Microsome Mediated in Vitro Metabolism: A Convenient Method for the Preparation of the PET Radioligand Metabolite [¹⁸F]FE-PE2I-OH for Translational Dopamine Transporter Imaging

Undesired radiometabolites can be detrimental to the development of positron emission tomography (PET) radioligands. Methods for quantifying radioligand metabolites in brain tissue include ex vivo studies in small animals or labeling and imaging of the radiometabolite(s) of interest. The latter is a time- and resource-demanding process, which often includes multistep organic synthesis. We hypothesized that this process could be replaced by making use of liver microsomes, an in vitro system that mimics metabolism. In this study, rat liver microsomes were used to prepare radiometabolites of the dopamine transporter radioligand [18F]FE-PE2I for in vitro imaging using autoradiography and in vivo imaging using PET in rats and nonhuman primates. The primary investigated hydroxy-metabolite [18F]FE-PE2I-OH ([18F]2) was obtained in a 2% radiochemical yield and >99% radiochemical purity. In vitro and in vivo imaging demonstrated that [18F]2 readily crossed the blood–brain barrier and bound specifically and reversibly to the dopamine transporter. In conclusions, the current study demonstrates the potential of liver microsomes in the production of radiometabolites for translational imaging studies and radioligand discovery

Discovery and Preclinical Validation of [¹¹C]AZ13153556, a Novel Probe for the Histamine Type 3 Receptor

The histamine type 3 receptor (H₃) is a G protein-coupled receptor implicated in several disorders of the central nervous system. Herein, we describe the radiolabeling and preclinical evaluation of a candidate radioligand for the H₃ receptor, 4-(1<i>S</i>,2<i>S</i>)-2-(4-cyclobutylpiperazine-1-carbonyl)­cyclopropyl]-<i>N</i>-methyl-benzamide (<b>5</b>), and its comparison with one of the frontrunner radioligands for H₃ imaging, namely, GSK189254 (<b>1</b>). Compounds <b>1</b> and <b>5</b> were radiolabeled with tritium and carbon-11 for in vitro and in vivo imaging experiments. The in vitro binding of [³H]<b>1</b> and [³H]<b>5</b> was examined by (i) saturation binding to rat and nonhuman primate brain tissue homogenate and (ii) in vitro autoradiography on tissue sections from rat, guinea pig, and human brain. The in vivo binding of [¹¹C]<b>1</b> and [¹¹C]<b>5</b> was examined by PET imaging in mice and nonhuman primates. <i>B</i>_max values obtained from Scatchard analysis of [³H]<b>1</b> and [³H]<b>5</b> binding were in good agreement. Autoradiography with [³H]<b>5</b> on rat, guinea pig, and human brain slices showed specific binding in regions known to be enhanced in H₃ receptors, a high degree of colocalization with [³H]<b>1</b>, and virtually negligible nonspecific binding in tissue. PET measurements in mice and nonhuman primates demonstrated that [¹¹C]<b>5</b> binds specifically and reversibly to H₃ receptors in vivo with low nonspecific binding in brain tissue. Whereas [¹¹C]<b>1</b> showed similar binding characteristics in vivo, the binding kinetics appeared faster for [¹¹C]<b>5</b> than for [¹¹C]<b>1</b>. Conclusions: [¹¹C]<b>5</b> has suitable properties for quantification of H₃ receptors in nonhuman primate brain and has the potential to offer improved binding kinetics in man compared to [¹¹C]<b>1</b>

Identification of a Novel Positron Emission Tomography (PET) Ligand for Imaging β‑Site Amyloid Precursor Protein Cleaving Enzyme 1 (BACE-1) in Brain

Alzheimer’s disease (AD) is characterized by accumulation of β-amyloid (Aβ) plaques and neurofibrillary tau tangles in the brain. β-Site amyloid precursor protein cleaving enzyme 1 (BACE1) plays a key role in the generation of Aβ fragments via extracellular cleavage of the amyloid precursor protein (APP). We became interested in developing a BACE1 PET ligand to facilitate clinical assessment of BACE1 inhibitors and explore its potential in the profiling and selection of patients for AD trials. Using a set of PET ligand design parameters, compound <b>3</b> (PF-06684511) was rapidly identified as a lead with favorable in vitro attributes and structural handles for PET radiolabeling. Further evaluation in an LC-MS/MS “cold tracer” study in rodents revealed high specific binding to BACE1 in brain. Upon radiolabeling, [¹⁸F]<b>3</b> demonstrated favorable brain uptake and high in vivo specificity in nonhuman primate (NHP), suggesting its potential for imaging BACE1 in humans

Identification of a Novel Positron Emission Tomography (PET) Ligand for Imaging β‑Site Amyloid Precursor Protein Cleaving Enzyme 1 (BACE-1) in Brain

Alzheimer’s disease (AD) is characterized by accumulation of β-amyloid (Aβ) plaques and neurofibrillary tau tangles in the brain. β-Site amyloid precursor protein cleaving enzyme 1 (BACE1) plays a key role in the generation of Aβ fragments via extracellular cleavage of the amyloid precursor protein (APP). We became interested in developing a BACE1 PET ligand to facilitate clinical assessment of BACE1 inhibitors and explore its potential in the profiling and selection of patients for AD trials. Using a set of PET ligand design parameters, compound <b>3</b> (PF-06684511) was rapidly identified as a lead with favorable in vitro attributes and structural handles for PET radiolabeling. Further evaluation in an LC-MS/MS “cold tracer” study in rodents revealed high specific binding to BACE1 in brain. Upon radiolabeling, [¹⁸F]<b>3</b> demonstrated favorable brain uptake and high in vivo specificity in nonhuman primate (NHP), suggesting its potential for imaging BACE1 in humans