G. Mann, A. Kaul, M. Thirumal, A.K. Mishra, A. Datta
University of Delhi,
Keywords: traumatic brain injury, SPECT, amyloid plaques, chalcone
Summary:Homodimeric SPECT Scintigraphy Radioligand for Imaging Traumatic Brain Injury Induced Amyloid Plaques Garima Mann1,2, Ankur Kaul1, M Thirumal2, Anil K Mishra1, Anupama Datta1* Department of Cyclotron and Radiopharmaceutical Sciences, INMAS DRDO, Delhi-110054, India Department of Chemistry, University of Delhi, Delhi-110007, India Background: Epidemiological evidence implicates formation of diffused amyloid plaques after incident of Traumatic Brain Injury as a risk factor for developing associated neurodegenerative disorders. Early detection of these plaques consequently remains vital for better regulation and management of these diseases. Scintigraphy using an amyloid plaque specific radioligand is an effective non-invasive imaging technique for early detection of neuropathologies. Considering this, we developed a cost effective SPECT radiotracer which will specifically bind to detrimental Aβ1-42 plaques. Objectives: A homodimeric chalcone scaffold based ligand was developed. Chalcone has previously shown high binding affinity to amyloid plaques and can cross the blood brain barrier easily. Owing to multiple binding sites on β amyloid aggregates, multivalent ligand leads to thermodynamically more favourable binding. Additionally, attachment of dimethyl amino substituted group on para position has also displayed highest binding efficiency over other functionalities. The chelating agent belonging to the ‘pa’ family, specifically, pentapa-en-NH2 was explored for the development of SPECT agent. We hypothesise application of the developed bivalent homodimeric molecule to display enhanced binding affinity and BBB penetration. Methods: The bifunctional chelator was synthesised through a series of steps in high purity and further conjugated to the chalcone derivative of substituted benzaldehyde and acetophenone precursors. The molecule was conjugated to 99mTc and further used for pre-clinical studies. A repetitive mild -TBI mouse model was developed and uptake of tracer in sham and treated mice was studied. Result and Conclusions: The molecular docking studies reported multiple binding of the tracer at the recognition sites of Aβ fibrils with a significantly high binding score (-12.64) as compared to known Aβ ligands. Bischalcone derivative, 6,6'-(((2-(bis(2-(4-((E)-3-(4 (dimethylamino)phenyl)acryloyl)phenoxy)ethyl)amino)ethyl)azanediyl)bis(methylene))dipicolinic acid, pa(Ch)2 was synthesized in 95% yield with high purity. Radiolabelling was carried out with 99mTc under mild conditions with 95.4% efficiency and 103-210 MBq μmol−1 specific activity. In vitro binding assay with Aβ1-42 aggregates displayed high binding affinity of pa(Ch)2 with an inhibition constant of 3.98 ± 0.62 nM. The fluorescent properties of the ligand with peaks of absorption/emission at 410/540 nm exhibited a blue shift with a 10-fold increase in emission intensity on binding with Aβ aggregates. Blood kinetics of the radiocomplex performed on normal rabbit displayed fast clearance (t1/2(F) = 32 ± 0.04 min; t1/2(S) = 1h 55min ± 0.03 min). In vivo scintigraphy displayed almost two fold increase of radioligand concentration in TBI model as compared to sham mice. Ex vivo biodistribution analysis demonstrated high blood brain barrier penetration in TBI model mice with brain uptake of 3.24 ± 0.31% ID g−1 at 2 min p.i. and rapid washout with negligible activity (0.28% ID g−1) left at 30 min p.i. These preliminary studies reveal that the bivalent approach enhanced the amyloid binding affinity and offers a new perspective in 99mTc probes for β-amyloid imaging.