Direct Plasma Metabolite Analysis of Positron Emission Tomography Radioligands by Micellar Liquid Chromatography with Radiometric Detection

Determination of radio-metabolites in plasma samples taken during a positron emission tomography (PET) study is an important component in the pharmacokinetic evaluation of PET radioligands. We have developed and validated a new analytical procedure for the plasma metabolite analysis of PET radioligands based on micellar liquid chromatography using an anionic surfactant mobile phase. Chromatographic separation was performed on an octadecyl semipreparative column (10 mm I.D. × 160 mm, 10 μm) using 100 mM sodium dodecyl sulfate (SDS) and 1-butanol in 10 mM sodium-phosphate (pH 7.2) at a flow rate of 5 mL/min. The samples taken from monkey or human plasma during PET measurements were directly injected into a liquid chromatographic (LC) system coupled to an online radiometric detector under micellar conditions using 1–2% (v/v) 1-butanol mobile phase to remove plasma proteins and concentrate the analytes at the column head. At 2 min, mobile phase was changed to elute and separate PET radioligand and its radiometabolites with high peak capacity under high submicellar conditions (10–25% 1-butanol). This procedure allowed direct plasma injection (up to 2 mL) into the LC column without any pretreatment with a short analysis-time of 8–10 min. Satisfactory reproducibility, linearity, sensitivity, accuracy and recovery were obtained in the validation study. The developed method was successfully applied to study the metabolism for diverse groups of PET radioligands and provided reliable determination of PET radioligands in human and monkey plasma. This method is advantageous in terms of simplifying and shortening the processes required to analyze short-lived radioligands as well as in providing a more accurate estimation of the metabolite corrected input function, especially for the radioligands with lower recoveries or degradation potential during the deproteination process in a conventional procedure

Direct Plasma Metabolite Analysis of Positron Emission Tomography Radioligands by Micellar Liquid Chromatography with Radiometric Detection

Determination of radio-metabolites in plasma samples taken during a positron emission tomography (PET) study is an important component in the pharmacokinetic evaluation of PET radioligands. We have developed and validated a new analytical procedure for the plasma metabolite analysis of PET radioligands based on micellar liquid chromatography using an anionic surfactant mobile phase. Chromatographic separation was performed on an octadecyl semipreparative column (10 mm I.D. × 160 mm, 10 μm) using 100 mM sodium dodecyl sulfate (SDS) and 1-butanol in 10 mM sodium-phosphate (pH 7.2) at a flow rate of 5 mL/min. The samples taken from monkey or human plasma during PET measurements were directly injected into a liquid chromatographic (LC) system coupled to an online radiometric detector under micellar conditions using 1–2% (v/v) 1-butanol mobile phase to remove plasma proteins and concentrate the analytes at the column head. At 2 min, mobile phase was changed to elute and separate PET radioligand and its radiometabolites with high peak capacity under high submicellar conditions (10–25% 1-butanol). This procedure allowed direct plasma injection (up to 2 mL) into the LC column without any pretreatment with a short analysis-time of 8–10 min. Satisfactory reproducibility, linearity, sensitivity, accuracy and recovery were obtained in the validation study. The developed method was successfully applied to study the metabolism for diverse groups of PET radioligands and provided reliable determination of PET radioligands in human and monkey plasma. This method is advantageous in terms of simplifying and shortening the processes required to analyze short-lived radioligands as well as in providing a more accurate estimation of the metabolite corrected input function, especially for the radioligands with lower recoveries or degradation potential during the deproteination process in a conventional procedure

Direct Plasma Metabolite Analysis of Positron Emission Tomography Radioligands by Micellar Liquid Chromatography with Radiometric Detection

Determination of radio-metabolites in plasma samples taken during a positron emission tomography (PET) study is an important component in the pharmacokinetic evaluation of PET radioligands. We have developed and validated a new analytical procedure for the plasma metabolite analysis of PET radioligands based on micellar liquid chromatography using an anionic surfactant mobile phase. Chromatographic separation was performed on an octadecyl semipreparative column (10 mm I.D. × 160 mm, 10 μm) using 100 mM sodium dodecyl sulfate (SDS) and 1-butanol in 10 mM sodium-phosphate (pH 7.2) at a flow rate of 5 mL/min. The samples taken from monkey or human plasma during PET measurements were directly injected into a liquid chromatographic (LC) system coupled to an online radiometric detector under micellar conditions using 1–2% (v/v) 1-butanol mobile phase to remove plasma proteins and concentrate the analytes at the column head. At 2 min, mobile phase was changed to elute and separate PET radioligand and its radiometabolites with high peak capacity under high submicellar conditions (10–25% 1-butanol). This procedure allowed direct plasma injection (up to 2 mL) into the LC column without any pretreatment with a short analysis-time of 8–10 min. Satisfactory reproducibility, linearity, sensitivity, accuracy and recovery were obtained in the validation study. The developed method was successfully applied to study the metabolism for diverse groups of PET radioligands and provided reliable determination of PET radioligands in human and monkey plasma. This method is advantageous in terms of simplifying and shortening the processes required to analyze short-lived radioligands as well as in providing a more accurate estimation of the metabolite corrected input function, especially for the radioligands with lower recoveries or degradation potential during the deproteination process in a conventional procedure

Discovery of a Novel Muscarinic Receptor PET Radioligand with Rapid Kinetics in the Monkey Brain

Positron emission tomography (PET), together with a suitable radioligand, is one of the more prominent methods for measuring changes in synaptic neurotransmitter concentrations in vivo. The radioligand of choice for such measurements on the cholinergic system is the muscarinic receptor antagonist <i>N</i>-[1-¹¹C]­propyl-3-piperidyl benzilate (PPB). In an effort to overcome the shortcomings with the technically cumbersome synthesis of [¹¹C]­PPB, we designed and synthesized four structurally related analogues of PPB, of which (<i>S</i>,<i>R</i>)-1-methylpiperidin-3-yl)­2-cyclopentyl-2-hydroxy-2-phenylacetate (<b>1</b>) was found to bind muscarinic receptors with similar affinity as PPB (3.5 vs 7.9 nM, respectively). (<i>S</i>,<i>R</i>)<b>-1</b> was radiolabeled via <i>N</i>-¹¹C-methylation at high radiochemical purity (>99%) and high specific radioactivity (>130 GBq/μmol). In vitro studies by autoradiography on human brain tissue and in vivo studies by PET in nonhuman primates demonstrated excellent signal-to-noise ratios and a kinetic profile in brain comparable to that of [¹¹C]­PBB. (<i>S</i>,<i>R</i>)-[¹¹C]<b>1</b> is a promising candidate for measuring changes in endogenous acetylcholine concentrations

Development of [<i>Carbonyl</i>-¹¹C]AZ13198083, a Novel Histamine Type‑3 Receptor Radioligand with Favorable Kinetics

The histamine subtype-3 receptor (H₃R) is implicated in a range of central nervous system disorders, and several radioligands have been developed for H₃R positron emission tomography imaging. However, a limitation of currently used PET radioligands for H₃R is the slow binding kinetics in high density brain regions. To address this, we herein report the development of three novel candidate H₃R radioligands, namely, [<i>carbonyl</i>-¹¹C]­AZ13153556 ([<i>carbonyl</i>-¹¹C]<b>4</b>), [<i>carbonyl</i>-¹¹C]­AZD5213­([<i>carbonyl</i>-¹¹C]<b>5</b>), and [<i>carbonyl</i>-¹¹C]­AZ13198083 ([<i>carbonyl</i>-¹¹C]<b>6</b>), and their subsequent preclinical evaluation in nonhuman primates (NHP). Radioligands [<i>carbonyl</i>-¹¹C]<b>4</b>–<b>6</b> were produced and isolated in high radioactivity (>1000 MBq), radiochemical purity (>99%), and moderate molar activity (19–28 GBq/μmol at time of injection) using a palladium-mediated ¹¹C-aminocarbonylation protocol. All three radioligands showed high brain permeability as well as a regional brain radioactivity distribution in accordance with H₃R expression (striatum > cortex > cerebellum). [<i>Carbonyl</i>-¹¹C]<b>6</b> displayed the most favorable in vivo kinetics and brain uptake, with an early peak in the striatal time–activity curve followed by a progressive washout from the brain. The specificity and on-target kinetics of [<i>carbonyl</i>-¹¹C]<b>6</b> were next investigated in pretreatment and displacement studies. After pretreatment or displacement with <b>5</b> (0.1 mg/kg), a uniformly low distribution of radioactivity across the NHP brain was observed. Collectively, this work demonstrates that [<i>carbonyl</i>-¹¹C]<b>6</b> is a promising candidate for H₃R imaging in human subjects

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

Development of [<i>Carbonyl</i>-¹¹C]AZ13198083, a Novel Histamine Type‑3 Receptor Radioligand with Favorable Kinetics

The histamine subtype-3 receptor (H₃R) is implicated in a range of central nervous system disorders, and several radioligands have been developed for H₃R positron emission tomography imaging. However, a limitation of currently used PET radioligands for H₃R is the slow binding kinetics in high density brain regions. To address this, we herein report the development of three novel candidate H₃R radioligands, namely, [<i>carbonyl</i>-¹¹C]­AZ13153556 ([<i>carbonyl</i>-¹¹C]<b>4</b>), [<i>carbonyl</i>-¹¹C]­AZD5213­([<i>carbonyl</i>-¹¹C]<b>5</b>), and [<i>carbonyl</i>-¹¹C]­AZ13198083 ([<i>carbonyl</i>-¹¹C]<b>6</b>), and their subsequent preclinical evaluation in nonhuman primates (NHP). Radioligands [<i>carbonyl</i>-¹¹C]<b>4</b>–<b>6</b> were produced and isolated in high radioactivity (>1000 MBq), radiochemical purity (>99%), and moderate molar activity (19–28 GBq/μmol at time of injection) using a palladium-mediated ¹¹C-aminocarbonylation protocol. All three radioligands showed high brain permeability as well as a regional brain radioactivity distribution in accordance with H₃R expression (striatum > cortex > cerebellum). [<i>Carbonyl</i>-¹¹C]<b>6</b> displayed the most favorable in vivo kinetics and brain uptake, with an early peak in the striatal time–activity curve followed by a progressive washout from the brain. The specificity and on-target kinetics of [<i>carbonyl</i>-¹¹C]<b>6</b> were next investigated in pretreatment and displacement studies. After pretreatment or displacement with <b>5</b> (0.1 mg/kg), a uniformly low distribution of radioactivity across the NHP brain was observed. Collectively, this work demonstrates that [<i>carbonyl</i>-¹¹C]<b>6</b> is a promising candidate for H₃R imaging in human subjects

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>