PMC4549186_01
The effect of tafamidis on an ATTRwt patient's plasma TTR tetramer stability. Plasma samples were obtained from an ATTRwt patient before treatment (0, blue), and after treatment with 20 (red), 40 (green), or 60 (purple) mg of once daily oral dose of tafamidis. (A) Plasma tafamidis concentration in the patient plasma samples was measured by HPLC. Each bar graph represents an average of three measurements, error bars represent standard deviation (SD). (B & C) Subunit exchange was quantified at 48 (B) and 96 (C) h, and the fraction exchange was calculated as described in Materials and Methods. (D) The rate of tetramer dissociation (kex) was calculated using the equation kex, t=t = -ln(1-FEt=t)/t and is expressed as the average of the rates obtained at 48 and 96 h subunit exchange incubation time. Each bar graph is an average of 4 or more individual technical replicates, error bars represent SD. Differences between groups were assessed by t-test (Prism 6). (E) Stability of plasma TTR was assessed as a function of plasma tafamidis concentrations. For the last two points on the graph, excess tafamidis (50 muM or 100 muM) was added ex vivo to the 60 mg dose plasma sample and subunit exchange was initiated with 1.8 muM FT2-WT TTR. (F) TTR tetramer concentrations in the patient plasma samples were assessed by measuring TTR A2 conjugate fluorescence at 0 h subunit exchange incubation time. Each bar graph is an average of 4 or more individual technical replicates, error bars represent SD. Differences between groups were assessed by t-test (Prism 6).
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F1
After 6 weeks of once daily dosing at 20, 40, and 60 mg of tafamidis, the tafamidis concentrations in the patient's blood were determined to be 6.6, 13.9, and 31.6 muM, respectively, based on a standard curve generated by adding increasing concentrations of tafamidis to plasma from a healthy donor (Figure 1A and S1A). As expected, the fraction of subunit exchange at 48 h (Figure 1B) was lower than the fraction of subunit exchange at 96 h (Figure 1C) without tafamidis treatment, and as a function of oral tafamidis dosing. The rate of subunit exchange was calculated from data at the 48 and 96 h time points separately and the averages of the two rates are graphed as a function of the daily dose of tafamidis (Figure 1D), as done previously. The kinetic stability of endogenous TTR in the patient's plasma (Figure 1D) at the 60 mg dose was significantly higher than at the 40 and 20 mg doses (P<0.0001). Furthermore, the measured kinetic stability of TTR tetramers exhibited a strong correlation with the plasma concentration of tafamidis (Figure 1E). This was demonstrated by the fact that ex vivo addition of 50 or 100 muM more tafamidis to the 60 mg tafamidis plasma sample did not significantly further decrease the rate of subunit exchange (increase kinetic stability; Figure 1E). The plasma concentration of normally folded TTR tetramers increased with increasing doses of oral tafamidis. At the 60 mg dose, the WT TTR tetramer concentration increased by 38% relative to the WT TTR tetramer plasma concentration before tafamidis treatment, as calculated from TTR A2 conjugate fluorescence peak intensity (Figure 1F and S1B).
A comparative plot detailing the effect of tafamidis on TTR tetramer stability of an ATTRwt patient's plasma with that of multiple SSA patients' plasma treated with 20 mg, once daily tafamidis. Patient X's kex values at a daily dose of 0 (blue), 20 (red), 40 (green), and 60 mg tafamidis (purple) are overlaid with previously reported data from five other tafamidis-treated SSA patients (grey filled circles) as a function of the measured blood concentration of tafamidis. The dotted line represents a linear regression fit of the 5 previously reported SSA patients' data.
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F2
TTR levels after tafamidis treatment, total TTR levels measured by quantitative Western blotting also increased in the tafamidis-treated samples (Figure S2). This is due to a pharmacologic chaperoning effect-tafamidis enters the endoplasmic reticulum of liver cells, binds to the WT TTR tetramer in the endoplasmic reticulum and shifts the linked folding and assembly process toward tetramer formation, thus enabling more WT TTR tetramer to be formed and secreted.