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.2019 Jun 13:10:534.
doi: 10.3389/fphar.2019.00534. eCollection 2019.

In vitro andin vivo Human Metabolism of (S)-[18F]Fluspidine - A Radioligand for Imaging σ1 Receptors With Positron Emission Tomography (PET)

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In vitro andin vivo Human Metabolism of (S)-[18F]Fluspidine - A Radioligand for Imaging σ1 Receptors With Positron Emission Tomography (PET)

Friedrich-Alexander Ludwig et al. Front Pharmacol..

Abstract

(S)-[18F]fluspidine ((S)-[18F]1) has recently been explored for positron emission tomography (PET) imaging of sigma-1 receptors in humans. In the current report, we have used plasma samples of healthy volunteers to investigate the radiometabolites of (S)-[18F]1 and elucidate their structures with LC-MS/MS. For the latter purpose additionalin vitro studies were conducted by incubation of (S)-[18F]1 and (S)-1 with human liver microsomes (HLM).In vitro metabolites were characterized by interpretation of MS/MS fragmentation patterns from collision-induced dissociation or by use of reference compounds. Thereby, structures of corresponding radio-HPLC-detected radiometabolites, bothin vitro andin vivo (human), could be identified. By incubation with HLM, mainly debenzylation and hydroxylation occurred, beside further mono- and di-oxygenations. The product hydroxylated at the fluoroethyl side chain was glucuronidated. Plasma samples (10, 20, 30 min p.i.,n = 5-6), obtained from human subjects receiving 250-300 MBq (S)-[18F]1 showed 97.2, 95.4, and 91.0% of unchanged radioligand, respectively. In urine samples (90 min p.i.) the fraction of unchanged radioligand was only 2.6% and three major radiometabolites were detected. The one with the highest percentage, also found in plasma, matched the glucuronide formedin vitro. Only a small amount of debenzylated metabolite was detected. In conclusion, our metabolic study, in particular the high fractions of unchanged radioligand in plasma, confirms the suitability of (S)-[18F]1 as PET radioligand for sigma-1 receptor imaging.

Keywords: fluspidine; liquid chromatography-mass spectrometry; liver microsomes; positron emission tomography; radiometabolites; sigma-1 receptors.

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Figures

FIGURE 1
FIGURE 1
Chemical structures of both fluspidine and [18F]fluspidine enantiomers ((S)- and (R)-1 and (S)- and (R)-[18F]1) and selectedin vitro metabolites detected after incubation of (S)-1 with rat liver microsomes (RLM). Synthesized racemic compoundsrac-2,rac-3, andrac-4 were used as reference compounds in this study (Holl et al., 2013).
FIGURE 2
FIGURE 2
Microsomal metabolic transformation of (S)-[18F]1 at different concentrations of carrier ((S)-1) by HLM and NADPH in PBS at 37°C (Section “Time- and Concentration-Dependent Metabolic Transformation of (S)-[18F]1”) determined by radio-HPLC (system II in Section “Radio-HPLC”).
FIGURE 3
FIGURE 3
MS/MS data of (S)-1 (exact mass: 325.18).(A) enhanced product ion (EPI) spectrum, precursor ion at m/z 326.2 (CE 40).(B) MS3 spectrum of m/z 326.2/179.1 (AF2 0.125).(C) Proposed fragmentation pathway for (S)-1 (in some of the structures of fragment ions positive charges were placed at specific atoms for illustrative purposes).
FIGURE 4
FIGURE 4
(A)Multiple reaction monitoring (MRM) chromatograms (Section “LC-MS/MS Analyses”) recorded after incubation of (S)-1 (2 μM) with HLM in presence of NADPH in PBS at 37°C for 120 min (Section “Incubation of (S)-[18F]1 and Non-radioactive References for Identification ofin vitro Metabolites and Radiometabolites”).(B) enlarged detail ofA. MRM transitions as provided in the legend. Data are summarized in Table 1.
FIGURE 5
FIGURE 5
Proposed fragmentation pathways (EPI, MS3) for(A) M3 and(B) M5 (in some of the structures of fragment ions positive charges were placed at specific atoms for illustrative purposes).
FIGURE 6
FIGURE 6
Multiple reaction monitoring (MRM) chromatograms (m/z 358.2/107.1) (Section “LC-MS/MS Analyses,” LC temperature 15°C instead of 40°C) recorded after incubation of (S)-1 andrac-3 (intensity reduced by factor 15) with HLM in presence of NADPH (Section “Incubation of (S)-[18F]1 and Non-radioactive References for Identification ofin vitro Metabolites and Radiometabolites”).
FIGURE 7
FIGURE 7
Multiple reaction monitoring (MRM) chromatograms (m/z 252.1/141.0) (Section “LC-MS/MS Analyses,” LC temperature 15°C instead of 40°C) recorded after incubation of (S)-1 andrac-2 with HLM in presence of NADPH (Section “Incubation of (S)-[18F]1 and Non-radioactive References for Identification ofin vitro Metabolites and Radiometabolites”).
FIGURE 8
FIGURE 8
(A)Multiple reaction monitoring (MRM) chromatograms (Section “LC-MS/MS Analyses”) recorded after incubation of (S)-1 (2 μM) with HLM in presence of NADPH and UDPGA in PBS at 37°C for 120 min (Section “Incubation of (S)-[18F]1 and Non-radioactive References for Identification ofin vitro Metabolites and Radiometabolites”).(B) enlarged detail ofA, showing MRM chromatograms for glucuronide conjugates of mono- and di-oxygenated metabolites.(C) detail according toB, but after incubation of (S)-1 at 200 μM. MRM transitions as provided in the legends. Data are summarized in Table 1.
FIGURE 9
FIGURE 9
Multiple reaction monitoring (MRM) chromatograms (Section “LC-MS/MS Analyses”) recorded before(A) and after(B) incubation ofM12 withβ-glucuronidase (Helix pomatia type H-3) (Section “β-Glucuronidase Cleavage of Microsomal-Formed MetaboliteM12”). MRM transitions as provided in the legend.
FIGURE 10
FIGURE 10
Structures ofin vitro metabolites detected after incubation of (S)-1 with HLM (NADPH, UDPGA).
FIGURE 11
FIGURE 11
Radio-HPLC chromatograms (system I, Section “Radio-HPLC”) recorded after incubation of (S)-[18F]1 (carrier-added, (S)-1, 2 μM) with HLM (NADPH and UDPGA as stated in the legend) (Section “Incubation of (S)-[18F]1 and Non-radioactive References for Identification ofin vitro Metabolites and Radiometabolites”) combined with an UV-HPLC chromatogram (210 nm) of a mixture of references.
FIGURE 12
FIGURE 12
Overview of structures of identifiedin vitro radiometabolites found after in incubation of (S)-[18F]1 with HLM (NADPH, UDPGA).
FIGURE 13
FIGURE 13
Fractions of intact (S)-[18F]1 in plasma and urine (Section “Investigation of the Metabolism of (S)-[18F]1 in Humans”) determined by radio-HPLC (system I, Section “Radio-HPLC”), mean values ± SD (error bars).
FIGURE 14
FIGURE 14
Identification of radiometabolites of (S)-[18F]1 formed in humans (Sections “Investigation of the Metabolism of (S)-[18F]1 in Humans” and “Incubation of (S)-[18F]1 and Non-radioactive References for Identification ofin vitro Metabolites and Radiometabolites”).(A) radio-HPLC chromatogram from plasma (30 min post injection).(B) radio-HPLC chromatogram from urine (90 min post injection).(C) comparison of radio-HPLC chromatograms from urine (120 min post injection) and HLM incubation (NADPH, UDPGA), including enlarged section.(D) structures of identified main radiometabolites formed in humans ([18F]M12: plasma and urine, [18F]M1: urine). All chromatograms were recorded using radio-HPLC system I (Section “Radio-HPLC”).
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