Optimization of a Collagen-Targeted PET Probe for Molecular Imaging of Pulmonary Fibrosis

Abstract

There is a large unmet need for a simple, accurate, noninvasive, quantitative, and high-resolution imaging modality to detect lung fibrosis at early stage and to monitor disease progression. Overexpression of collagen is a hallmark of organ fibrosis. Here, we describe the optimization of a collagen-targeted PET probe for staging pulmonary fibrosis. Methods: Six peptides were synthesized, conjugated to a copper chelator, and radiolabeled with 64Cu. The collagen affinity of each probe was measured in a plate-based assay. The pharmacokinetics and metabolic stability of the probes were studied in healthy rats. The capacity of these probes to detect and stage pulmonary fibrosis in vivo was assessed in a mouse model of bleomycin-induced fibrosis using PET imaging. Results: All probes exhibited affinities in the low micromolar range (1.6 μM < Kd < 14.6 μM) and had rapid blood clearance. The probes showed 2- to 8-fold-greater uptake in the lungs of bleomycin-treated mice than sham-treated mice, whereas the distribution in other organs was similar between bleomycin-treated and sham mice. The probe 64Cu-CBP7 showed the highest uptake in fibrotic lungs and the highest target-to-background ratios. The superiority of 64Cu-CBP7 was traced to a much higher metabolic stability compared with the other probes. The specificity of 64Cu-CBP7 for collagen was confirmed by comparison with a nonbinding isomer. Conclusion:64Cu-CBP7 is a promising candidate for in vivo imaging of pulmonary fibrosis.

Keywords: 64Cu; PET imaging; fibrosis; lung; type I collagen.

© 2017 by the Society of Nuclear Medicine and Molecular Imaging.

Figures

FIGURE 1.

(A) General protocol for synthesis of CBPs. (B) Structures of CBP1, CBP3, CBP5, CBP6, and CBP7. Bip = 4,4′-biphenylalanine; 2-Nal = 2-napthylalanine.

FIGURE 2.

(A) Metabolic stability of each probe estimated from HPLC analysis of serum samples in rat at 15 and 60 min after probe injection. (B) Mean whole lung PET activity values in sham and bleomycin-treated mice (2 wk after bleomycin instillation) for CBP1, CBP3, CBP5, CBP6, and CBP7 (100–120 min after injection). (C) Ex vivo lung uptake in sham and bleomycin-treated mice (2 wk after bleomycin instillation) for CBP1, CBP3, CBP5, CBP6, and CBP7 at 150 min after probe injection. ****P < 0.0001. BM = bleomycin.

FIGURE 3.

(A) Representative images of lung tissue stained with hematoxylin and eosin and picrosirius red (×10; scale bar, 100 μm) for sham mice and for bleomycin-treated mice (2 wk after bleomycin instillation). (B–E) Correlation between hydroxyproline content as measure of total lung collagen and %ID/lung in sham and bleomycin-treated mice, 2 wk after instillation of bleomycin or vehicle and 150 min after probe injection.

FIGURE 4.

(A) Representative fused PET/CT images (axial view) of sham and bleomycin-treated animals injected with CBP7 (top) and CBP11 (bottom). Color scale image shows PET image from integrated data 100–120 min after probe injection. (B) Mean lung PET activity values (%ID/cm3) in sham and bleomycin-treated mice for CBP7 and CBP11 from data 100 to 120 min after injection. (C) Ex vivo lung uptake in sham and bleomycin-treated mice at 150 min after CBP7 and CBP11 injection. BM = bleomycin.

FIGURE 5.

Biodistribution for CBP7 (n = 7/group) (A) and CBP11 (n = 4/group) (B) in sham and bleomycin-treated mice, 120 min after probe injection. BM = bleomycin.