Metabolism of the A1 Adenosine Receptor Positron Emission Tomography Ligand [f]8-cyclopentyl-3-(3-fluoropropyl)-1-propylxanthine ([f]cpfpx) in Rodents and Humans

Studies of plasma from mice, rats, and human volunteers evaluated methods for the extraction and quantification of the positron emission tomography ligand [F]8-cyclopentyl-3-(3-fluoropropyl)1-propylxanthine ([F]CPFPX) and identification of its metabolites in plasma by thin-layer chromatography and high-performance liquid chromatography (HPLC). Analysis of human, mouse, and rat plasma extracts by HPLC identified four identical radioactive metabolites in each species. The low mass of radioligand administered to humans (0.5 5 nmol) prevented direct identification of metabolites. However, incubating liver microsomes with CPFPX and analysis by means of liquid chromatography-mass spectrometry (LC-MS) identified seven compounds, four having the same retention times as the metabolites in human plasma. Analysis of microsomal metabolites by LC-MS identified five [M H] ions of m/z equivalent to hydroxy derivatives, 339, one of m/z equivalent to an oxo derivative, m/z 337, and one of m/z equivalent to a difunctionalized oxo-desaturation species, m/z 335, which is prominent in rat and mouse plasma and is the main metabolite in human plasma. An [M H] ion corresponding to a N-dealkylated derivative was not detected. Thus, like the natural methylxanthines, CPFPX seems to undergo oxidation by liver microsomes but, unlike those methylxanthines, dealkylation did not occur. LC-MS experiments with “in source” fragmentation identified the cyclopentyl moiety to be the most functionalized part of the molecule by liver microsomes and in vivo oxidations. Except for two metabolites, hydroxylated at the N1 propyl chain, all oxidative modifications found took place at the cyclopentyl ring. Positron emission tomography (PET) is a useful procedure for assessing the density (Phelps, 2000) and pharmacological properties of receptors in vivo (Merlet et al., 1993; Beugel et al., 1999). However, receptor-PET studies favor radioligands that are either stable in the body or that give rise to metabolites that do not interfere with the imaging of specifically bound ligand. The ligand [F]8-cyclopentyl-3-(3-fluoropropyl)-1-propylxanthine ([F]CPFPX) (Holschbach et al., 2002) is used to image the A1 adenosine receptor (A1AR) in human brain (Bauer et al., 2003). Because this ligand does not undergo degradation in the central nervous system, specifically bound ligand accounts for a very large fraction of brain radioactivity. However, such is not the case in peripheral tissues. The metabolism of [F]CPFPX in primates (Boy et al., 1998) and humans gives rise to at least three polar metabolites in blood, and studies illustrated the confounding effects of these metabolites. For example, the intravenous administration of [F]CPFPX to experimental animals caused intense labeling of the heart that was unaffected by the administration of unlabeled ligand, evidence for high unspecific binding of a metabolite (Holschbach et al., 1998). The physiological importance of the A1AR, its wide tissue distribution, and the success of PET-imaging A1ARs in the central nervous system urge extension of this technique to other organs. The design of more stable radioligands to achieve that end requires the kind of information about the metabolism of [F]CPFPX provided by this study. Measurements of receptor density by PET depend on compartmental analysis by mathematical models that are very sensitive to the concentration of native radioligand in blood perfusing the organ (the “input function”). Such measurements on the plasma of human subjects (Meyer et al., 2004) identified several [F]CPFPX metabolites in addition to unchanged ligand. Because the radiotracers for PET studies are prepared under no-carrier-added conditions, the amount of compound administered is in the low to subnanomolar range, making direct spectrometric identification of metabolites impossible. As this report describes, incubating CPFPX with human liver microsomes generated compounds that by HPLC had the same mobilities as the metabolites in plasma, and LC-MS tentatively identified them by measuring m/z of the [M H] ions. The literature contains little information about the metabolism of synthetic xanthines. The use of CPFPX in humans for diagnostic and research PET imaging necessitates the knowledge of its metabolism in vivo. To our knowledge, the present study of the biotransformation of CPFPX in humans is the first of its kind. Article, publication date, and citation information can be found at http://dmd.aspetjournals.org. doi:10.1124/dmd.105.006411. ABBREVIATIONS: CPFPX, 8-cyclopentyl-3-(3-fluoropropyl)-1-propylxanthine; A1AR, A1 adenosine receptor; DPCPX, 8-cyclopentyl-1,3-dipropylxanthine; PET, positron emission tomography; HPLC, high-performance liquid chromatography; LC-MS, liquid chromatography-mass spectrometry; TLC, thin-layer chromatography; p.i., postinjection. 0090-9556/06/3404-570–576$20.00 DRUG METABOLISM AND DISPOSITION Vol. 34, No. 4 Copyright © 2006 by The American Society for Pharmacology and Experimental Therapeutics 6411/3098806 DMD 34:570–576, 2006 Printed in U.S.A. 570 at A PE T Jornals on O cber 4, 2017 dm d.aspurnals.org D ow nladed from Materials and Methods Animal experiments used female Wistar rats and NMRI mice obtained, cared for, and used in accordance with German law, which is consonant with the Declaration of Helsinki, under institutionally approved protocol 50.203.2KFA 12/02. The studies of human plasma were part of larger studies of PET (Bauer et al., 2003; Meyer et al., 2004) reviewed and approved by the Ethics Committee of the Medical Faculty of the University of Düsseldorf and the German Federal Office for Radiation Protection. Sterile, pyrogen-free physiological saline (Braun, Melsungen, Germany) was the solvent for solutions administered intravenously. Blood collection from rodents was by cardiac puncture into syringes moistened with heparin. The in-house syntheses of CPFPX (Holschbach et al., 1998b), [H]CPFPX (Holschbach et al., 2003), and [F]CPFPX (Holschbach et al., 2002) followed published methods. Solvents were HPLC grade and were used as supplied by Sigma-Aldrich Chemie GmbH (Steinheim, Germany). TLC of blood extracts used glass-backed silica gel sheets (G 25 UV254; Macherey-Nagel, Düren, Germany); scanning of the plates by an InstantImager (Canberra-Packard GmbH, Dreieich, Germany) measured radioactivity. Calibration showed that the response of the device to fluorine-18 was linear over the range 23 to 5900 Bq (0.63–160 nCi). Human liver microsomes pooled from different human donors were obtained from Sigma-Aldrich Chemie GmbH, BD Biosciences (Woburn, MA), XenoTech (Kansas City, KS), and In Vitro Technologies (Baltimore, MD). All biochemicals were obtained from Fluka/Sigma-Aldrich Chemie GmbH (Steinheim, Germany). Microsomes from three rats and three mice, respectively, were prepared by differential centrifugation using a procedure slightly modified from the method described by Sugiura et al. (1974). Rats were sacrificed by decapitation; livers were removed rapidly and homogenized with 3 volumes of 1.5% (isotonic) KCl solution in a Potter homogenizer on ice. The homogenate was centrifuged (10,000g, 20 min, 4°C). The lipophilic layer on top was carefully removed. The pellet was discarded and the microsomes sedimented by ultracentrifugation (105,000g, 60 min, 4°C). The pellet was washed three times with ice-cold KCl solution and finally suspended in 1 volume of 1.5% KCl. The homogenate was stored in 50l portions at 70°C. Protein estimation used a commercial assay (Bio-Rad DC Protein Assay; Bio-Rad, Hercules, CA) after solubilization in 15% NH4OH containing 2% SDS (w/v); human serum albumin served as a standard. Extraction and Metabolite Profile of [F]CPFPX in Mice. Preliminary experiments used mice to determine an extraction solvent. Groups of three mice each were bled 1, 2, 5, 10, 15, 30, or 60 min after the tail vein injection of [F]CPFPX (3700 KBq, 100 Ci) in a volume of 50 l. The addition of [H]CPFPX (74 KBq, 2 Ci) to the blood samples served for evaluating the effect of extraction solvents to determine whether residual protein interfered with chromatography (Blanchard, 1981) and for calculation of tracer recovery. Centrifugation (1250g, 3 min, 18°C) separated the plasma. Triplicate aliquots (20 l each) were mixed with an equal volume of either ethanol, methanol, acetonitrile, methanol/acetonitrile, 50:50 (v/v), or a mixture of methanol/ dichloromethane, 80:20 (v/v) and vigorously shaken for 2 min on a vortex mixer. After centrifugation (20,800 g, 2 min, 18°C), triplicate samples (2 l each) of the supernatant were spotted on each of two TLC sheets. One sheet was developed with ethyl acetate/hexane, 75:25 (v/v). The other sheet, which also contained samples (2 l each) of whole blood and native plasma, was not developed, and served for measuring total radioactivity in each sample. Scanning both sheets immediately with the InstantImager measured fluorine-18 activity. After 48 h (27 half-lives of fluorine-18, when 10 % of the original radioactivity of fluorine-18 remained), the sheets were exposed for 5 days to an imaging plate (TR2025; Fuji, Tokyo, Japan), which is sensitive to the low-energy -radiation of tritium, and were scanned with a Fujifilm BAS 5000 system (Fuji) to measure tritium activity. Recovery of radiotracer by the extraction solvent was calculated as the ratio of radioactivity in an aliquot of extract to that in an aliquot of plasma of an equivalent volume, expressed as a percentage. Metabolite Profile of [F]CPFPX in Rats. [F]CPFPX (3700 KBq, 100 Ci in 300 l) was injected into rats (n 7) via the tail vein, 1, 2, 5, 10, 15, 30, or 60 min (one rat per time point) before exsanguination. Centrifugation of the blood samples (1250g, 3 min, 18°C) yielded ca. 2 ml of plasma per rat. Aliquots (2 l) were spotted on TLC sheets as standards for calculation of the recovery of radioactivity from plasma extracts. Aliquots of plasma (100 ml) were then mixed with 100 l of methanol/acetonitrile, 50:50 (v/v), and a sample (2 l) of the supernatant after centrifugation (20,800g, 2 min, 18°C) was applied to a TLC sheet for subsequent chromatography. Triplicate samples (2 l each) were spotted on a second TLC sheet for measurement of radioactivity. The first TLC sheet was developed with ethyl acetate/hexane, 75:25 (v/v). Scanning both sheets immediately with the InstantImager measured fluorine-18 activity. Pharmacokinetics and Metabolite Profile of [F]CPFPX in Human Volunteers. The PET studies were as described previously (Bauer et al., 2003; Meyer et al., 2004). The dose of [F]CPFPX was 273 12 GBq (7.4 0.3 mCi, 0.5–5 nmol) in saline (10 ml), injected i.v. over approximately 20 s. Blood sampling ( 4 ml) into heparinized syringes was at every 6 s for 90 s, then at 1, 2, 3, 4, 6, 8, 10, 15, 20, 30, 45, 60, 75, and 90 min after the beginning of tracer injection. Assays of metabolites in plasma were made on samples collected at 1 and 2 min and on each sample collected thereafter. The blood samples were centrifuged (1000g, 3 min, 18°C) and the plasma was collected. Samples of plasma (100 l each) were extracted with 100 l of acetonitrile/ methanol, 50:50 (v/v). Aliquots (5 l each) of both plasma and, separately, plasma extract were spotted directly above the TLC lane beyond the zone of TLC development (see Fig. 1A). TLC and analyses were as described above for rodent plasma. High-Performance Liquid Chromatography. Analysis of plasma extracts used a Kromasil 100-3 C18 column (250 4.6 mm) (CS-Chromatographie Service GmbH, Langerwehe, Germany) in a HPLC system consisting of a WellChrom K-1001 pump (Knauer, Berlin, Germany), a K-2001 UV detector (Knauer), and a manual sample injector (type 7125; Rheodyne, Bensheim, Germany) fitted with a 500l sample loop. Isocratic elution with water/ methanol/acetic acid, 45:55:0.2 (v/v/v) was at a flow rate of 1 ml/min. UV monitoring at 275 nm detected CPFPX and its metabolites. For measurement of radioactivity, the outflow of the UV detector was connected in series to an on-line NaI(Tl) well-type scintillation detector with a 250l detection loop. Chromatograms were corrected for the transit time between the detectors and the retention time of CPFPX. Mass Spectrometry. For LC-MS measurements, the outlet of the UVdetector was coupled via an electrospray interface to a mass spectrometer (Surveyor MSQ; Thermo Electron Corporation, San Jose, CA). Nebulizer gas pressure was 4 bar and desolvation temperature was 450°C. Positive ion electrospray ionization detected CPFPX and its metabolites. The sprayer voltage and the cone voltage were 3000 and either 60 or 110 V, respectively. Positive ion spectra were recorded over an m/z range of 150 to 450 at a scan time of 1 s. The Xcalibur software, version 1.3, provided with the instrument permitted scans of the chromatograms for ions of a desired m/z over the range m/z 0.5. Oxidation of [F]CPFPX by Liver Microsomes. Liver microsomes (0.8 mg of protein) were dispersed in 0.1 M phosphate buffer (pH 7.4) containing 3.3 mM MgCl2 and a NADPH-generating system consisting of 1.3 mM NADP, 3.3 mM glucose 6-phosphate, and 0.4 U of glucose-6-phosphate dehydrogenase in a final volume of 1 ml. Incubation was at 37°C. The addition of [F]CPFPX, alone or with carrier CPFPX (3 g, 9 nmol) in dimethyl sulfoxide (1 l), initiated the reaction, which was stopped after 20 min (rat and mouse microsomes) or 4 h (human microsomes) by the addition of acetonitrile (1 ml). After 2 min of vortex mixing and centrifugation (20,800g, 1 min, 4°C), the supernatant was vacuum evaporated to dryness at ambient temperature. A solution of the residue in HPLC eluent (100 l) was centrifuged (20,800g, 1 min, 4°C) to remove sediment. Control experiments testing the possibility that the addition of carrier affected the metabolism of [F]CPFPX were identical except that the reaction mixture contained no carrier CPFPX. Preparation of Human Blood Samples for HPLC. HPLC analysis of the [F]CPFPX metabolites in human blood required an 8-ml sample of whole blood. The samples, which contained 30 KBq ( 0.8 Ci) of fluorine-18 radioactivity, were centrifuged (1000g, 5 min, 18°C) to obtain approximately 3 ml of plasma. After the addition of 2 volumes of acetonitrile, the mixture was shaken (vortex mixer) for 1 min. The supernatant separated from precipitated protein by centrifugation (5000g, 5 min) was evaporated to dryness in vacuo at ambient temperature. A solution of the residue in HPLC eluent (500 l) was filtered through a 2m membrane filter before injection. 571 HUMAN METABOLISM OF [F]CPFPX at A PE T Jornals on O cber 4, 2017 dm d.aspurnals.org D ow nladed from