Pharmacodynamic End Points

PD properties of AG10 were assessed ex vivo in serum or plasma samples obtained before and after administration of AG10 by 3 established assays of TTR stabilization: fluorescent probe exclusion assay (FPE), western blot, and measurement of circulating TTR concentration. FPE is a competitive binding assay measuring occupancy of TTR's T4 binding site; the western blot is a measure of a bound ligand's ability to prevent the accelerated dissociation of tetrameric TTR under denaturing conditions. Serum TTR (prealbumin) concentration reflects an individual balance between overall nutritional status and the intrinsic stability of the TTR tetramer, and thus at a given nutritional state reflects the pharmacological effect of a TTR stabilizer in vivo.

The FPE assay was performed using serum from timed blood samples according to an established method.13 Briefly, the probe is a small molecule that becomes fluorescent only when covalently bound to the T4 binding site of TTR. The time‐dependent development of the fluorescence signal is reduced in direct proportion to the percentage of occupancy of the T4 binding site by a competing ligand. Relative fluorescence units at t = 60 minutes were normalized to t = 60 minutes relative fluorescence units from the predose serum sample from each individual subject to determine the percentage of target occupancy by the competing ligand AG10.

The western blot assay was performed on plasma from timed blood draw samples as previously described.14 Briefly, plasma samples were acidified to pH 3.8 and incubated for 72 hours prior to crosslinking with glutaraldehyde. “Time zero” replicates from each plasma sample were similarly acidified immediately prior to crosslinking. All replicates were subjected to denaturing gel electrophoresis and immunoblotting using antihuman TTR polyclonal antibody (DAKO‐A0002, DAKO, Carpinteria, California). Immunoblots were quantified using a ChemiDoc MP Imaging System (Biorad Laboratories, Richmond, California). Band intensities for tetrameric TTR (either TTR alone or retinol binding protein–bound TTR) were compared between zero‐ and 72‐hour denaturation for each sample to determine the percentage of stabilization.

Serum TTR (prealbumin) concentrations were determined using a qualified enzyme‐linked immunosorbent assay (ELISA) according to the manufacturer's instructions (Prealbumin ELISA kit [human]; Aviva Systems Biology, San Diego, California). Because of differences in methodology compared to a widely available clinical prealbumin assay, the results are not directly comparable to results generated by the clinical assay and should be considered qualitative in nature.