The positron emission tomography ligand DAA1106 binds with high affinity to activated microglia in human neurological disorders

Abstract

Chronic microglial activation is an important component of many neurological disorders, and imaging activated microglia in vivo will enable the detection and improved treatment of neuroinflammation. 1-(2-chlorphenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinoline-carbox-amide (PK11195), a peripheral benzodiazepine receptor ligand, has been used to image neuroinflammation, but the extent to which PK11195 binding distinguishes activated microglia and reactive astrocytes is unclear. Moreover, PK11195 may lack sufficient sensitivity for detecting mild neuroinflammation. We hypothesized that N-(2,5-dimethoxybenzyl)-N-(4-fluoro-2-phenoxyphenyl) acetamide (DAA1106), a new ligand that binds specifically to peripheral benzodiazepine receptor, binds to activated microglia in human neurological diseases with higher affinity than does PK11195. We therefore compared the pharmacological binding properties of [3H](R)-PK11195 and [3H]DAA1106 in postmortem tissues from patients with cerebral infarcts, amyotrophic lateral sclerosis, Alzheimer disease, frontotemporal dementia, and multiple sclerosis (n=10 each). In all diseases, [3H]DAA1106 showed a higher binding affinity as reflected by lower dissociation constant (KD) values than that of [3H](R)-PK11195. Moreover, specific binding of both ligands correlated with the presence of activated microglia identified by immunohistochemistry in situ. We conclude that 1) ligands that bind peripheral benzodiazepine receptor mainly label activated microglia in human neurological disorders and that 2) DAA1106 may possess binding characteristics superior to those of PK11195, which may be beneficial for in vivo positron emission tomography imaging.

FIGURE 1

Microglial activation and reactive astrocytosis in various CNS disorders. Activated microglia (CD68, green) and reactive astrocytosis (GFAP, green) were assessed in various CNS diseases. Samples included the core of an acute cerebral infarct core (A), an active multiple sclerosis plaque (B), frontal cortex in a case of frontotemporal dementia (C), primary motor cortex in a case of amyotrophic lateral sclerosis (D), and frontal cortex in a case of Alzheimer disease (E). Individual (left and middle panels) and merged images (right panels) are shown. Scale bar = 50 μm.

FIGURE 2

Assessment of microglial activation and reactive astrocytosis in human CNS disorders. Activated microglia and reactive astrocytosis were assessed as described in the Materials and Methods section in brain tissue samples from patients diagnosed postmortem with acute cerebral infarcts (Infarct, core of infarct), multiple sclerosis (MS, active plaques), frontotemporal dementia (FTD, frontal cortex), amyotrophic lateral sclerosis (ALS, primary motor cortex), and Alzheimer disease (AD, frontal cortex) and compared with those in controls (Con). Both microglial activation (A) and reactive astrocytosis (B) were higher in patients with the disease conditions assessed compared with those in controls. Data were analyzed using analysis of variance (***, p < 0.0001; **, p < 0.001; *, p < 0.05).

FIGURE 3

Quantitative film autoradiography with [³H](R)-1-(2-Chlorphenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinoline-carbox-amide (PK11195) and [³H]N-(2,5-dimethoxybenzyl)-N-(4-fluoro-2-phenoxyphenyl) acetamide (DAA1106). Quantitative film autoradiography with tritium microscales was performed on frozen brain tissue samples from cases of acute cerebral infarct, multiple sclerosis (MS) plaques, frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS), and Alzheimer disease (AD) and compared with that in controls (Con), as described in the Materials and Methods section.(A) Representative images from ALS brain tissue samples incubated with [³H]DAA1106 and [³H](R)-PK11195 (left images). Sections were incubated with DAA1106 and PK11195 in parallel (right images) to assess nonspecific binding. Scale bar = 1 cm. (B) Quantitative film autoradiography was assessed in all the above conditions with [³H](R)-PK11195 (open bars) and [³H]DAA1106 (black bars). Data were analyzed using Student t-test (*, p < 0.05).

FIGURE 4

Saturation binding curves and representative Scatchard plots with [³H](R)-1-(2-Chlorphenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinoline-carbox-amide (PK11195) and [³H]N-(2,5-dimethoxybenzyl)-N-(4-fluoro-2-phenoxyphenyl) acetamide (DAA1106). Saturation binding curves with [³H]DAA1106 (A, B) and [³H](R)-PK11195 (C, D) (insets show representative Scatchard plots with x intercepts representing B_max values and slopes representing K_D values) in brain tissues derived from patients diagnosed postmortem with amyotrophic lateral sclerosis (ALS, black squares), acute cerebral infarct core (Infarct, gray squares), frontotemporal dementia (FTD) cortex (clear squares), multiple sclerosis (MS) active plaque (black circles), Alzheimer disease (AD, gray circles), and controls (Con, clear circles).

FIGURE 5

[³H](R)-1-(2-Chlorphenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinoline-carbox-amide (PK11195) and [³H]N-(2,5-dimethoxybenzyl)-N-(4-fluoro-2-phenoxyphenyl) acetamide (DAA1106) show higher B_max values compared with those in controls (Con), with [³H]DAA1106 binding with higher affinity in human neurological disorders. Saturation filtration binding experiments using [³H](R)-PK11195 and [³H]DAA1106 were performed on acute cerebral infarct core, multiple sclerosis (MS) active plaque, frontotemporal dementia (FTD) frontal cortex, amyotrophic lateral sclerosis (ALS) primary motor cortex, and Alzheimer disease (AD) frontal cortex samples and compared with those performed on the Con samples. (A) The B_max values (in femtomoles per milligram), which reflect the total number of binding sites, were significantly higher in patients with the disease conditions assessed compared with those in Con with both [³H](R)-PK11195 (clear bars) and [³H]DAA1106 (black bars). B_max values with [3H]DAA1106 and [³H](R)-PK11195 did not differ significantly within the same disease condition. (B) The K_D values (in nanomoles per liter) of [³H]DAA1106 were significantly lower than those of [³H]-(R)-PK11195 in each disease condition, indicating higher ligand binding affinities. The K_D values did not differ from the Con with either [³H]DAA1106 or [³H]-(R)-PK11195. Data were analyzed using Student t-test (***, p < 0.0001; **, p < 0.001; *, p < 0.05).

FIGURE 6

[³H](R)-1-(2-Chlorphenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinoline-carbox-amide (PK11195) and [³H]N-(2,5-dimethoxybenzyl)-N-(4-fluoro-2-phenoxyphenyl) acetamide (DAA1106) autoradiography overlaps mainly with CD68-labeled cells. Combined autoradiography and immunostaining were performed on brain tissue from acute infarcts (INFARCT, A), multiple sclerosis (MS, B), frontotemporal dementia (FTD, C), amyotrophic lateral sclerosis (ALS, D), and Alzheimer disease (AD, E) samples. Autoradiograms with [³H](R)-PK11195 (PK) and [³H]DAA1106 (DAA) are shown in the column on the left; the white boxes indicate the regions from which high-resolution images were taken (scale bar = 1 cm). Immunostaining was performed for astrocytes (GFAP, red) and activated microglia (CD68, green). [³H](R)-PK11195 (PK)- and [³H]DAA1106 (DAA)-specific binding ([³H]-, black grains) of both ligands overlapped mainly with CD68-labeled activated microglia (merge). Scale bar = 100 μM.

FIGURE 7

[³H](R)-1-(2-Chlorphenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinoline-carbox-amide (PK11195) binding in human CNS disorders correlates with CD68-labeled but not GFAP-labeled cells. (A) [³H](R)-PK11195 B_max values (y axis, in femtomoles per milligram) derived from tissues from patients with the disease conditions assessed and controls correlated with the abundance of CD68-labeled activated microglia (x axis, CD68 units) assessed in the same area in these same patients (r = 0.8213, p < 0.0001). (B) [³H](R)-PK11195 B_max values (y axis, in femtomoles per milligram) did not correlate significantly with the extent of reactive astrocytosis (r = 0.2666, p = 0.0839).

FIGURE 8

[³H]N-(2,5-dimethoxybenzyl)-N-(4-fluoro-2-phenoxyphenyl) acetamide (DAA1106) binding in human CNS disorders correlates with CD68-labeled but not GFAP-labeled cells. (A) [³H]DAA1106 B_max values (y axis, in femtomoles per milligram) derived from tissues from patients with the disease conditions assessed and controls correlated with the abundance of CD68-labeled activated microglia (x axis, CD68 units) assessed in the same area in these same patients (r = 0.8259, p < 0.0001). (B) [³H]DAA1106 B_max values (y axis, in femtomoles per milligram) did not correlate significantly with the extent of reactive astrocytosis (r = 0.1822, p = 0.2480).