Radiochemical examination of transthyretin (TTR) brain penetration assisted by iododiflunisal, a TTR tetramer stabilizer and a new candidate drug for AD

Abstract

It is well settled that the amyloidogenic properties of the plasma protein transporter transthyretin (TTR) can be modulated by compounds that stabilize its native tetrameric conformation. TTR is also present in cerebrospinal fluid where it can bind to Aβ-peptides and prevent Aβ aggregation. We have previously shown that treatment of Alzheimer's Disease (AD) model mice with iododiflunisal (IDIF), a TTR tetramer stabilizing compound, prevents AD pathologies. This evidence positioned IDIF as a new lead drug for AD. In dissecting the mechanism of action of IDIF, we disclose here different labeling strategies for the preparation of ¹³¹I-labeled IDIF and ¹³¹I- and ¹²⁴I-labeled TTR, which have been further used for the preparation of IDIF-TTR complexes labeled either on the compound or the protein. The biodistribution of all labeled species after intravenous administration has been investigated in mice using ex vivo and in vivo techniques. Our results confirm the capacity of TTR to cross the blood brain barrier (BBB) and suggest that the formation of TTR-IDIF complexes enhances BBB permeability of both IDIF and TTR. The increased TTR and IDIF brain concentrations may result in higher Aβ-peptide sequestration capacity with the subsequent inhibition of AD symptoms as we have previously observed in mice.

Figure 1

(a) Amount of radioactivity in the different fractions after elution of [¹³¹I]IDIF (black dots) and [¹³¹I]IDIF-TTR complex (red dots) through the Illustra NAP-5 column; (b) Percentage of [¹³¹I]IDIF displaced at different concentrations of IDIF.

Figure 2

Accumulation of radioactivity in different organs and fluids obtained at different time points after intravenous administration of [¹³¹I]IDIF (black bars), TTR-[¹³¹I]IDIF (grey bars), [¹³¹I]TTR (red bars) and [¹³¹I]TTR-IDIF (pink bars). Values of percentage of injected dose (% ID) per gram of tissue are expressed as mean ± standard deviation, n = 3 per compound and time point.

Figure 3

(a) Autoradiography studies of brain slices collected at 15 min, 1 h and 6 h after intravenous administration of [¹³¹I]TTR at different Bregma values. The contour of the brain is delineated in all cases for clarity; (b) Picture of a representative slice corresponding to Bregma = −0.94 mm. The cortex (red), hippocampus (green), striatum (yellow) and third ventricles (dark blue) are delineated; three consecutive slices containing the regions mentioned above were analyzed per animal and time point, and the results are represented as concentration of radioactivity in each region, relative to the concentration in the whole brain slice; (c) Schematic representation of the mouse brain (sagittal view). The positions of the different Bregma values for which autoradiography images are shown in (a) are indicated with red lines.

Figure 4

PET-CT images obtained at different time points after intravenous administration of [¹²⁴I]TTR. Representative coronal slices have been co-registered with CT images of the same animal for better localization of the radioactive signal. On the right, major organs are schematically identified.