(M0930-06-32) Development of Quantitative MALDI Mass Spectrometry Imaging Methods for Studying Distribution of Antituberculosis Drugs at Their Site of Action

Purpose: As plasma drug concentration often poorly correlates with tissue concentration, determination of active drug concentrations at the site of action is becoming increasingly important to build accurate and predictive physiologically-based pharmacokinetics (PBPK) models to eventually improve dose selection in first-in-human trials. This observation is even more important in anti-tuberculosis (TB) drug development, as Mycobacterium tuberculosis (MTb) creates very specific granulomatous lesions, difficult for the drug to penetrate, resulting in discrepancies between exposure of healthy and diseased tissues. As a result, we developed MALDI Mass Spectrometry Imaging methods for multiple antituberculosis drugs currently being investigated by the antiTB community.

Methods: Untreated frozen rat lungs were cryosectioned using a cryostat and mounted on Indium-Tin-Oxide glass slides (Delta Tech, Loveland, CO, USA). Dilution series of each of the drug were prepared and spotted on the control tissues before coating the slide with various MALDI matrices, solvents, and derivatization agents when necessary, using an automated sprayer (TM sprayer, HTX Imaging, Chapel Hill, NC, USA). Slides were imaged using a MALDI FTICR (Solarix 2XR, Bruker, Bremen, Germany) using QuadruPolar Detection to maximize spectral resolution and method specificity. Calibration curves were obtained using Multimaging 1.3.2.1 software (Aliri, Loos, France).

Results: Limit of detection (LOD), Lower Limit of Quantification (LLOQ) and Upper Limit of Quantification (ULOQ) were determined for several antituberculosis drugs as illustrated in table 1. Rifampicin, Ethambutol, Moxifloxacin, Rifapentin, Bedaquiline and Pretomanid methods have been developed using a simple matrix application, either 2,5-dihydroxybenzoic acid (DHB) or 1,5-diaminonaphtalene (DAN) with optimized solvent composition and led to LLOQ in the low µg/g range. Due to the low molecular weight and high polarity of Isoniazid, we optimized an on-tissue chemical derivatization with trans-cinnamaldehyde based on Manier et al [1] to which we added an extra washing step of the tissue section, to remove hydrophobic interfering species and decrease ion suppression to eventually obtain a sensitivity of 1.29 µg/g, compatible with expected tissue exposure at therapeutic doses. MALDI MSI calibration curve of isoniazid is presented in figure 1 and shows good linearity. Finally, as the sensitivity of pyrazinamide parent drug was unsatisfactory (81.4 µg/g), we have developed an alternative strategy based on the derivatization of its active metabolite (pyrazinoic acid) using CAX-B derivatization agent. Although the active metabolite cannot recapitulate by itself the entire drug-related exposure, as it is considered to be the active form of pyrazinamide, produced by Mycobacterium tuberculosis pyrazidamidase, this analytical method offers an opportunity to better understand the mechanism of the drug at its site of action, in the granulomatous lesion.

Conclusion: A complete set of MALDI QMSI methods has been developed to serve the need of the consortium ERA4TB to study the distribution of multiple drugs to be tested either alone or in combination regimens in multiple preclinical models of infected or non-infected animals. This platform offers opportunities for studying pharmacokinetics at the site of action, refining PBPK models, studying mechanism of action, drug-drug interactions, or host-pathogen interactions under treatment.

References: [1] Manier et al. Reagent Precoated Targets for Rapid In-Tissue Derivatization of the Anti-Tuberculosis Drug Isoniazid Followed by MALDI Imaging Mass Spectrometry. J. Am. Soc. Mass Spectrom. (2011) 22:1409-1419

Acknowledgements: The project leading to this publication has received funding from the Innovative Medicines Initiative 2 Joint Undertaking (JU) under grant agreement No 853989. The JU receives support from the European Union’s Horizon 2020 Research and Innovation Programme and EFPIA and Global Alliance for TB Drug Development Non-Profit Organisation, Bill & Melinda Gates Foundation, University of Dundee. This work reflects only the author's views, and the JU is not responsible for any use that may be made of the information it contains.


Table 1: method performance for several anti-TB drugs in MALDI MSI


Figure 1: calibration curve of isoniazid (zoom on lower end)