The association between sterilizing activity and drug distribution into tuberculosis lesions

Abstract

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.

Figure 1

Quantitative drug distribution in human pulmonary lesions. Closed nodules, cavity wall and cavity caseum samples from each subject were homogenized prior to measuring the concentrations of INH (a), RIF (b), PZA (c), and MXF (d). Each subject contributed an average of 11 to 12 lesions. The major metabolites of INH (acetyl-INH), RIF (desacetyl-RIF) and PZA (POA) are shown as black empty symbols in a,b,c. Note that INH is a prodrug activated by M. tuberculosis’ catalase, the product of which forms transient NAD adducts that may display a distinct distribution pattern but cannot be captured by LCMS or MALDI imaging. The dotted line indicates the lower limit of quantitation. The clear and grey shaded boxes (a,b,d) represent clinical MIC and MAC ranges, respectively. The orange shaded box in c is the range of PZA activity in acidic conditions.

Figure 2

Two-dimensional imaging of MXF and PZA by MALDI mass spectrometry. (a) Schematic of MALDI mass spectrometry imaging of small molecules in TB infected lung tissue, from tissue sectioning to data acquisition and overlay with histology stain of an adjacent tissue section. The relative ion abundance of specific analytes in regions of interest delineated based on histology staining can be measured to provide semi-quantitative data. (b)Ion maps of PZA and MXF in representative lung lesions sampled throughout the dosing interval; the signal intensity is fixed for each drug and indicated by the rainbow color scale bar to the right. Hematoxylin and Eosin (H&E) staining of adjacent sections is shown below the ion maps. Black/white contour lines highlight the necrotic center of each lesion. Asterisks indicate lesions from patients at steady state for MXF^(*) or PZA^(**). Black scale bar: 5mm. (c) Diffusion of MXF in caseum as a function of caseum cellularity. Two-tailed unpaired t-tests were used to analyze the correlation between moxifloxacin caseum/cellular concentration ratios and caseum cellularity scores. Means +/− s.d. are shown (n=3). ** p <0.05. A typical H&E example representative of each cellularity score is shown on the right.

Figure 3

Penetration of RIF in TB lesions. (a) Ion maps of RIF in lung lesions collected following a single drug dose; (b) RIF accumulates in necrotic foci at steady-state. H&E staining of adjacent sections are shown below each set of ion maps. Black/white contour lines highlight the necrotic center of each lesion. Black scale bar: 5mm. (c) Average RIF concentration in plasma, uninvolved lung tissue, and lesions in one subject at steady state. The samples were collected 26h after the last of 180 RIF doses was administered. Means +/− s.d. of 3 to 5 lung pieces or distinct lesions are shown.

Figure 4

Factors affecting drug diffusion into caseum. Relationship between caseum binding (orange bars), accumulation into macrophages (red bars), lipophilicity (cLogP) and diffusion into caseum in vivo for the study drugs. Each assay was performed in triplicates, with means +/− s.d. shown. Scale bar: 5mm