Dynamic PET Reveals Compartmentalized Brain and Lung Tissue Antibiotic Exposures

Abstract

Tuberculosis (TB) remains a leading cause of death, but antibiotic treatments for tuberculous meningitis, the deadliest form of TB, are based on those developed for pulmonary TB and not optimized for brain penetration. Here, we performed first-in-human dynamic ¹⁸F-pretomanid positron emission tomography (PET) studies in eight human subjects for three-dimensional, multi-compartmental in situ visualization of antibiotic concentration-time exposures (area under the curve - AUC), demonstrating preferential brain (AUC_{tissue/plasma} 2.25) versus lung (AUC_{tissue/plasma} 0.97) tissue partitioning. Preferential, antibiotic-specific partitioning into brain or lung tissues of antibiotics active against MDR strains were confirmed in experimentally-infected mice and rabbits, using dynamic PET with chemically identical antibiotic radioanalogs, and postmortem mass spectrometry measurements. PET-facilitated pharmacokinetic modeling predicted human dosing necessary to attain therapeutic brain exposures in human subjects. These data were used to design optimized, pretomanid-based regimens which were evaluated at human equipotent dosing in a mouse model of TB meningitis, demonstrating excellent bactericidal activity without an increase in intracerebral inflammation or brain injury. Importantly, several antibiotic regimens demonstrated discordant activities in brain and lung tissues in the same animal, correlating with the compartmentalized tissue exposures of the component antibiotics. These data provide a mechanistic basis for the compartmentalized activities of antibiotic regimens, with important implications for the development of antimicrobial regimens for meningitis and other infections in compartments with unique antibiotic penetration.

Figure 1. First-in-human dynamic ¹⁸F-pretomanid PET/CT studies in human subjects.

Eight subjects were prospectively enrolled and imaged in accordance with the U.S. Food and Drug Administration guidelines (NCT05609552). a, ¹⁸F-Pretomanid PET area under the curve (AUC) heatmap overlay on computed tomography (CT) of a representative subject with pulmonary TB (subject 7). b, Coronal CT section from the same subject showing the volumes of interest (VOI) used to quantify the ¹⁸F-pretomanid PET signal and obtain time-activity curves from different tissues (panel c). d, ¹⁸F-Pretomanid AUC heatmap overlay on CT of representative healthy subject (subject 2). e, Coronal CT section from the same subject showing the volumes of interest (VOI) used to quantify the ¹⁸F-pretomanid PET signal and obtain time-activity curves from different tissues (panel f). g, ¹⁸F-Pretomanid (tissue-to-plasma) AUC_{tissue/plasma} ratios from all subjects demonstrate significantly higher brain versus lung tissue exposures. Circles and squares represent female and male subjects respectively. Empty shapes represent TB patients. n = 2 VOI per tissue per subject. Data are represented as median ± interquartile range. Statistical comparisons were made using a two-tailed Mann-Whitney U test.

Figure 2. Compartmentalized antibiotic exposures in mouse studies.

Radioanalog of four antibiotics active against multidrug-resistant (MDR) Mycobacterium tuberculosis strains were utilized in mouse studies. Each radioanalog is chemically identical to the respective parent antibiotic (panels a, d, g, j) and the radioisotope is retained within the major metabolite. Coronal, sagittal and transverse CT with PET AUC heatmap overlays (representing AUC_{tissue/plasma} ratio) from representative mice, demonstrating spatially compartmentalized antibiotic exposures in lung and brain compartments are shown for (b) ¹⁸F-pretomanid (AUC_0–60min), (e) ¹⁸F-sutezolid (AUC_0–60min), (h) ¹⁸F-linezolid (AUC_0–60min), and (k) ⁷⁶Br-bedaquiline (AUC_0–48h). Tissue-to-plasma ratios obtained using dynamic PET in live animals (AUC ratio) and by mass spectrometry (MS) from postmortem (tissue-to-plasma ratio) brain (red) and lung (blue) tissues are respectively shown for (c) ¹⁸F-pretomanid PET [AUC_0–60min ratio, data from four animals; n = 8 brain volumes of interest (VOIs), n = 8 lung VOIs], pretomanid MS (tissue/plasma ratio, data from six animals; n = 6 brain, n = 6 lung samples), (f) ¹⁸F-sutezolid PET (AUC_0–60min ratio, data from four animals; n = 8 brain VOIs, n = 8 lung VOIs), sutezolid MS (data from six animals; n = 6 brain, n = 6 lung samples), (i) ¹⁸F-linezolid PET (AUC_0–60min ratio, data from four animals; n = 8 brain VOIs, n = 8 lung VOIs), linezolid MS (data from six animals; n = 5 brain, n = 6 lung samples), and (l) ⁷⁶Br-Bedaquiline PET (AUC_0–48h ratio, data from three animals; n = 6 brain VOIs, n = 6 lung VOIs), bedaquiline MS (data from five animals; n = 5 brain, n = 5 lung samples). The horizontal dotted lines indicate tissue/plasma ratio of 1. Data are represented as median ± interquartile range. Statistical comparisons were made using a two-tailed Mann-Whitney U test.

Figure 3. Pharmacokinetic model to predict human tissue exposures for pretomanid.

a, Schematic representation of the model for pretomanid tissue penetration based on a physiology-based pharmacokinetic (mPBPK) framework. b, Observed (black dots) and individual model-predicted (purple line) for ¹⁸F-pretomanid exposures in different tissues [brain, CSF (measured in cerebral ventricles), lung and plasma (measured in left heart ventricle)] for the human subjects (S1-S8). Y-axis shows antibiotic exposure and x-axis shows time (hours). CL = plasma clearance, Kp = partition coefficient for the tissue compartment, Qt = blood flow rate to tissue, Qlung = blood flow rate to lungs, Qbrain = blood flow rate to brain, QCSF = blood flow rate to brain ventricles (CSF), Rbrain = penetration ratio for brain, RCSF = penetration ratio for CSF, Rlung = penetration ratio for lungs.

Figure 4. Monte Carlo simulations to predict humans tissue exposures.

Monte Carlo simulations were performed in 1,000 virtual subjects for each antibiotic to predict tissue exposure area under the curve (AUC) in plasma (green), brain (red) and lung (blue) at various oral doses are shown for a, pretomanid, b, sutezolid, c, linezolid and, d, bedaquiline. The horizontal dotted black line represents target AUC for each antibiotic and represents the lung tissue exposures achieved with standard oral dosing (bolded red) in patients with pulmonary TB. A second dotted line is shown for pretomanid, representing a dose of 600 mg/day. Data are represented as median ± interquartile range.

Figure 5. Optimized pretomanid-based multidrug regimens at human equipotent dosing.

a, Two weeks after infection with Mycobacterium tuberculosis, mice with TB meningitis were randomly allocated to receive multidrug regimens via oral gavage at human equipotent dosing (R, rifampin; H, isoniazid; Z, pyrazinamide; B, bedaquiline; Pa₅₀ or Pa₁₀₀, pretomanid corresponding to a human dose of 200 mg/day or 400–600 mg/day respectively; L, linezolid; S, sutezolid; Mx, moxifloxacin). Selected pyrazinamide-containing regimens were tested in mice infected with pyrazinamide-resistant M. tuberculosis (pncA mutant). Red bars represent optimized regimens, grey bars represent other regimens and black bars represent reference regimens. All animals also received adjunctive dexamethasone, which is the standard of care for TB meningitis. b, Bacterial burden [colony-forming unit (CFU) per gram of brain tissue (log₁₀) from whole brain] after six weeks of treatment (n = 3–14 mice/regimen). The bacterial burdens for reference regimens are shown as horizontal dotted lines for comparison; top grey is for untreated animals representing the starting bacterial burden, middle black is for the approved MDR regimen for pulmonary TB (BPa₅₀L) and lower black is for the first-line, standard TB treatment for drug-susceptible TB meningitis (R₁₀HZ). Regimens tested in mice infected with the pncA mutant are indicated by empty rectangles. c, Bacterial burden after six weeks comparing regimens with and without bedaquiline and/or pyrazinamide (n = 5–9 mice/regimen). d, Bacteria disseminate to the lung after the brain infection. Therefore, bactericidal activities of several antibiotic regimens in brain and lung tissues of the same animal were assessed. Data are shown as the reduction in whole brain (solid filled rectangle) and lung (checkered filled rectangle) organ CFU two weeks after treatment initiation (n = 4–5 mice/regimen). e, Mice underwent live imaging at two weeks after treatment initiation. Transverse ¹²⁴I-DPA-713 PET/CT images of representative mice from different treatment regimens and untreated mice (left) and quantification of ¹²⁴I-DPA-713 PET signal in the brain represented as standard uptake value mean (SUV_mean) (n = 4–8 mice/group) (right) are shown. For CFU, data are represented as mean ± standard deviation and statistical comparisons were made using a two-tailed student t-test. For imaging, data are represented as median ± interquartile range. Statistical comparisons were made using a two-tailed Mann-Whitney U test.