The Plebiscite and the S.R.M
Authors: R. P. Liston
Journal: BMJ (1947)

<b>1178</b> <h3><b>Objectives:</b></h3> Tetramethylpiperidine (TEMPO) is a compound that has long been used to detect redox state with MR signal. TEMPO compounds contain a nitroxide-radical group that gives T1 contrast with MR when oxidized, but as TEMPO is reduced its ability to yield a T1 contrast is lost. A major limitation of <i>in vivo</i> use of these compounds is that washout from tissue is indistinguishable from reduction, making quantitative imaging impossible. However, these compounds can be labeled with a positron-emitting radionuclide, and simultaneous PET-MRI imaging can be used synergistically to discriminate between washout and reduction, allowing for non-invasive quantification of redox state in various tissues. Here we show the development of untargeted radiolabeled TEMPO compounds: <sup>11</sup>C-methylamino-TEMPO (<sup>11</sup>C-MA-TEMPO) and <sup>18</sup>F-fluoroethylamino-TEMPO (<sup>18</sup>F-FEA-TEMPO), allowing for quantitative whole-body imaging of redox status. <h3><b>Methods:</b></h3> <sup>11</sup>C-MA-TEMPO was produced by methylation of 4-amino-TEMPO with [11C]MeI . Prior to production of [11C]MeI, 1 mg of 4-amino-TEMPO in 100 μL DMF was loaded directly onto the HPLC loop used for purification. [11C]MeI was passed from the GE FX-C directly onto the semi-prep HPLC loop for 4 minutes and allowed to react at room temperature for an additional 4 minutes. Purification was performed using semi-preparative HPLC. <sup>11</sup>C-TEMPO eluted at 10.5 minutes and was then diluted with 30 mL of water containing 5 mg potassium bicarbonate (pH ~8) and loaded onto a C18 Sep-Pak. The product was eluted with 1 mL of EtOH, diluted with saline, and passed through a 0.22 μm filter. <sup>18</sup>F-FEA-TEMPO was synthesized by reacting 2-[18F]fluoroethyltosylate with 4-amino-TEMPO (20 mg) in DMF (0.5 mL) and heating at 120°C for 10 min. The resultant solution of 4-[18F]fluoroethylamino-TEMPO was purified by HPLC. The fraction containing the radiotracer was collected and diluted with sterile water (30 mL) and the activity was immobilized on a Sep Pak C18 cartridge, washed with water (5 mL) and eluted with ethanol (2 mL) and diluted with saline solution. To confirm that fluorination did not alter the MR properties of TEMPO, several rod phantoms were constructed to measure the T1 relaxation time of <sup>18</sup>F-FEA-TEMPO at varying concentrations and at varying concentrations the reducing agent glutathione (GSH). <i>In vivo</i> biodistribution of <sup>11</sup>C-MA-TEMPO was measured in mice using dynamic microPET/CT imaging, after a 10MBq injection through the tail vein. Lastly, <sup>18</sup>F-FEA-TEMPO was injected into healthy rabbits which then underwent PET/MRI imaging for measurement of redox status across multiple healthy soft tissues. <h3><b>Results:</b></h3> Rod phantoms imaged by PET/MRI showed that T1 contrast of oxidized FEA-TEMPO was retained after fluorination, and that reduction of FEA-TEMPO by GSH resulted in shortening of T1 contrast. <i>In vivo</i> imaging in mice showed that although the radiotracer entered soft tissues systemically, it did not cross the blood brain barrier. In PET-MRI imaging of rabbits, <sup>18</sup>F-FEA-TEMPO was able to distinguish differences in redox status between healthy tissues. <sup>18</sup>F-FEA-TEMPO uptake occurred rapidly in the highly perfused kidney, and PET signal remained high for up to 20 minutes. However, MRI signal in the kidney was rapidly lost, indicating a highly reductive environment. Skeletal muscle also showed rapid uptake and prolonged retention of <sup>18</sup>F-FEA-TEMPO by PET, but in contrast to the renal cortex, displayed a much slower loss of MRI signal, indicating a more oxidative environment. <h3><b>Conclusions:</b></h3> The TEMPO compound is a promising platform for PET/MRI agents for non-invasive quantification of redox status. This work demonstrates the ability of these radiolabeled TEMPO compounds to noninvasively and quantitatively assay redox status in the whole body. <b>Research Support:</b> P41EB022544