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Allysine-targeting gadolinium-based MRI probes for imaging lung fibrogenesis1Radiology, A. A. Martinos Center for Biomedical Imaging and the Institute for Innovation in Imaging, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, United States, 2Department of Medicine: Pulmonary, Allergy, Sleep and Critical Care Medicine, Boston University School of Medicine, Boston, MA, United States
Synopsis
There is an enormous unmet medical need for non-invasive methods to assess lung fibrosis that can distinguish between active disease and stable scar. Here, we report on a gadolinium-containing contrast agent Gd-CHyd that targets fibrogenesis (active fibrosis) by undergoing a condensation reaction with allysine, an abundant aldehyde motif on fibrosing tissue. This probe reacts with aldehydes at a rate that is an order of magnitude faster than the parent probe Gd-Hyd. This faster reactivity leads to higher accumulation of the probe in the lungs of bleomycin treated mice (mouse model of lung fibrosis) and two-fold contrast enhancement relative to Gd-Hyd.
Introduction
Fibrosis results in the formation of scar tissue following chronic tissue injury and fibrotic diseases account for half of the deaths in the industrialized world.1 Lung fibrosis remains a major cause of mortality and idiopathic pulmonary fibrosis (IPF, the most common fibrosing lung disease) is responsible for 40,000 deaths per year in the U.S.2 Invasive biopsy remains the current gold standard for diagnosis of early stages of fibrosis and non-invasive methods that can distinguish between active disease and stable scar remain largely unavailable. Development of molecular imaging agents that can non-invasively target fibrogenesis (active fibrosis) and distinguish between active disease and stable scar tissue is crucial for monitoring disease progression and response to therapeutics. We demonstrated that the small-molecule, gadolinium-containing MR probe Gd-Hyd binds reversibly to allysine and enables detection of pulmonary fibrosis in bleomycin-injury mouse model.3 Gd-Hyd undergoes a condensation reaction with allysine residues, which are abundant in actively fibrosing tissue. Here we sought to modify the hydrazine targeting moiety to enhance the condensation reaction rate,4 in order to increase probe uptake in fibrogenic lung.Methods
Synthesis: Gd-Hyd was synthesized as previously reported.3 Gd-CHyd was synthesized by alkylation of benzylcarbazate with bis(2-chloroethyl)amine hydrochloride followed by amide coupling to t-butyl protected DOTAGA. Acid deprotection followed by gadolinium complexation gave the product. In vitro analyses: Relaxivity for Gd-Hyd and Gd-CHyd were measured at 1.41 T and 37 °C. Reaction kinetics of Gd-Hyd and Gd-CHyd was measured by monitoring the absorbance change when challenged with an excess of 2-formylpyridine. Binding affinity to allysine-rich aorta was determine by measuring the accumulation of the probes (by ICP-MS) in the tissue. Animal model: Mice were treated intratracheally with bleomycin, (BM, 1 U/kg) or PBS (Sham) and imaged 14 days later. In vivo MRI: Mice were imaged at 4.7T. A 2D T1-weighted RARE acquisition provided pulmonary anatomy. 3D T1-weighted UTE and 3D T1-weighted FLASH imaging was performed prior to and following i.v. injection of probe (100 nmol/g). The FLASH image was performed immediately after injection and was repeated 6 times to measure blood clearance of the probe, estimated from an exponential fit of signal decay in the vena cava with time. The UTE image was the repeated 16 min after probe injection. Contrast to noise ratios (CNR) were calculated from regions of interest in the lung relative to adjacent skeletal muscle. Mice were first scanned with Gd-Hyd and 24 hours later scanned with Gd-CHyd. Ex vivo analyses: Lung tissue was collected 75 minutes post probe injection and analyzed for gadolinium uptake via ICP-MSResults
Gd-CHyd, which features an alkyl hydrazine, reacts with aldehydes 11 times faster than the parent probe Gd-Hyd, which bears an acyl hydrazide. Gd-Hyd and Gd-CHyd bind similarly to allysine-rich porcine aorta with Kd values for Gd-Hyd and Gd-CHyd of 579 ± 67 μM and 456 ± 56 μM, respectively. The relaxivities of both compounds were also similar at 4.07 mM-1s-1 and 3.80 mM-1s-1 respectively. In mice, the blood clearance of both probes was very fast and similar. We then directly compared the ability of Gd-Hyd and Gd-CHyd to detect pulmonary fibrogenesis. We found that Gd-CHyd resulted in a ~2-fold enhancement in the change of contrast to noise ratio (ΔCNR, post Gd – pre Gd) compared to Gd-Hyd, indicating higher on-target accumulation. Ex vivo analyses of the gadolinium uptake in the lungs revealed 6 fold higher uptake in the lungs of BM injured mice compared to sham animals, demonstrating the specificity of the probe for actively fibrosing lung tissue.Conclusions
Collectively our data indicate that the fast rate of reactivity of the allysine-targeting MRI contrast agent Gd-CHyd results in a more effective probe for imaging of fibrosing lungs. Gd-CHyd an improved analogue of Gd-Hyd capable of imaging active lung fibrogenesis with superior sensitivity.Acknowledgements
No acknowledgement found.References
1. Urban, M. L.; Manenti, L.; Vaglio, A., Fibrosis--A Common Pathway to Organ Injury and Failure. N. Engl. J. Med. 2015, 373 (1), 95-6.
2. Olson, A. L.; Swigris, J. J.; Lezotte, D. C.; Norris, J. M.; Wilson, C. G.; Brown, K. K., Mortality from pulmonary fibrosis increased in the United States from 1992 to 2003. Am. J. Respir. Crit. Care Med. 2007, 176 (3), 277-84.
3. Chen, H. H.; Waghorn, P. A.; Wei, L.; Tapias, L. F.; Schu Hle, D. T.; Rotile, N. J.; Jones, C. M.; Looby, R. J.; Zhao, G.; Elliott, J. M.; Probst, C. K.; Mino-Kenudson, M.; Lauwers, G. Y.; Tager, A. M.; Tanabe, K. K.; Lanuti, M.; Fuchs, B. C.; Caravan, P., Molecular imaging of oxidized collagen quantifies pulmonary and hepatic fibrogenesis. JCI Insight 2017, 2 (11).
4. Kool, E.; Crisalli, P.; Chan, K., Fast Alpha Nucleophiles: Structures that Undergo Rapid Hydrazone/Oxime Formation at Neutral pH. Org. Lett. 2014, 16 (5), 1454-1457.
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