TB remains a major threat to human health22. TB meningitis is a serious, life threatening form of TB, and current treatments prevent death or disability in less than half5. Importantly, the central nervous system (CNS) has more than one compartment [e.g. brain parenchyma and CSF], which are separated from the circulation by the blood-brain barrier (BBB) that limit penetration of many drugs. Despite knowledge that many antibiotics do not penetrate into the brain adequately and that immunopathology is the critical pathologic process, current TB meningitis treatment is not optimized and continues to be based on those developed for pulmonary TB3,4. Importantly, the alarming rise of MDR strains of M. tuberculosis, poses further challenges in the management of TB meningitis, as tissue pharmacokinetics and activities of newer antibiotics effective against MDR strains are not known. In fact, TB meningitis due to MDR strains is associated with the highest mortality23,24, with drug-resistance being an independent predictor of death25. In one report, mortality in TB meningitis patients with drug-resistance (67%) using current regimens was significantly higher than in those with drug-susceptible disease (24%, P < 0.001)26. Therefore, effective treatments against TB meningitis due to MDR strains are urgently needed.
Using whole-body, noninvasive and unbiased, dynamic PET imaging, we were able to obtain a rich dataset of concentration-time profiles in multiple compartments in three-dimensional space simultaneously, demonstrating compartmentalized brain and lung tissue exposures. Animal studies confirmed the compartmentalized pretomanid brain and lung tissue exposures noted in the human studies but also demonstrated antibiotic-specific compartmentalization, e.g. while pretomanid had higher brain versus lung tissue exposures, the opposite was noted for bedaquiline. Direct antibiotic measurements from postmortem animal tissues confirmed the findings from the PET studies. PET-facilitated pharmacokinetic modeling and Monte Carlo simulations were then used to predict antibiotic exposures in brain and lung tissues. Only pretomanid achieved therapeutic brain tissue exposures at the standard human oral dosing and bedaquiline brain tissue exposures remained subtherapeutic even at a dose four times the standard human oral dose. It should be noted that pretomanid up to 1,200 mg/day is tolerated well in humans27. Given its concentration-time dependent activity as well as animal studies demonstrating better bactericidal activities at human equivalent dose of 400–600 mg/day (versus 200 mg/day)28, higher pretomanid dosing could be considered29, especially when the exposures of other antibiotics in the regimen are predicted to be suboptimal. Pyrazinamide has excellent CSF30,31, and lung penetration and exposures correlate with treatment response in patients with MDR pulmonary TB with pyrazinamide-susceptible strains32. However, one study has reported increased mortality and neurological toxicity in TB meningitis with elevated CSF pyrazinamide33, but a mechanistic basis for this finding is not known and therefore the significance of this association is unclear. Nonetheless, pyrazinamide continues to be used worldwide as the standard first-line regimen for drug-susceptible TB meningitis for the first two months of treatments. Pyrazinamide has strong sterilizing activity critical for shortening pulmonary TB treatments and one study in children has demonstrated that regardless of disease stage at presentation, a 6-month pyrazinamide-containing regimen was more efficacious than 9 or 12 month regimens without pyrazinamide34. Although brain tissue exposures for pyrazinamide are unknown in humans, PET imaging in non-human primates with a chemically identical radioanalog of pyrazinamide demonstrated excellent brain tissue exposures35, which were consistent with our data (mass spectrometry) from the mouse studies. Similarly, while moxifloxacin (Mx), active against several MDR strains of M. tuberculosis, has excellent CSF penetration36,37, brain tissue exposures are not known and our data from mouse studies demonstrated limited brain (tissue/plasma ratio 0.23) but excellent lung tissue penetration (tissue/plasma ratio 3.34). Overall, these data suggested that pretomanid and pyrazinamide based multidrug regimens could be highly effective in TB meningitis. It should be noted that concomitant use of pretomanid and pyrazinamide led to hepatotoxicity-related treatment discontinuations in 6–7% of participants in one study for the treatment of pulmonary TB,38 with treatment-emergent elevations of alanine transaminase (ALT) greater than three times the normal limits in 10.8% of those treated with pretomanid-pyrazinamide regimens versus 8.6% and 5.6% in those treated with BPaL, or the first-line TB regimens. However, given the substantial benefits of pyrazinamide32, and the high mortality associated with TB meningitis, especially due to MDR strains, the risk-benefit comparison likely favors its use in TB meningitis. Finally, antibiotics such as sutezolid, linezolid and moxifloxacin that are active against MDR strains and have moderate brain tissue exposures, could provide additive activity when combined with other highly effective antibiotics.
Data from the Monte Carlo simulations were used to design optimized, pretomanid-based multidrug regimens in the mouse model of TB meningitis7,14, administered at human equipotent doses. While addition of pyrazinamide substantially improved the bactericidal activities of all MDR-TB regimens, as predicted, addition of bedaquiline did not. The additive effects of sutezolid and linezolid were similar, but sutezolid has a better safety profile at higher dosing (up to 1,600 mg/day)39, required to achieve better brain tissue exposures. Importantly, we developed several pretomanid-based multidrug regimens (BPa50LZ, Pa100LZ and Pa50LMxZ) active against TB meningitis due to MDR strains, with bactericidal activities substantially better than R10HZ or BPa50L regimens. However, given the high rates of pyrazinamide resistance amongst MDR strains40, we also developed a pyrazinamide-sparing regimen (Pa100SMx), which while not as bactericidal as pyrazinamide-containing regimens, was still as effective as the first-line standard TB treatment, and substantially better than the BPa50L regimen. Finally, we were able to assess the activities of several antibiotic regimens in the brain and lung compartments in the same animal, which demonstrated discordant bactericidal activities, corresponding to the compartmentalized tissue exposures of the component antibiotics in the regimen. While bactericidal activity is an important endpoint for pulmonary TB treatments, clinical outcomes in TB meningitis may also be closely associated with intracerebral inflammatory responses41. This was assessed using live imaging as well as postmortem analyses demonstrating that the optimized, pretomanid-based multidrug regimens did not increase intracerebral inflammation or markers of brain metabolism and injury. Specifically, pyrazinamide-containing regimens had significantly lower intracerebral inflammation. High CSF tryptophan levels are associated with increased mortality in patients with TB meningitis42, and CSF and plasma tryptophan levels were lower in mice treated with the optimized regimens. Similarly, elevated levels of brain injury markers [GFAP, NEFL (or Nfl), Tau and S100B] in the CSF or plasma, are associated with poor outcomes in patients with brain damage43, and in TB meningitis44, and brain injury marker levels in CSF and plasma were lower in mice treated with the optimized regimens.
Our studies have some limitations. Healthy volunteers and patients with pulmonary TB were imaged with 18F-pretomanid PET, and pretomanid brain exposures were excellent in these subjects with presumably an intact BBB and healthy brain tissues. It is anticipated that pretomanid brain exposures would remain higher than those plasma in infected tissues7, or in the setting of a leaky BBB in patients with TB meningitis. Due to the high risks of work with MDR M. tuberculosis strains in the laboratory, antibiotic regimens active against MDR strains were evaluated in animals infected with the drug-susceptible M. tuberculosis H37Rv strain. This is the standard and accepted approach for TB drug development, even for antibiotic regimens active against MDR strains45,46. In fact, the vast majority of MDR-TB regimens currently being evaluated in clinical trials or in clinical use (BPaL) were originally developed in mouse models utilizing infections with the drug-susceptible M. tuberculosis H37Rv strain10,20. While 18F-pretomanid is chemically identical to the parent antibiotic, for PET studies it was administered at a microdose (ng-µg per subject) rather than at a therapeutic dose. However, several studies support that microdosing as a reliable predictor of the drug biodistribution at therapeutic doses47,48. Further, 18F-pretomanid and the other radiolabeled antibiotics were administered intravenously with the injection time corresponding to the plasma Tmax, and brain uptake reaching Cmax (maximum concentration) within the first few minutes, enabling the first 60 min to adequately capture the pharmacokinetic profile. Of note, bedaquiline has a much longer half-life than the other antibiotics evaluated here, but given the much longer physical half-life (16 hours) of Br-76 utilized as the radiolabel for bedaquiline (by replacing the endogenous Br with Br-76), imaging could be performed for 48 hours after tracer injection16. Importantly, all radiolabels were introduced into the antibiotic to keep them chemically identical to the parent compound and retained even after their metabolism7,15,16.
In summary, we report first-in-human dynamic 18F-pretomanid PET/CT in eight human subjects to simultaneously measure brain and lung tissue exposures that demonstrated compartmentalized exposures. PET studies in live animal and direct antibiotic measurements in postmortem tissue samples validated these findings but also demonstrated preferential (AUCtissue/plasma >1) antibiotic-specific partitioning into brain or lung tissues within the same subject, driven presumably by the physiochemical properties of each antibiotic. PET-facilitated pharmacokinetic modeling was used to design optimized, pretomanid-based multidrug regimens tested at human equipotent dosing in a mouse model of TB meningitis. We developed several multidrug regimens active against TB meningitis due to MDR strains, with bactericidal activities substantially better than R10HZ or BPa50L regimens, without an increase in intracerebral inflammation or markers of brain metabolism and injury. These optimized, pretomanid-based multidrug MDR regimens comprise antibiotics either already approved for human use or being assessed in clinical trials. Therefore, these regimens could be readily evaluated in clinical studies for TB meningitis. Importantly, several antibiotic regimens demonstrated discordant bactericidal activities in brain versus lung tissues in the same animal, correlating with compartmentalized tissue exposures of the component antibiotics visualized with PET and confirmed by postmortem mass spectrometry. Our data provide a mechanistic basis for the discordant activities of antibiotic regimens for pulmonary TB and TB meningitis. Our approach is highly generalizable and has major implications for antimicrobial drug development and compartment-specific optimization of regimens, especially for meningitis and other infections in compartments with unique antibiotic penetration.