Evaluation of Bone-Seeking Agent Reactivity in AA mice and with Human ATTR Amyloid Extracts

Objectives The goal of this preliminary study was to examine the reactivity of clinically available ^99mTc-HDP, MDP, and PYP bone seeking radiopharmaceuticals with human ATTR and AL amyloid extracts in vitro and with a well characterized murine model of inflammation-associate (AA) amyloidosis in vivo..

Methods Each radiopharmaceutical was assayed in a solution-phase assay. Briefly, ~50 µg of human ATTR or AL extract was incubated with the radiotracer tracer for 1 h at room temperature. The unbound tracer was removed by centrifugation. The bound and free tracer was measured by using a gamma counter and the data expressed as “% bound”. Three AA and three WT mice were administered IV ~200 µCi each of ^99mTc-HDP, MDP, or PYP in the lateral tail vein. After a 1.5 h uptake period for MDP and HDP and ~15 min uptake for PYP the mice were imaged postmortem by SPECT/CT. After imaging a necropsy was performed on the heart, liver, spleen, kidney, pancreas, upper intestines and muscle harvested. Each organ was measured for tissue radioactivity (% injected dose/gram) by gamma counting. A second piece of tissue was fixed and stained with Congo red.

Results In microSPECT imaging studies we observed accumulation of radiotracer in the heart of both AA-laden mice as well as the healthy control mice, in equivalent amounts. Using the in vitro “pull down” assay each radiotracer bound human cardiac amyloid extracts at a level comparable with background levels with the highest recorded binding to ALκ extract, at 8.87%.

Conclusions The low binding in the pull down assays and the insignificant uptake in the mouse hearts we conclude that the ligand recognized by the bone-seeking agents likely resides in the patient cardiac tissue and not associated with the amyloid material itself. Murine cardiac AA amyloid deposits lack the ligand that resides in human patient cardiac tissue. More studies will be necessary to evaluate the mechanism underlying uptake of bone seeking agents in patients with cardiac ATTR.