Orally administered diflunisal stabilizes transthyretin against dissociation required for amyloidogenesis

Abstract

Objective: Rate-limiting transthyretin (TTR) tetramer dissociation and monomer misfolding enable misassembly into numerous aggregate morphologies including amyloid, a process genetically linked to and thought to cause amyloid pathology. T119M TTR trans-suppressor subunit inclusion into tetramers otherwise composed of disease-associated subunits ameliorates human amyloidosis by increasing the tetramer dissociation barrier. Diflunisal binding to the 99% unoccupied L-thyroxine binding sites in TTR also increases the tetramer dissociation barrier; hence, we investigated the feasibility of using diflunisal for the treatment of human TTR amyloidosis using healthy volunteers.

Methods: Diflunisal (125, 250 or 500 mg bid) was orally administered to groups of 10 subjects for 7 days to evaluate serum diflunisal concentration, diflunisal binding stoichiometry to TTR, and the extent of diflunisal imposed TTR kinetic stabilization against urea- and acid-mediated TTR denaturation in human serum. The rates of urea-mediated tetramer dissociation and acid-mediated aggregation as a function of diflunisal concentration were also evaluated in vitro, utilizing physiologically relevant concentrations identified by the above experiments.

Results: In the 250 mg bid group, 12 h after the 13th oral dose, the diflunisal serum concentration of 146 +/- 39 microM was sufficient to afford a TTR binding stoichiometry exceeding 0.95 +/- 0.13 ( approximately 1.75 corrected). Diflunisal binding to TTR at this dose slowed urea-mediated dissociation and acid-mediated TTR aggregation at least, threefold (p < 0.05) in serum and in vitro, consistent with kinetic stabilization of TTR.

Conclusion: Diflunisal-mediated kinetic stabilization of TTR should ameliorate TTR amyloidoses, provided that the nonsteroidal anti-inflammatory drug liabilities can be managed clinically.