Styrylbenzoxazole derivatives for in vivo imaging of amyloid plaques in the brain

Abstract

Progressive deposition of senile plaques (SPs) is one of the major neuropathological features of Alzheimer's disease (AD) that precedes cognitive decline. Noninvasive detection of SPs could, therefore, be a potential diagnostic test for early detection of AD patients. For imaging SPs in the living brain, we have developed a series of styrylbenzoxazole derivatives that achieve high binding affinity for amyloid-beta (Abeta) fibrils. One of these compounds, 6-(2-Fluoroethoxy)-2-[2-(4-methylaminophenil) ethenyl]benzoxazole (BF-168), selectively binds SPs in AD brain sections and recognizes Abeta1-42-positive diffuse plaques as well as neuritic plaques in AD brain sections. Intravenous injection of BF-168 in PS1/APP and APP23 transgenic mice resulted in specific in vivo labeling to both compact and diffuse amyloid deposits in the brain. In addition, (18)F-radiolabeled BF-168 demonstrated abundant initial brain uptake (3.9% injected dose/gm at 2 min after injection) and fast clearance (t(1/2) = 24.7 min) after intravenous administration in normal mice. Furthermore, autoradiograms of brain sections from APP23 transgenic mice at 180 min after intravenous injection of [(18)F]BF-168 showed selective labeling of brain amyloid deposits with little nonspecific binding. These findings strongly suggest that styrylbenzoxazole derivatives are promising candidate probes for positron emission tomography and single-photon emission computed tomography imaging for early detection of amyloid plaque formation.

Figure 1.

Chemical structure and radiolabeling of BF-168 and BF-180.

Figure 2.

Scatchard plots of [¹²⁵I]BF-180 binding to aggregates of Aβ1-40 and Aβ1-42. A K_d value of 6.8 nm (Aβ1-40) and 10.6 nm (Aβ1-42) indicate high binding affinity for Aβ aggregates.

Figure 3.

Neuropathological staining of 6 μm AD brain sections from the temporal cortex (A-G, M, N) and hippocampus (H-L) and an aged normal temporal brain section (O). A-D are frozen sections, and E-O are paraffin-embedded sections. Many neuritic plaques are clearly stained with BF-168 (A). Intense fluorescence can be seen in the core of neuritic plaques. Aβ immunostaining with antibodies 6F/3D (B) in the adjacent section show an identical staining pattern of plaques. The brain section stained with BF-168 (C) and the adjacent section immunostained with a mAb against Aβ (D) demonstrate specific binding of BF-168 to diffuse amyloid plaques. Aβ1-40 (E) and Aβ1-42 (G) immunostaining and BF-168 staining (F) in serial sections reveal specific binding of BF-168 to not only Aβ1-40-positive plaques (arrows) but also Aβ1-42-positive diffuse plaques (arrowheads). H, BF-168-stained NFTs, and this staining correlated well with tau immunostaining (I) in the adjacent section. Clear staining of cerebrovascular amyloid (J) can also be observed. There is no obvious difference in the BF-168 staining ability in ethanol solution (K) and in the PBS solution (L) in the serial sections. BF-168 could not stain any plaques and tangles after the treatment with formic acid (N), in contrast to the staining without pretreatment with formic acid (M). Finally, no apparent staining was observed in aged normal brain sections (O). Scale bars: A-D, 50 μm; E-O, 100 μm.

Figure 4.

Brain sections from a PS1/APPsw transgenic mouse (A, C, D) and a wild-type mouse (B) after intravenous administration of 4 mg/kg BF-168, showing specific binding of BF-168 to amyloid deposits (A, C) and no nonspecific binding in the brain of the wild-type mouse (B). The same brain section from the transgenic mouse was subsequently immunostained with Aβ-specific mAb 6F/3D (D), revealing specific binding of BF-168 to amyloid deposits in vivo. Both compact amyloid deposits (arrows) and diffuse deposits (arrowhead) were labeled with intravenously administered BF-168. Scale bar, 50 μm.

Figure 5.

Brain sections from an APP23 transgenic mouse (13 months of age) after intravenous administration of 4 mg/kg BF-168, showing specific binding of BF-168 to early amyloid deposits in the entorhinal cortex and hippocampus (A). The fluorescence of BF-168 in the entorhinal cortex (B) showed identical distribution to immunostaining with Aβ-specific mAb 6F/3D (C) in the same section. Scale bar, 100 μm.

Figure 6.

Brain uptake and brain/blood ratio of [¹⁸F]BF-168 in normal mice. Each data point was obtained from three or four separate experiments and plotted as value of mean ± SD.

Figure 7.

In vivo labeling of amyloid deposits in brain sections from an APP23 transgenic mouse (A) and a wild-type mouse (B) with [¹⁸F]BF-168. An autoradiographic study was performed using brain sections at 180 min after intravenous injection of [¹⁸F]BF-168. Numerous hot spots of [¹⁸F]BF-168 can be detected in the cerebral cortex, hippocampus, and entorhinal cortex of transgenic mouse brain (A), in contrast to no apparent uptake in wild-type mouse brain (B). Fluorescences of amyloid deposits appeared after in vitro staining with thioflavin-S in the same section (C). Arrows show amyloid deposits labeled with both [¹⁸F]BF-168 and thioflavin-S, indicating specific labeling of amyloid plaques with intravenously administered [¹⁸F]BF-168. The percentage of areas of [¹⁸F]BF-168 uptake in nine autoradiographic images were significantly correlated with the percentage of areas of in vitro thioflavin-S staining in the same sections (r = 0.923; p < 0.001) (D), suggesting high binding specificity of [¹⁸F]BF-168 to amyloid plaques.