Dynamic imaging in patients with tuberculosis reveals heterogeneous drug exposures in pulmonary lesions

Abstract

Tuberculosis (TB) is the leading cause of death from a single infectious agent, requiring at least 6 months of multidrug treatment to achieve cure¹. However, the lack of reliable data on antimicrobial pharmacokinetics (PK) at infection sites hinders efforts to optimize antimicrobial dosing and shorten TB treatments². In this study, we applied a new tool to perform unbiased, noninvasive and multicompartment measurements of antimicrobial concentration-time profiles in humans³. Newly identified patients with rifampin-susceptible pulmonary TB were enrolled in a first-in-human study⁴ using dynamic [¹¹C]rifampin (administered as a microdose) positron emission tomography (PET) and computed tomography (CT). [¹¹C]rifampin PET-CT was safe and demonstrated spatially compartmentalized rifampin exposures in pathologically distinct TB lesions within the same patients, with low cavity wall rifampin exposures. Repeat PET-CT measurements demonstrated independent temporal evolution of rifampin exposure trajectories in different lesions within the same patients. Similar findings were recapitulated by PET-CT in experimentally infected rabbits with cavitary TB and confirmed using postmortem mass spectrometry. Integrated modeling of the PET-captured concentration-time profiles in hollow-fiber bacterial kill curve experiments provided estimates on the rifampin dosing required to achieve cure in 4 months. These data, capturing the spatial and temporal heterogeneity of intralesional drug PK, have major implications for antimicrobial drug development.

Extended Data Fig. 1 |. Study outline and patient characteristics.

(a) Patients with pulmonary TB receiving a rifampin-based TB treatment were enrolled and ¹¹C-rifampin PET/CT performed within 6-weeks of treatment initiation. A subset of patients (n = 2) was also imaged at least 20 weeks after starting treatment. (b) All patients were HIV negative and were receiving an oral regimen of isoniazid, rifampin, pyrazinamide, and ethambutol at the time of imaging, except for patient 5 that received moxifloxacin instead of ethambutol. Patient 7 was excluded from the study due to significant motion artifact during the ¹¹C-rifampin PET/CT. Gender: male (M) and female (F).

Extended Data Fig. 2 |. Heterogeneous pulmonary lesions in different lung regions of TB patients.

(a) Transverse CT sections from the TB patients demonstrate the simultaneous presence of multiple pulmonary lesions. (b) TB lesions and cavities with different sizes (volumes) were noted in all lung regions, although there was a preference for the upper lung lobes. Data from all 12 patients is shown. (c and d) Using the CT as a reference, the volumes of interest (VOIs) were drawn and applied to the PET data. VOIs for unaffected lung were placed at the same location in the contralateral unaffected lung.

Extended Data Fig. 3 |. ¹¹C-Rifampin measurements in the liver and brain of TB patients.

(a) Time-activity curve of ¹¹C-rifampin measured in the liver of patients. (b) AUC liver to plasma ratios (c) Time-activity curve of ¹¹C-rifampin measured in the brain of patients. (d) AUC brain to plasma ratios. The red dots represent individual measurements [n = 12 VOIs (measured at 10 time-points; total 120 VOIs per organ) corresponding to the 12 patients], with the black line and grey region representing the median and interquartile range, respectively. Tissue to plasma AUCs are represented as median and interquartile range.

Extended Data Fig. 4 |. Intrasubject heterogeneity of ¹¹C-rifampin exposure.

¹¹C-Rifampin area under the concentration-time curve (AUC) obtained by integrating the area under the dynamic ¹¹C-rifampin PET time-activity curves (middle panels) as well as the AUC ratio (tissue to plasma) (right panels) from selected patients, are shown as heatmap overlays on the CT (left panels). The dotted white lines outline the lesion/cavity. Tissue density [X-ray attenuation value (Hounsfield Unit)] obtained via CT was used to correct the corresponding PET data to represent ¹¹C-rifampin concentrations per mass of tissue.

Extended Data Fig. 5 |. The rabbit model of cavitary TB mimics human disease.

(a) Scheme describing the experiments performed in the rabbit model. (b) After aerosol infection with M. tuberculosis, the rabbits developed a heterogeneous pulmonary disease similar to humans. Transverse CT sections from rabbits at week 18 post-infection are shown. (c) Noninvasive longitudinal monitoring of disease progression using CT. Cavities/lesions are indicated with red arrows. (d) Histopathological analysis of infected-rabbit tissues demonstrates acid-fast bacilli (AFB) in the necrotic areas of the granuloma and cavity wall. Surrounding fibrosis (blue regions) can be noted with Mason’s trichrome staining. Histological analyses were repeated independently four times with similar results.

Extended Data Fig. 6 |. ¹¹C-Rifampin PET in rabbits.

Time-activity curves for individual lesions (a) cavity walls, (b) TB lesions and (c) unaffected lung regions from M. tuberculosis-infected rabbits.

Extended Data Fig. 7 |. Direct tissue measurements of rifampin (mass spectrometry) in rabbits.

(a) Gross pathology demonstrating the M. tuberculosis-infected lungs and the (b) areas selected for quantification of rifampin by mass spectrometry. U, unaffected lung (white); L, lesion (blue); C, cavity wall (red). (c) Tissue concentrations of rifampin and its metabolite 25-desacetyl rifampin. ND, not detectable. The lung tissues from each rabbit were processed independently (n = 5 animals).

Extended Data Fig. 8 |. Rifampin concentrations in rabbit tissues before and after treatment.

All rabbits received five oral doses of rifampin prior to PET imaging to achieve steady state. No differences were identified in (a) ¹¹C-rifampin PET AUC tissue/plasma ratios (n = 11 cavity wall lesions, 13 TB lesions, and 21 unaffected lung regions are shown), (b) absolute rifampin concentrations measured by mass spectrometry (n = 10 cavity wall lesions, 19 TB lesions, 20 unaffected lung regions, caseum from 10 lesions, and five plasma samples are shown), or (c) the tissue/plasma ratios for TB lesions using mass spectrometry quantification in animals before or after treatment (n = 10 cavity wall lesions, 19 TB lesions, 20 unaffected lung regions and caseum from 10 lesion are shown). Data represented as median ± interquartile range. Statistical comparisons performed using a one-tailed Mann-Whitney U test.

Extended Data Fig. 9 |. Pharmacokinetic lung-biodistribution model.

(a) Model schematic. Maximal elimination rate (V_max), rifampin concentration at which the elimination is half-maximal (K_m) and volume of distribution (V_c) of the central compartment of therapeutic dose, clearance (CL_mic) and volume of distribution (V_c,mic) of the central compartment of the ¹¹C-rifampin dose, partition coefficient for left ventricle (PC_LV), unaffected lung (PC_UL), pulmonary lesion (PC_PL), cavity wall (PC_CW), equilibration rate constant (K_eq) between lung and venous plasma, equilibration rate constant (K_eq-lv) between left ventricle and venous plasma, transit rate constant (K_tr), maximal increase in the enzyme production rate (S_max), rifampin concentration at which half the S_max is reached (SC₅₀), and rate constant for the first-order degradation of the enzyme pool (k_enz). (b) Individual fittings of observed (red dots) and model-predicted ¹¹C-rifampin concentrations (black lines) in plasma and pulmonary tissues from all patients. Patient 7 was excluded from the study due to significant motion artifact during the ¹¹C-rifampin PET/CT.

Extended Data Fig. 10 |. Pharmacokinetic model with observed and previously published data.

(a) Observed and model-predicted ¹¹C-rifampin exposure AUC in plasma, unaffected lung, TB lesions and cavity walls of all patients. (b) External data digitized from these studies on TB patients receiving varying doses of intravenous rifampin were used to enrich the dataset. (c, d) Model-predicted rifampin plasma AUC_24h and C_max at steady state compared to the data previously reported by Boree et al. (Am J Respir Crit Care Med. 2015).

Fig. 1 |. First-in-human dynamic [¹¹C]rifampin PET–CT studies in patients with TB.

Twelve patients with confirmed pulmonary TB were prospectively enrolled and imaged in accordance with FDA guidelines. a, Three-dimensional maximum-intensity projection (MIP) of [¹¹C]rifampin PET–CT from a representative patient with TB. The CT is represented in blue, while the [¹¹C]rifampin PET signal is represented in orange. b, A coronal CT section from the same patient demonstrates the selection of volumes of interest (VOIs) to quantify [¹¹C]rifampin PET signal. c, Data from VOIs are represented as time–activity curves, showing that the plasma concentrations were fivefold higher than those found in cavity walls and noncavitary lesions. Data are represented as median ± interquartile range. At each time point, there were n = 2 VOIs from cavity walls, n = 4 VOIs from lesions, n = 2 VOIs from unaffected lung and n = 1 VOI from plasma.

Fig. 2 |. Heterogeneity of disease and intralesional rifampin exposure.

a, A three-dimensional MIP and coronal CT section from a representative patient with cavitary TB. The cavity in the lung apex is outlined in white (left). The [¹¹C]rifampin AUC is also shown as a heat map overlay in the selected transverse section. b, [¹¹C]rifampin (tissue-to-plasma) AUC ratios in patients with TB (data derived from 12 patients; n = 14 cavity wall lesions, n = 24 TB lesions and n = 21 unaffected lung regions are shown) demonstrate limited [¹¹C]rifampin exposure in lesions, with the lowest exposure noted in cavity walls, which paradoxically also have the highest bacterial burden (10^7–10⁹ bacteria). c, A three-dimensional MIP and transverse CT section from a representative rabbit with cavitary TB. The cavity in the lung apex is outlined in white (left). d, Gross pathology, H&E staining and acid-fast bacilli histology of a necrotic pulmonary granuloma and cavity from rabbits. e, Tissue-to-plasma AUC ratios confirm the limited [¹¹C]rifampin exposures in rabbit pulmonary lesions (data were derived from five animals; n = 9 cavity wall lesions, n = 11 TB lesions and n = 14 unaffected lung regions are shown). f, Bacterial burden, as c.f.u. per ml (log₁₀), was high in the cavity walls of rabbit tissues (data were derived from four animals; n = 6 TB lesions, n = 5 cavity wall lesions and caseum from n = 4 different cavities). g,h, Absolute rifampin concentrations (g) and tissue-to-plasma ratios (h) measured by mass spectrometry demonstrate a trend similar to the PET data (data were derived from five animals; n = 10 cavity wall lesions, n = 19 TB lesions, n = 20 unaffected lung regions, caseum from n = 10 lesions and n = 5 plasma samples). Data are represented as median ± interquartile range. Statistical comparisons were made using a one-tailed Mann–Whitney U test (except for in f, where a two-tailed Mann–Whitney U test was used).

Fig. 3 |. Heterogeneous temporal changes in [¹¹C]rifampin exposure during treatment.

a, [¹¹C]rifampin PET–CT was performed at 2 and 36 weeks after initiation of TB treatment in a 45-year-old male patient with cavitary TB. Although no differences were noted in the plasma time–activity curves at these two time points, lower [¹¹C]rifampin exposure was noted in the lesion at 36 weeks. b, Similar results were noted in a 26-year-old female patient with noncavitary TB who was imaged at 2 and 25 weeks after initiation of treatment. Although no difference was noted in plasma levels at these time points, lesion 1 and lesion 2 showed opposite changes in [¹¹C]rifampin exposure. Red arrows point to the location of the selected lesions.

Fig. 4 |. Effect of intralesional rifampin concentrations on treatment shortening.

a, Simulated AUC_0–24 at steady state after oral administration of daily doses from 10–50 mg kg⁻¹ in plasma and lung regions based on the PK lung biodistribution model. Oral bioavailability was assumed to be 90%. b, M. tuberculosis bacterial kill curves achieved at the different rifampin AUC exposures (in combination with isoniazid and pyrazinamide) used in the hollow-fiber model (HFS-TB). The solid lines (median) represent the rapidly (red) and slowly (black) growing bacterial subpopulations, while the shaded regions represent the 95% confidence intervals. The red and black dots represent intracellular and extracellular bacteria in log phase and slow (semidormant/nonreplicating) growth phases, respectively. For these AUC studies, mg × h / kg is equivalent to mg × h / l (n = 3 HFS-TB cartridges per regimen). c, The corresponding TTE of the bacterial population in the HFS-TB model (red bars) used to predict the time to cure in patients (gray bars) is shown below each corresponding AUC exposure in b. The dotted red line represents estimates of the time after treatment initiation (in days) to achieve cure in 95% of the patients.