Abstract
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Objectives: Adrenocortical cells and macrophages express acyl-coenzyme A:cholesterol acyltransferase 1 (ACAT1) to convert free cholesterol into cholesterol esters for storage. This process protects the cell from free cholesterol-associated toxicity.1 Persistent cholesterol esterification by ACAT1 leads to cholesterol ester accumulation, the formation of macrophage foam cells and consequently, atherosclerotic plaques.1 PET imaging of ACAT1 would enable the detection of foam cells and atherosclerotic lesions as unstable atherosclerotic plaques should have the highest concentration of macrophages expressing ACAT1. A selective ACAT1 inhibitor, ATR-101,2 was originally intended to be a cholesterol lowering drug. ATR-101 has since been repurposed as a treatment for congenital adrenal hyperplasia and Cushing’s syndrome by Millendo.3 The binding to ACAT1 expression in adrenocortical cells and macrophages is demonstrated in the clinic; therefore, the development of a radiolabeled analogue of ATR-101 would provide imaging of ACAT1 expression in adrenal indications as well as atherosclerosis.
Methods: ATR-101 and its desmethyl precursor were prepared. In addition, a known fluoro-analogue of ATR-101 developed by Parke-Davis (with the same affinity for ACAT1), and its aryl stannane precursor were prepared (ATR-F). Radiolabelling of ATR-101 was accomplished via 11C-methylation of the desmethyl precursor. Copper-mediated fluorination was used to prepare ATR-F.4 Biodistribution studies were conducted in female Sprague Dawley rats to determine adrenal uptake as a measure of ACAT1 binding. In addition, imaging of C57BL6 and ApoE-/- mice was conducted to determine the ability to image foam cells of the aortic arch and carotid arteries.
Results: [11C]ATR-101 was synthesized from the desmethyl precursor via loop chemistry with [11C]MeOTf. Subsequent HPLC purification and formulation yielded 464±92 MBq of [11C]ATR-101 (RCY, decay-corrected from [11C]MeI = 2.7±1%, molar activity = 197±95 GBq/µmol, synthesis time = 34 ± 1 minutes, n = 4). [18F]ATR-F was prepared, purified in formulated to give 2.41 GBq (RCY, decay corrected = 7.6±3.0%, molar activity = 188±138 GBq/µmol, synthesis time = 120 min). Uptake of the radioligands was highest in the adrenal glands (2.3 %ID/g at 10 min for both) as expected (Fig 1). Dynamic 1 hr PET studies were conducted after administering C57BL6 and ApoE-/- mice with [11C]ATR-101. The ApoE-/- mice show high tracer uptake in the aortic arch and carotid artery, where foam cells are known to accumulate (Fig 1). Conclusion: ACAT1 inhibitors [11C]ATR-101 and [18F]ATR-F were synthesized. Biodistribution studies showed good adrenal uptake for both agents. [11C]ATR-101 when administered to (healthy) C57BL6 and (atherosclerotic) ApoE-/- mice could easily distinguish the diseased animal from the healthy control. Future studies will use PET/CT for additional structural information, and [18F]ATR-F will be used in longer imaging studies. References: (1) Yu, X.-H.; Fu, Y.-C.; Zhang, D.-W.; Yin, K.; Tang, C.-K. Foam Cells in Atherosclerosis. Clin. Chim. Acta 2013, 424, 245-252.(2) Trivedi, B. K.; Purchase, T. S.; Holmes, A.; Augelli-szafran, C. E.; Essenburg, A. D.; Hamelehle, K. L.; Stanfield, R. L.; Bousley, R. F.; Krauset, B. R. Inhibitors of Acyl-CoA:Cholesterol Acyltransferase (ACAT). 7. Development of a Series of’ Substituted N-Phenyl-"-[ (l-Phenylcyclopentyl)Methyl]Ureas with Enhanced Hypocholesterolemic Activity1. 1994, 51 (Scheme 2), 1652-1659.(3) Nevanimibe selectively targets adrenal steroidogenesis to treat serious orphan diseases https://millendo.com/pipeline/nevanimibe/ (accessed Nov 15, 2019).(4) Makaravage, K. J.; Brooks, A. F.; Mossine, A. V; Sanford, M. S.; Scott, P. J. H. Copper-Mediated Radiofluorination of Arylstannanes with [18F]KF. Org. Lett. 2016, 18 (20), 5440-5443. Acknowledgements: We thank Jenelle Stauff, Janna Arteaga, and Phillip Sherman for conducting animal imaging studies