Imaging pulmonary inducible nitric oxide synthase expression with PET

Abstract

Inducible nitric oxide synthase (iNOS) activity increases in acute and chronic inflammatory lung diseases. Imaging iNOS expression may be useful as an inflammation biomarker for monitoring lung disease activity. We developed a novel tracer for PET that binds to iNOS in vivo, (18)F-NOS. In this study, we tested whether (18)F-NOS could quantify iNOS expression from endotoxin-induced lung inflammation in healthy volunteers.

Methods: Healthy volunteers were screened to exclude cardiopulmonary disease. Qualifying volunteers underwent a baseline, 1-h dynamic (18)F-NOS PET/CT scan. Endotoxin (4 ng/kg) was then instilled bronchoscopically in the right middle lobe. (18)F-NOS imaging was performed again approximately 16 h after endotoxin instillation. Radiolabeled metabolites were determined from blood samples. Cells recovered by bronchoalveolar lavage (BAL) after imaging were stained immunohistochemically for iNOS. (18)F-NOS uptake was quantified as the distribution volume ratio (DVR) determined by Logan plot graphical analysis in volumes of interest placed over the area of endotoxin instillation and in an equivalent lung region on the left. The mean Hounsfield units (HUs) were also computed using the same volumes of interest to measure density changes.

Results: Seven healthy volunteers with normal pulmonary function completed the study with evaluable data. The DVR increased by approximately 30%, from a baseline mean of 0.42 ± 0.07 to 0.54 ± 0.12, and the mean HUs by 11% after endotoxin in 6 volunteers who had positive iNOS staining in BAL cells. The DVR did not change in the left lung after endotoxin. In 1 volunteer with low-level iNOS staining in BAL cells, the mean HUs increased by 7% without an increase in DVR. Metabolism was rapid, with approximately 50% of the parent compound at 5 min and 17% at 60 min after injection.

Conclusion: (18)F-NOS can be used to image iNOS activity in acute lung inflammation in humans and may be a useful PET tracer for imaging iNOS expression in inflammatory lung disease.

Trial registration: ClinicalTrials.gov NCT01407796.

Keywords: endotoxin; inducible nitric oxide synthase; lung inflammation; positron emission tomography.

FIGURE 1

Study design. Postendotoxin ¹⁸F-NOS PET scan occurred at approximately 16 h after endotoxin instillation.

FIGURE 2

Immunohistochemical staining for iNOS (green) in cells obtained by BAL in endotoxin-challenged airway. Only 1 individual had negative iNOS staining (iNOS (−)). iNOS (+) image is representative of positive staining results obtained on BAL cells from 6 volunteers. Neutrophils (white arrowheads) and macrophages (yellow arrowheads) were identified by nuclear morphology from 4′,6-diamidino-2-phenylindole staining (blue). Images taken at ×20 magnification.

FIGURE 3

Representative Logan parametric ¹⁸F-NOS PET/CT images (DVR scale, mL lung/mL blood) obtained before and after bronchoscopic instillation of endotoxin in right middle lobe. VOIs are shown in white.

FIGURE 4

Time–activity curves and Logan plots from images (VOI) in Figure 3. Last 12 data points (last 50 min of image acquisition) were used for Logan plot linear regression for all scans. Inset shows later time points of curve focused on lower range activity to better illustrate differences in activity among different VOIs. Units for Logan plot axes: x-axis = mL blood/mL lung × min; y-axis = min.

FIGURE 5

¹⁸F-NOS DVR and mean HUs in right and left lung VOIs before and after endotoxin instillation in right middle lobe. White circles denote 1 volunteer without iNOS in BAL cells. Arrowheads denote data from 1 volunteer with heterozygosity for 2 TLR4 single nucleotide polymorphisms reported to predict endotoxin hyporesponsiveness. *P < 0.05, compared with either left lung after endotoxin or right middle lobe before endotoxin. Before = before endotoxin instillation; After = 16 h after endotoxin instillation.

FIGURE 6

¹⁸F-NOS metabolism showing percentage of parent compound in plasma. Values are shown as mean ± SD bars.