Radioiodinated Styrylbenzenes and Thioflavins as Probes for Amyloid Aggregates

Abstract

We report for the first time that small molecule-based radiodiodinated ligands, showing selective binding to Aβ aggregates, cross the intact blood−brain barrier by simple diffusion. Four novel ligands showing preferential labeling of amyloid aggregates of Aβ(1−40) and Aβ(1−42) peptides, commonly associated with plaques in the brain of people with Alzheimer's disease (AD), were developed. Two 125I-labeled styrylbenzenes, (E,E)-1-iodo-2,5-bis(3-hydroxycarbonyl-4-hydroxy)styrylbenzene, 12 (ISB), and (E,E)-1-iodo-2,5-bis(3-hydroxycarbonyl-4-methoxy)styrylbenzene, 13 (IMSB), and two 125I-labeled thioflavins, 2-[4‘-(dimethylamino)phenyl]-6-iodobenzothiazole, 18a (TZDM), and 2-[4‘-(4‘ ‘-methylpiperazin-1-yl)phenyl]-6-iodobenzothiazole, 18b (TZPI), were prepared at a high specific activity (2200 Ci/mmol). In vitro binding studies of these ligands showed excellent binding affinities with K d values of 0.08, 0.13, 0.06, and 0.13 nM for aggregates of Aβ(1−40) and 0.15, 0.73, 0.14, and 0.15 nM for aggregates of Aβ(1−42), respectively. Interestingly, under a competitive-binding assaying condition, different binding sites on Aβ(1−40) and Aβ(1−42) aggregates, which are mutually exclusive, were observed for styrylbenzenes and thioflavins. Autoradiography studies of postmortem brain sections of a patient with Down's syndrome known to contain primarily Aβ(1−42) aggregates in the brain showed that both [125I]18a and [125I]18b labeled these brain sections, but [125I]13, selective for Aβ(1−40) aggregates, exhibited very low labeling of the comparable brain section. Biodistribution studies in normal mice after an iv injection showed that [125I]18a and [125I]18b exhibited excellent brain uptake and retention, the levels of which were much higher than those of [125I]12 and [125I]13. These findings strongly suggest that the new radioiodinated ligands, [125I]12 (ISB), [125I]13 (IMSB), [125I]18a (TZDM), and [125I]18b (TZPI), may be useful as biomarkers for studying Aβ(1−40) as well as Aβ(1−42) aggregates of amyloidogenesis in AD patients.

A Novel Tool for Detecting Amyloid Deposits in Systemic Amyloidosis In Vitro and In Vivo

“…HPLC analysis (Wakosil II 5C18HG) with a 4:1 (v/v) mixture of acetonitrile and 10 mM phosphate buffer (pH 7.0) as an eluent showed 99% purity, as detected at 254 nm. IR (KBr): 3500, 1680 cm Ϫ1 , FAB-MS: m/z 481 (M ϩ 1), and 1 H-NMR (300 MHz, DMSO-d 6 ) spectrum data were in good agreement with results reported by Zhuang et al (2001aZhuang et al ( , 2001b. …”

“…Recently, the thioflavin T test has been used to evaluate the degree of amyloid formation in vitro (LeVine, 1993). However, we did not analyze the effect of BSB on amyloid formation in vitro as assessed by the thioflavin t test because that method examines the competitive inhibition between thioflavin T and BSB (Zhuang et al, 2001a(Zhuang et al, , 2001b, and we are not aware of the true effect of BSB on amyloid formation of the TTR molecule with this method. Therefore, we used electron microscopy for our analysis.…”

“…Chrysamine G, a Congo red derivative with a similar molecular structure used for the same purpose as Congo red, has the same limitations (Dezutter et al, 1999(Dezutter et al, , 2001aKlunk et al, 1995Klunk et al, , 1998. To overcome these problems, we must have a new tool for examination of amyloid deposition in tissues.(trans,trans)-1-bromo-2,5-bis-(3-hydroxycarbonyl-4-hydroxy)styrylbenzene (BSB), which has been found to bind to amyloid plaques in postmortem samples of brains from patients with Alzheimer's disease, is also a Congo red derivative and has been the focus of recent attention (Klunk et al, 1994;Schmidt et al, 2001;Skovronsky et al, 2000;Zhuang et al, 2001aZhuang et al, , 2001b. Because this compound is lipophilic, it can traverse the blood-brain barrier, and it binds to amyloid fibrils in the brain when it is injected intravenously into transgenic mice with Alzheimer's disease (Skovronsky et al, 2000).…”

Synthesis and Evaluation of Two 18F-Labeled 6-Iodo-2-(4‘-N,N-dimethylamino)phenylimidazo[1,2-a]pyridine Derivatives as Prospective Radioligands for β-Amyloid in Alzheimer's Disease

“…5 This is one of three binding sites reported in the literature for thioflavin-type ligand series. The other two are for a Congo red series 5 and an FDDNP series.…”

“…5 This is one of three binding sites reported in the literature for thioflavin-type ligand series. The other two are for a Congo red series 5 and an FDDNP series. 21 The K i values decrease and the experimentally determined log D values (log P at pH 7.4) increase with increasing length of the side chain on amino group (Table 1).…”