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Pharmacology and autoradiography of human DP prostanoid receptors using [(3)H]-BWA868C, a DP receptor-selective antagonist radioligand.

ABSTRACT: 1. A potent and highly selective DP prostanoid receptor antagonist radioligand, [(3)H]-cyclohexyl-N-BWA868C (3-benzyl-5-(6-carboxyhexyl)-1-(2-cyclohexyl-2-hydroxyethyl-amino) hydantoin, ([(3)H]-BWA868C)), has been generated for receptor binding and autoradiographic studies. 2. Specific [(3)H]-BWA868C binding to human platelet membranes achieved equilibrium within 60 min at 23 degrees C and constituted up to 95% of the total binding. The association (K(+1)) and dissociation (K(-1)) rate constants of binding were 0.758+/-0.064 min(-1), mmol and 0.0042+/-0.0002 min(-1), respectively, yielding dissociation constants (K(D)s) of 5.66+/-0. 44 nM (n=4). 3. Specific [(3)H]-BWA868C bound to DP receptors with a high affinity (K(D)=1.45+/-0.01 nM, n=3) and to a finite, saturable number of binding sites (B(max)=21.1+/-0.6 nmol g(-1) wet weight). 4. DP receptor class prostanoids (e.g. ZK118182, BW245C, BWA868C, PGD(2)) exhibited high (nanomolar) affinities for [(3)H]-BWA868C binding, while prostanoids selective for EP, FP, IP and TP receptors showed a low (micromolar) affinity. 5. Specific DP receptor binding sites were autoradiographically localized on the ciliary epithelium/process, longitudinal and circular ciliary muscles, retinal choroid and iris in human eye sections using [(3)H]-BWA868C. While [(3)H]-PGD(2) yielded similar quantitative distribution of DP receptors as [(3)H]-BWA868C, the level of non-specific binding observed with [(3)H]-PGD(2) was significantly greater than that observed with [(3)H]-BWA868C. 6. It is concluded that [(3)H]-BWA868C is a high-affinity and very specific DP receptor radioligand capable of selectively labelling the DP receptor. [(3)H]-BWA868C may prove useful for future homogenate-based and autoradiographic studies on the DP receptor.

SUBMITTER: Sharif NA

PROVIDER: S-EPMC1572442 | biostudies-other | 2000 Nov

REPOSITORIES: biostudies-other

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Project description:BackgroundReactive microglia and recruited peripheral macrophages contribute to the pathogenesis of Alzheimer's dementia (AD). Monocytes, macrophages and microglia all express the marker colony-stimulating factor 1 receptor (CSF1R). 4-Cyano-N-(4-(4-methylpiperazin-1-yl)-2-(4-methylpiperidin-1-yl)phenyl)-1H-pyrrole-2-carboxamide (1) is a high-affinity antagonist for CSF1R. We report the radiosynthesis of both [3H]1 and [11C]1. The PET imaging properties of [11C]1 in mice and baboon were investigated. [3H]1 was studied in Bmax measurement in post-mortem autoradiography in the frontal cortex, inferior parietal cortex and hippocampus from donors diagnosed with AD and age-matched controls. In vitro binding affinity of 1 was measured commercially. Nor-methyl-1 precursor was radiolabeled with [11C]iodomethane or [3H]iodomethane to produce [11C]1 and [3H]1, respectively. Ex vivo brain biodistribution of [11C]1 was compared in normal mice versus lipopolysaccharide-administered (LPS) murine model of neuroinflammation. Dynamic PET imaging was performed in a healthy male Papio anubis baboon. Post-mortem autoradiography with [3H]1 was performed in frozen sections using a standard saturation binding technique.ResultsCompound 1 exhibits a high in vitro CSF1R binding affinity (0.59 nM). [11C]1 was synthesized with high yield. [3H]1 was synthesized similarly (commercially). Biodistribution of [11C]1 in healthy mice demonstrated moderate brain uptake. In LPS-treated mice the brain uptake of [11C]1 was ~ 50% specific for CSF1R. PET/CT [11C]1 study in baboon revealed low brain uptake (0.36 SUV) of [11C]1. Autoradiography with [3H]1 gave significantly elevated Bmax values in AD frontal cortex versus control (47.78 ± 26.80 fmol/mg vs. 12.80 ± 5.30 fmol/mg, respectively, P = 0.023) and elevated, but not significantly different binding in AD hippocampus grey matter and inferior parietal cortex (IPC) white matter.ConclusionsCompound 1 exhibits a high in vitro CSF1R binding affinity. [11C]1 specifically labels CSF1R in the mouse neuroinflammation, but lacks the ability to efficiently cross the blood-brain barrier in baboon PET. [3H]1 specifically labels CSF1R in post-mortem human brain. The binding of [3H]1 is significantly higher in the post-mortem frontal cortex of AD versus control subjects.

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Pharmacology and autoradiography of human DP prostanoid receptors using [(3)H]-BWA868C, a DP receptor-selective antagonist radioligand.

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