(W1030-02-08) Distribution of Standard-of-Care Antituberculosis Drugs in Cynomolgus Macaque Lungs

Purpose: The non-human primate (NHP) provides the most clinically relevant model of human tuberculosis. The aim of this study was to determine the tissue pharmacokinetics of orally delivered isoniazid (H), rifampicin (R), pyrazinamide (Z) and ethambutol (E) combination therapy in the lung of cynomolgus macaques in order to prepare following studies in Mycobacterium tuberculosis-infected animals. To do so, as part of the ERA4TB consortium, a IMI-funded initiative aiming at developing new drugs and drug regimens to treat TB, we applied Quantitative Mass Spectrometry Imaging (QMSI) methods for determining tissue PK of HRZE in NHP lungs.

Methods: Six cynomolgus macaques received a single oral delivery of isoniazid at 15 mg/kg, rifampicin at 15 mg/kg, pyrazinamide at 200 mg/kg and ethambutol at 75 mg/kg. Necropsies were conducted  2h45, 3h15, 3h30, 5h30, 6h15, 24h after drug administration at which the upper right lung lobe was collected, inflated with agarose, snap frozen and shipped to MSI lab. Lung samples 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 and Solarix 2XR, Bruker, Bremen, Germany). Calibration curves were obtained using Multimaging 1.3.2.1 software (Aliri, Loos, France). Adjacent sections collected on Superfrost slides were stained with Hematoxylin-Eosin to produce histological images which have been overlayed with the MSI datasets.

Results: Despite the use of a specific derivatization protocol with a limit of detection of around 2 µg/g of tissue, isoniazid was barely detected in the samples investigated. Conversely, QMSI revealed a wide distribution of ethambutol up to 24h after administration of around 30 µg/g. As the pyrazinamide limit of detection was too high to enable imaging at therapeutic doses, its active metabolite pyrazinoic acid was imaged as a surrogate, pending further analytical development on the parent drug. Pyrazinoic acid was detected in lung tissue collected at each of the timepoints investigated (Fig 1), the concentration-time profile was built (Fig 2) and PK parameters obtained (Table 1). Although pyrazinoic acid was thought to be produced in the granulomatous lesions by bacterial pyrazidamidase activity, MALDI-FTICR revealed the presence of pyrazinoic acid in non-infected NHP, suggesting an additional production of this metabolite by a hydrolytic enzyme of the host. Whilst the study was limited to the evaluation of a single animal at each time point was used, for obvious ethical reasons, it has highlighted the potential for variation in the response to drug treatment as some animals (animal ID I050CAB, T 6h15) exhibited very different ADME behaviour toward HRZE combination therapy, with exposure much higher than expected from the concentration-time profile built with the other animals. This consideration should be taken into account when building PBPK models of antituberculosis drugs with limited number of NHP.

Conclusion: This study produced reference HRZE distribution data in cynomolgus macaque lung and contributes to a better understanding of the PK of the standard-of-care antituberculosis drugs that will assist the optimization of dosing regimens in antiTB combination therapies. From a methodological perspective, this imaging experiment also paved the way for future determination of drug concentration in lesions (granuloma, inner and outer caseum, etc) in infected cynomolgus macaque to support PK/PD investigations.

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.
Institutional Review Board Statement: All animal procedures and study design were approved by the UK Health Security Agency, Porton Down Establishment Animal Welfare and Ethical Review Body, and authorized under an appropriate UK Home Office project license.


Fig 1:Distribution of pyrazinoic acid in NHP lung obtained by MALDI MSI


Fig 2: Concentration-Time profile of pyrazinoic acid in NHP lung (T 6h15 excluded)


Table 1: PK parameters of pyrazinoic acid in NHP lung