Quantification of Transthyretin Kinetic Stability in Human Plasma Using Subunit Exchange

Abstract

The transthyretin (TTR) amyloidoses are a group of degenerative diseases caused by TTR aggregation, requiring rate-limiting tetramer dissociation. Kinetic stabilization of TTR, by preferential binding of a drug to the native tetramer over the dissociative transition state, dramatically slows the progression of familial amyloid polyneuropathy. An established method for quantifying the kinetic stability of recombinant TTR tetramers in buffer is subunit exchange, in which tagged TTR homotetramers are added to untagged homotetramers at equal concentrations to measure the rate at which the subunits exchange. Herein, we report a subunit exchange method for quantifying the kinetic stability of endogenous TTR in human plasma. The subunit exchange reaction is initiated by the addition of a substoichiometric quantity of FLAG-tagged TTR homotetramers to endogenous TTR in plasma. Aliquots of the subunit exchange reaction, taken as a function of time, are then added to an excess of a fluorogenic small molecule, which immediately arrests further subunit exchange. After binding, the small molecule reacts with the TTR tetramers, rendering them fluorescent and detectable in human plasma after subsequent ion exchange chromatography. The ability to report on the extent of TTR kinetic stabilization resulting from treatment with oral tafamidis is important, especially for selection of the appropriate dose for patients carrying rare mutations. This method could also serve as a surrogate biomarker for the prediction of the clinical outcome. Subunit exchange was used to quantify the stabilization of WT TTR from senile systemic amyloidosis patients currently being treated with tafamidis (20 mg orally, once daily). TTR kinetic stability correlated with the tafamidis plasma concentration.

Personalized medicine approach for optimizing the dose of tafamidis to potentially ameliorate wild-type transthyretin amyloidosis (cardiomyopathy)

“…The kinetic stability of endogenous TTR in patient plasma was assessed using the TTR subunit exchange method [25]. We measured the subunit exchange rate of WT TTR in the patient plasma samples before and after each 6-week treatment with the increasing oral doses of tafamidis.…”

“…Tafamidis levels in the tafamidis-treated patient plasma samples were quantified by HPLC following the previously described method [25]. For each experiment, a standard curve was generated by adding tafamidis (in DMSO; final concentration ranging from 0–36 μM) to a healthy donor plasma sample.…”

“…Subunit exchange assays were carried out as previously described [25] with the following minor modifications: (i) in order to reduce the sudden pressure jump during sample injection, and to achieve a complete separation of TTR from other plasma proteins, the first 6 min of the TTR separation on UPLC was done at a low flow rate (0.2 mL/min), and then the flow rate was increased to 0.6 mL/min for the rest of the run (total run time = 39 min); and (ii) 1 μL of a 3 mM A2 stock solution (final concentration of 300 μM) was used instead of 3 μL of a 130 μM solution (final concentration was 90 μM) to minimize the effect of DMSO and to account for high tafamidis concentrations. For analysis of TTR kinetic stability in plasma, a frozen ATTRwt plasma sample aliquot (200 μL) was thawed on ice.…”

“…The significance of these observations will only be clear after the placebo-controlled clinical trial being enrolled by Pfizer is finished (ClinicalTrials.gov Identifier: NCT01994889). In a recent study on 6 of these ATTRwt (cardiomyopathy) patients continuing to take tafamidis, the plasma tafamidis concentration and the extent of TTR kinetic stabilization varied 4-fold between patients receiving a 20 mg once daily dose of tafamidis [25]. While the number of patients in this study is small, the variability in plasma concentration and kinetic stabilization using a single 20 mg dose of tafamidis supports the development of an approach for determining the optimal dose of tafamidis for each cardiomyopathy patient [25].…”

“…In a recent study on 6 of these ATTRwt (cardiomyopathy) patients continuing to take tafamidis, the plasma tafamidis concentration and the extent of TTR kinetic stabilization varied 4-fold between patients receiving a 20 mg once daily dose of tafamidis [25]. While the number of patients in this study is small, the variability in plasma concentration and kinetic stabilization using a single 20 mg dose of tafamidis supports the development of an approach for determining the optimal dose of tafamidis for each cardiomyopathy patient [25]. Herein, we study a single male ATTRwt patient before treatment and 6 weeks after daily doses of 20 or 40 or 60 mg of tafamidis, in a dose escalation paradigm.…”