Summary

Tuberculosis (TB) remains one of the leading killers from a single infectious agent and TB meningitis is the most devastating extrapulmonary form, especially in the young and immunocompromised. Multidrug-resistant (MDR)-TB, caused by Mycobacterium tuberculosis resistant to first-line antibiotics (i.e., isoniazid and rifampin), is on the rise. TB meningitis due to MDR strains is associated with high mortality, and drug-resistance is an independent predictor of death. In a recent retrospective cohort study among 237 patients with TB meningitis, mortality was significantly higher among patients with drug-resistant (67%) than drug-susceptible disease (24%, P < 0.001). Moreover, mortality was significantly higher (adjusted hazards ratio of 7.2) in patients with drug-resistant TB meningitis after 90 days of initiation of treatment (P < 0.001). New drugs and more efficacious treatments against MDR-TB are therefore urgently needed to combat this public health threat. Pretomanid (formerly PA-824) is a small molecule belonging to the nitroimidazole class of antimicrobial agents, approved by the U.S. Food and Drug Administration (FDA) in 2019 for the treatment of pulmonary MDR-TB, in combination with bedaquiline and linezolid (BPaL - bedaquiline, pretomanid, linezolid). Pretomanid is active against both replicating and non-replicating M. tuberculosis, which contributes to its excellent bactericidal activity.
With few exceptions, current antibiotic dosing recommendations are based on plasma concentrations, without information on drug concentrations at the site of infection. Since inappropriate antibiotic levels in target tissues can lead to selection of resistant organisms, toxicity or organ injury, and ultimately treatment failure, a growing number of studies and the U.S. FDA increasingly support measuring antibiotic concentrations in infected tissues. Therefore, the authors have developed positron emission tomography (PET)-based clinically-translatable technologies for noninvasive, simultaneous, and unbiased, multi-compartment in situ measurements of antibiotic concentration-time curves in animals and humans. In this study, the authors report the development of ¹⁸F-pretomanid as a molecular imaging tool to noninvasively assess whole-body drug biodistribution utilizing detailed animal studies in mouse and rabbit models of TB meningitis. In brief, infected animals undergo dynamic PET/computed tomography (CT) with ¹⁸F-pretomanid to obtain time-activity curves (TACs) and areas under the curve (AUCs) by quantifying the PET signal in volumes of interest (VOI). Post-mortem autoradiography and histology are also performed in all animal models. Given the unknown potential of pretomanid-containing regimens for TB meningitis, the bedaquiline (B), pretomanid (P) and linezolid (L) regimen is tested in a mouse model of TB meningitis. Mass spectrometry and traditional microbiology techniques are employed to evaluate intraparenchymal drug levels and bactericidal efficacy (bacterial burden quantified with colony-forming units [CFU]) longitudinally. Radiosynthesis of ¹⁸F-pretomanid under current Good Manufacturing Practices (cGMP) facilitates translation into humans, and first-in-human dynamic ¹⁸F-pretomanid PET studies are performed in accordance with U.S. FDA guidelines.

Results from the nanoScan PET/CT

For the PET/CT imaging, M. tuberculosis-infected mice and rabbits were used inside transparent and sealed biocontainment containers, compliant with biosafety level (BSL)-3 containment and capable of delivering O₂-anesthetic mixture to sustain live animals during imaging as previously described (43). PET acquisition and subsequent CT were performed using the nanoScan PET/CT and images were automatically co-registered. ¹⁸F-Pretomanid PET/CT: Mice were injected with ¹⁸F-pretomanid (pulmonary TB, 3.77 ± 1.78 MBq; TB meningitis, 3.88 ± 1.44 MBq) via the tail vein. Anesthetized rabbits were injected with ¹⁸F-pretomanid (4.65 ± 0.75 MBq) through the ear vein. The injection time coincided with the start of the dynamic PET acquisition. ¹⁸F-FDG PET/CT: Animals were imaged as previously described (20). 

Figure 3. shows the imaging and quantification of the PET/CT.  The authors' initial studies characterizing the whole body biodistribution of ¹⁸F-pretomanid in mouse models of pulmonary TB revealed that the penetration into the brain parenchyma was high, with a median AUC ratio (brain/plasma) of 1.73 (IQR, 1.41–2.04), even in healthy brains. This prompted detailed investigation of ¹⁸F-pretomanid penetration into infected brains in two mammalian models of TB meningitis also showing excellent brain penetration of ¹⁸F-pretomanid [AUC ratios (brain/plasma) >1]. However, in vivo 3D PET/CT and 2D ex vivo autoradiography in the mouse model of TB meningitis showed reduced uptake of ¹⁸F-pretomanid with filling defects at the center of the brain lesion (visible in live animals with ¹⁸F-FDG PET/CT and ex vivo histopathology, respectively) (Fig. 3a, b). AUC ratio (brain/plasma) was 1.35 (median; IQR, 0.81–1.52) in brain lesions and 1.56 (median; IQR, 1.22–1.69) in unaffected brain regions (Fig. 3c, d). Similar findings were noted in a rabbit model of TB meningitis (Fig. 3e-h), with median AUC ratio (brain/plasma) of 1.87 (IQR, 1.66–4.63) into brain lesions and 2.75 (IQR, 1.64–5.73) into the unaffected brain.

• The use of radiolabeled antimicrobials, combined with conventional microbiology techniques and novel animal models, offers unmatched potential to noninvasively assess PK profiles in infected tissues, characterized by complex, heterogeneous lesions existing simultaneously in the same host.
• Importantly, the authors' studies demonstrate that pretomanid has excellent penetration into the CNS, confirmed in first-in-human ¹⁸F-pretomanid studies, suggesting that novel pretomanid-based regimens in combination with other antibiotics active against MDR strains and with excellent brain penetration should be considered for the treatment of MDR-TB meningitis.
• Finally, the authors' studies reaffirm the compartmentalized and discordant antibiotic penetration into the CNS, which are related to the physiochemical properties of the antibiotic.

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