High-sensitivity MALDI-MRM-MS imaging of moxifloxacin distribution in tuberculosis-infected rabbit lungs and granulomatous lesions

Abstract

MALDI-MSI is a powerful technology for localizing drug and metabolite distributions in biological tissues. To enhance our understanding of tuberculosis (TB) drug efficacy and how efficiently certain drugs reach their site of action, MALDI-MSI was applied to image the distribution of the second-line TB drug moxifloxacin at a range of time points after dosing. The ability to perform multiple monitoring of selected ion transitions in the same experiment enabled extremely sensitive imaging of moxifloxacin within tuberculosis-infected rabbit lung biopsies in less than 15 min per tissue section. Homogeneous application of a reference standard during the matrix spraying process enabled the ion-suppressing effects of the inhomogeneous lung tissue to be normalized. The drug was observed to accumulate in granulomatous lesions at levels higher than that in the surrounding lung tissue from 1.5 h postdose until the final time point. MALDI-MSI moxifloxacin distribution data were validated by quantitative LC/MS/MS analysis of lung and granuloma extracts from adjacent biopsies taken from the same animals. Drug distribution within the granulomas was observed to be inhomogeneous, and very low levels were observed in the caseum in comparison to the cellular granuloma regions. In this experiment the MALDI-MRM-MSI method was shown to be a rapid and sensitive method for analyzing the distribution of anti-TB compounds and will be applied to distribution studies of additional drugs in the future.

Figures

Figure 1

Tuberculomas as observed 6–7 weeks post aerosol infection of a NZW rabbit with MTB strain HN878. A. Dorsal aspect of the lung showing cream-colored lesions (arrows); inset, cross-section of the left upper lobe prepared for cryosectioning (arrowhead 1) and half of one matched lesion (arrowhead 2) prior to processing for drug quantitation by LC/MS/MS analysis. B. Small necrotizing lesions identified by central necrosis with peripheral macrophages and lymphocytes in ball-like lesions stained with H&E. C. Cellular lesion containing epithelioid macrophages but little or no central necrosis, similarly stained. Scale bar = 0.5 mm in length.

Figure 2

MALDI-MS/MS spectra for levofloxacin (A) and moxifloxacin (B). The dominant product ions at m/z 344 and m/z 384 correspond to the [M + H – H2O]+ fragments of each respective fluoroquinolone.

Figure 3

Figure showing the MALDI-MS images at each stage of the normalization process. Lower LEV reference standard signals were observed from the granulomas (large central granuloma indicated by the arrow in the H&E reference image) compared to that of the surrounding normal lung tissue. Greater LEV signal suppression occurred in the viable granuloma compared to that in the caseum(identifiable as the light pink center of the large central granuloma). Ion signal intensities were individually scaled for each image. Scale bar = 5 mm.

Figure 4

MS images showing MXF distributions within the rabbit lung biopsy sections at a defined range of postdose times. A subsequent H&E stained reference tissue section is displayed below these images. MXF is uptaken rapidly into the lung, and accumulation within granulomas occurs from 1.5 h. Granuloma drug levels remain higher than surrounding lung tissue over the remaining time points monitored. Lower levels of MXF are observed within the central caseous necrotic areas of the granulomas in the 1.5 and 2.17 h tissues (necrosis is visible as a light pink center in the H&E stained reference tissue). Signal intensities are shown as a fixed scale. Scale bar = 5 mm.

Figure 5

Mean postnormalization granuloma (n = 2–6) and normal lung (n = 3) MXF ion signal intensities taken from a selected MALDI-MS image for each postdose time point. Clear accumulation of MXF within granulomas above surrounding lung begins to occur between 0.5 and 1.5 h. Granuloma MXF levels are consistently higher than those in normal lung over the 1.5–5.14 h period. Viable granuloma and necrotic granuloma areas were not resolved when defining the granuloma region of interest.

Figure 6

Graph showing the concentrations of MXF in plasma, lung, and granuloma lesion tissues at a range of time points postdose. Plasma was sampled immediately prior to euthanasia, lesion, and lung extracts were taken from several (n = 2–5) biopsies from the lungs of each animal. From 1.5 h postdose lesion, concentrations of MXF were higher than that of normal lung. The highest concentration of MXF in granuloma was recorded at 2.17 h postdose.