercle bacilli reside. In contrast, moxifloxacin, which is active in vitro against a subpopulation of Mycobacterium tuberculosis that persists in specific niches under drug pressure and has achieved treatment shortening in mice, does not diffuse well in caseum, concordant with its failure to shorten therapy in recent clinical trials. We suggest that such differential spatial distribution and kinetics of accumulation in lesions may create temporal and spatial windows of monotherapy in specific niches, allowing the gradual development of multidrug-resistant TB. We propose an alternative working model to prioritize new antibiotic regimens based on quantitative and spatial distribution of TB drugs in the major lesion types found in human lungs. The finding that lesion penetration may contribute to treatment outcome has wide implications for TB.

Prediction of Drug Penetration in Tuberculosis Lesions.

The penetration of antibiotics in necrotic tuberculosis lesions is heterogeneous and drug-specific, but the factors underlying such differential partitioning are unknown. We hypothesized that drug binding to macromolecules in necrotic foci (or caseum) prevents passive drug diffusion through avascular caseum, a critical site of infection. Using a caseum binding assay and MALDI mass spectrometry imaging of tuberculosis drugs, we showed that binding to caseum inversely correlates with passive diffusion into the necrotic core. We developed a high-throughput assay relying on rapid equilibrium dialysis and a caseum surrogate designed to mimic the composition of native caseum. A set of 279 compounds was profiled in this assay to generate a large data set and explore the physicochemical drivers of free diffusion into caseum. Principle component analysis and modeling of the data set delivered an in silico signature predictive of caseum binding, combining 69 molecular descriptors. Among the major positive drivers of binding were high lipophilicity and poor solubility. Determinants of molecular shape such as the number of rings, particularly aromatic rings, number of sp(2) carbon counts, and volume-to-surface ratio negatively correlated with the free fraction, indicating that low-molecular-weight nonflat compounds are more likely to exhibit low caseum binding properties and diffuse effectively through caseum. To provide simple guidance in the property-based design of new compounds, a rule of thumb was derived whereby the sum of the hydrophobicity (clogP) and aromatic ring count is proportional to caseum binding. These tools can be used to ensure desirable lesion partitioning and guide the selection of optimal regimens against tuberculosis.

Ethambutol partitioning in tuberculous pulmonary lesions explains its clinical efficacy

ABSTRACT Clinical trials and practice have shown that ethambutol is an important component of the first-line tuberculosis (TB) regime. This contrasts the drugs rather modest potency and lack of activity against nongrowing persister mycobacteria. The standard plasma-based pharmacokinetic-pharmacodynamic profile of ethambutol suggests that the drug may be of limited clinical value. Here, we hypothesized that this apparent contradiction may be explained by favorable penetration of the drug into TB lesions. First, we utilized novel in vitro lesion pharmacokinetic assays and predicted good penetration of the drug into lesions. We then employed mass spectrometry imaging and laser capture microdissection coupled to liquid chromatography and tandem mass spectrometry (LCM and LC/MS-MS, respectively) to show that ethambutol, indeed, accumulates in diseased tissues and penetrates the major human-like lesion types represented in the rabbit model of TB disease with a lesion-to-plasma exposure ratio ranging from 9 to 12. In addition, ethambutol exhibits slow but sustained passive diffusion into caseum to reach concentrations markedly higher than those measured in plasma at steady state. The results explain why ethambutol has retained its place in the first-line regimen, validate our in vitro lesion penetration assays, and demonstrate the critical importance of effective lesion penetration for anti-TB drugs. Our findings suggest that in vitro and in vivo lesion penetration evaluation should be included in TB drug discovery programs. Finally, this is the first time that LCM with LC-MS/MS has been used to quantify a small molecule at high spatial resolution in infected tissues, a method that can easily be extended to other infectious diseases.

High Systemic Exposure of Pyrazinoic Acid Has Limited Antituberculosis Activity in Murine and Rabbit Models of Tuberculosis

ABSTRACT Pyrazinamide (PZA) is a prodrug requiring conversion to pyrazinoic acid (POA) by an amidase encoded by pncA for in vitro activity. Mutation of pncA is the most common cause of PZA resistance in clinical isolates. To determine whether the systemic delivery of POA or host-mediated conversion of PZA to POA could circumvent such resistance, we evaluated the efficacy of orally administered and host-derived POA in vivo. Dose-ranging plasma and intrapulmonary POA pharmacokinetics and the efficacy of oral POA or PZA treatment against PZA-susceptible tuberculosis were determined in BALB/c and C3HeB/FeJ mice. The activity of host-derived POA was assessed in rabbits infected with a pncA-null mutant and treated with PZA. Median plasma POA values for the area under the concentration-time curve from 0 h to infinity (AUC0–∞) were 139 to 222 μg·h/ml and 178 to 287 μg·h/ml after doses of PZA and POA of 150 mg/kg of body weight, respectively, in mice. Epithelial lining fluid POA concentrations in infected mice were comparable after POA and PZA administration. In chronically infected BALB/c mice, PZA at 150 mg/kg reduced lung CFU counts by >2 log10 after 4 weeks. POA was effective only at 450 mg/kg, which reduced lung CFU counts by ∼0.7 log10. POA had no demonstrable bactericidal activity in C3HeB/FeJ mice, nor did PZA administered to rabbits infected with a PZA-resistant mutant. Oral POA administration and host-mediated conversion of PZA to POA producing plasma POA exposures comparable to PZA administration was significantly less effective than PZA. These results suggest that the intrabacillary delivery of POA and that producing higher POA concentrations at the site of infection will be more effective strategies for maximizing POA efficacy.

Extreme drug tolerance ofMycobacterium tuberculosisin caseum

ABSTRACT Tuberculosis (TB) recently became the leading infectious cause of death in adults, while attempts to shorten therapy have largely failed. Dormancy, persistence, and drug tolerance are among the factors driving the long therapy duration. Assays to measure in situ drug susceptibility of Mycobacterium tuberculosis bacteria in pulmonary lesions are needed if we are to discover new fast-acting regimens and address the global TB threat. Here we take a first step toward this goal and describe an ex vivo assay developed to measure the cidal activity of anti-TB drugs against M. tuberculosis bacilli present in cavity caseum obtained from rabbits with active TB. We show that caseum M. tuberculosis bacilli are largely nonreplicating, maintain viability over the course of the assay, and exhibit extreme tolerance to many first- and second-line TB drugs. Among the drugs tested, only the rifamycins fully sterilized caseum. A similar trend of phenotypic drug resistance was observed in the hypoxia- and starvation-induced nonreplicating models, but with notable qualitative and quantitative differences: (i) caseum M. tuberculosis exhibits higher drug tolerance than nonreplicating M. tuberculosis in the Wayne and Loebel models, and (ii) pyrazinamide is cidal in caseum but has no detectable activity in these classic nonreplicating assays. Thus, ex vivo caseum constitutes a unique tool to evaluate drug potency against slowly replicating or nonreplicating bacilli in their native caseous environment. Intracaseum cidal concentrations can now be related to the concentrations achieved in the necrotic foci of granulomas and cavities to establish correlations between clinical outcome and lesion-centered pharmacokinetics-pharmacodynamics (PK-PD) parameters.

An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions

The eradication of tuberculosis disease requires drug regimens that can penetrate the multiple layers of complex pulmonary lesions. Drug distribution in the caseous cores of cavities and lesions is especially crucial because they harbor subpopulations of drug-tolerant bacteria also commonly referred to as persisters. Existing methods for the measurement of drug penetration in tuberculosis lesions involve costly and time-consuming in vivo pharmacokinetic studies coupled to bioanalytical or imaging techniques. The in vitro measurement of drug binding to caseum macromolecules was proposed as an alternative to such techniques since this binding hinders the passive diffusion of drug molecules through caseum. Rapid equilibrium dialysis is a fast and reliable system for performing plasma protein and tissue binding studies. In this protocol, we used a rapid equilibrium dialysis (RED) device to measure drug binding to homogenates of caseum that is excised from the lesions and cavities of tuberculosis-infected rabbits. The protocol also describes how to generate a surrogate matrix from lipid loaded THP-1 macrophages to use in place of caseum. This caseum/surrogate binding assay is an important tool in tuberculosis drug discovery and can be adapted to help study drug distribution in lesions or abscesses caused by other diseases.

In Vivo-Selected Pyrazinoic Acid-Resistant Mycobacterium tuberculosis Strains Harbor Missense Mutations in the Aspartate Decarboxylase PanD and the Unfoldase ClpC1

Through mutant selection on agar containing pyrazinoic acid (POA), the bioactive form of the prodrug pyrazinamide (PZA), we recently showed that missense mutations in the aspartate decarboxylase PanD and the unfoldase ClpC1, and loss-of-function mutation of polyketide synthases Mas and PpsA-E involved in phthiocerol dimycocerosate synthesis, cause resistance to POA and PZA in Mycobacterium tuberculosis. Here we first asked whether these in vitro-selected POA/PZA-resistant mutants are attenuated in vivo, to potentially explain the lack of evidence of these mutations among PZA-resistant clinical isolates. Infection of mice with panD, clpC1, and mas/ppsA-E mutants showed that whereas growth of clpC1 and mas/ppsA-E mutants was attenuated, the panD mutant grew as well as the wild-type. To determine whether these resistance mechanisms can emerge within the host, mice infected with wild-type M. tuberculosis were treated with POA, and POA-resistant colonies were confirmed for PZA and POA resistance. Genome sequencing revealed that 82 and 18% of the strains contained missense mutations in panD and clpC1, respectively. Consistent with their lower fitness and POA resistance level, independent mas/ppsA-E mutants were not found. In conclusion, we show that the POA/PZA resistance mechanisms due to panD and clpC1 missense mutations are recapitulated in vivo. Whereas the representative clpC1 mutant was attenuated for growth in the mouse infection model, providing a possible explanation for their absence among clinical isolates, the growth kinetics of the representative panD mutant was unaffected. Why POA/PZA resistance-conferring panD mutations are observed in POA-treated mice but not yet among clinical strains isolated from PZA-treated patients remains to be determined.

Impact of immunopathology on the antituberculous activity of pyrazinamide

In the 1970s, inclusion of pyrazinamide (PZA) in the drug regimen of tuberculosis (TB) patients for the first 2 mo achieved a drastic reduction of therapy duration. Until now, however, the mechanisms underlying PZA’s unique contribution to efficacy have remained controversial, and animal efficacy data vary across species. To understand how PZA kills bacterial populations present in critical lung lesion compartments, we first characterized a rabbit model of active TB, showing striking similarities in lesion types and fates to nonhuman primate models deemed the most appropriate surrogates of human TB. We next employed this model with lesion-centric molecular and bacteriology readouts to demonstrate that PZA exhibits potent activity against Mycobacterium tuberculosis residing in difficult-to-sterilize necrotic lesions. Our data also indicate that PZA is slow acting, suggesting that PZA administration beyond the first 2 mo may accelerate the cure. In conclusion, we provide a pharmacodynamic explanation for PZA’s treatment-shortening effect and deliver new tools to dissect the contribution of immune response versus drug at the lesion level.