In vitro study: binding of ^99mTc-DPD to synthetic amyloid fibrils

Abstract

This paper is an report of the investigation of the in vitro binding of 99mTc-DPD for synthetic amyloid fibrils used for the diagnosis of cardiac amyloidosis (CA), as compared with the use of 99mTc-HMDP and 99mTc-PPI. It also includes an inquiry into the role played by Ca2+ ions and serum proteins on binding to amyloid like materials, as well as the saturability and specificity of DPD for fibrils versus amorphous precipitates (AP). In the work, synthetic insulin fibrils (SIF) and AP were characterized by Congo red staining and TEM imaging. An equal amount of three radiopharmaceuticals were then added to fibrils in Ca2+ (0-4.2 mmol/L) or human serum (HS) adjoined samples and radiopharmaceutical uptake was assessed. To test the saturability of amyloid binding sites, a displacement assays with cold DPD was performed, while adding 50-1500 nmol of 99mTc-DPD to SIF or AP, saturation binding tests were subsequently carried out for evaluating its specificity for amyloid. Herein, synthetic fibrils and AP showed conformational differences at TEM and polarized microscopy analysis. In our study, 99mTc-DPD fibrils uptake was seen to be the highest and increased with calcium ions concentration. What is more, serum proteins reduced the bound fraction to the amyloid deposits of about 15%, and the Kd values of 90 nM and 114 nM relative to SIF and AP, respectively, did not significantly differ. We saw that 99mTc-DPD is the best seeker for amyloid fibrils in cardiac amyloidosis, and that Ca2+ concentration positively influenced DPD fibrils binding. Furthermore, the radioactivity bound to the serum protein clear up the idea of nuclide exchanging dynamic balance between amyloid and circulating proteins. Moreover, non-labeled DPD did not exert a competition for 99mTc-DPD binding sites, and, finally, DPD cannot be defined a radiopharmaceutical specific for amyloid deposits.

Bone tracers for transthyretin amyloid cardiomyopathy: are [99mTc]Tc-DPD and [99mTc]Tc-HMDP truly equivalent?

“…A study showed that [ 99m Tc]Tc-HMDP uptake is significantly correlated with histological amyloid load in endomyocardial biopsy [24]. Also, an in-vitro study showed a higher affinity of [ 99m Tc]Tc-DPD for amyloid fibrils than [ 99m Tc]Tc-HMDP or [ 99m Tc]Tc-PYP [25]. Wollenweber and al. suggest that [ 99m Tc]Tc-DPD scintigraphy is useful to detect patients with a higher risk for polyneuropathy associated to ATTR-CM [26].…”

A Narrative Review of 99mTc-Aprotinin in the Diagnosis of Cardiac Amyloidosis and a New Life for an Unfairly Abandoned Drug

“…However, the binding mechanism of labeled aprotinin is highly specific for fibrils and very different from that observed with 99m Tc-DPD and –PPi, which bind amorphous precipitates with the same kinetics of fibrils [ 38 ]. More recently, Thelander et al demonstrated that the binding of bone tracers to amyloid-containing hearts depends on an irregular presence of clouds of very tiny calcifications, which seem not to be directly associated with amyloid fibrils [ 39 ].…”