Introduction: Transthyretin amyloid cardiomyopathy (ATTR-CM) caused by deposition of toxic TTR amyloid aggregates in myocardium is an uncommon cause of heart failure. Pathologic TTR variants such as V122I form less stable tetramers than wild-type TTR, and increase the risk and severity of ATTR-CM. The beneficial mutation T119M forms more stable tetramers than wild-type and prevents ATTR amyloidosis.
Hypothesis: A small molecule TTR stabilizer with binding characteristics mimicking the T119M mutation will stabilize TTR tetramer to a high degree in vitro and in vivo.
Methods: AG10 occupies both T4 binding sites of TTR with high affinity (Kd1 = 4.8nM, Kd2 = 314nM). A fluorescence probe exclusion (FPE) assay was used to assess efficacy and selectivity of AG10 binding in serum and after oral dosing of AG10 with parallel measures of circulating plasma levels in dogs. Immunoblots of serum samples pre-incubated under denaturing conditions measured AG10’s ability to stabilize tetrameric TTR. Dosing of AG10 in mice, rats and dogs assessed bioavailability. Bioactivity in circulation, as measured ex vivo by FPE, was quantified after single and 7 day repeat oral dosing in dogs.
Results: Due to a lack of informative animal models of ATTR-CM, FPE and immunoblot assays were used to test efficacy of AG10 in stabilizing TTR. There was a significant correlation between AG10’s T4 binding site occupancy and tetrameric TTR stability. High oral bioavailability (%F) with sustained plasma exposures, low systemic clearance (Cl) and volume of distribution (Vss) were seen in mouse, rat and dog (TABLE). After oral dosing in dogs, ex vivo assays correlated plasma concentration, T4 binding site occupancy, and TTR tetramer stabilization (FIGURE).
Conclusion: In vitro studies, multispecies oral bioavailability and dog studies of AG10 reveal AG10’s unique and potent mode of TTR binding, mimicking the T119M mutation. These data support further investigation of AG10 as a potential treatment for ATTR-CM.