OtherBASIC SCIENCE INVESTIGATIONS

Novel PET Probes Specific for Deoxycytidine Kinase

Chengyi J. Shu, Dean O. Campbell, Jason T. Lee, Andrew Q. Tran, Jordan C. Wengrod, Owen N. Witte, Michael E. Phelps, Nagichettiar Satyamurthy, Johannes Czernin and Caius G. Radu
Journal of Nuclear Medicine July 2010, 51 (7) 1092-1098; DOI: https://doi.org/10.2967/jnumed.109.073361

Article Figures & Data

Figures

  • FIGURE 1. 
    FIGURE 1. 

    In vivo metabolite analysis of 18F-FAC. (A) Chemical structures of 18F-FAC and 18F-FAU and schematic showing extracellular and intracellular routes by which 18F-FAC can be deaminated to 18F-FAU. (B) Percentage of total detected radioactivity in plasma that is attributable to 18F-FAC over time as determined by HLPC analysis. Analysis is performed after intravenous injection of 18F-FAC in mice. 5′-NT = 5′nucleotidase; DCTD = deoxycytidylate deaminase (catabolic enzymes are shown in red fonts); MP = monophosphate.

  • FIGURE 2. 
    FIGURE 2. 

    18F-FAC and 18F-clofarabine (18F-CA) small-animal PET/CT scans of C57BL/6J mice (images are representative of pattern observed in 3 mice scanned with each probe). %ID/g = percentage injected dose per gram of tissue; Bl = urinary bladder; BF = brown fat; BM = bone marrow; GI = gastrointestinal tract; K = kidney; L = liver; S = spleen; Thy = thymus.

  • FIGURE 3. 
    FIGURE 3. 

    Candidate dCK-specific PET probes that resist deamination. (A) Chemical structures of deoxycytidine analogs amenable to 18F labeling. (B) In vitro deamination assay. After incubation at 37°C for 1 h in presence (blue dashed traces) or absence (red solid traces) of recombinant purified CDA, candidate probes were analyzed on HPLC. d-analogs are shown in top row, and l-analogs are shown in bottom row. abs = absorbance. R = H (FAC); R = CH3 (FMAC); R = F (FFAC); R = Cl (FCAC); R = Br (FBAC).

  • FIGURE 4. 
    FIGURE 4. 

    Analyses of affinity of l-analogs for dCK. l-analogs were tested for their ability to competitively inhibit phosphorylation (red bars; left axis) and uptake (blue bars; right axis) of tritium-labeled deoxycytidine (3H-dC) using dCK-expressing L1210 cells. Results represent 2 independent experiments. P values are calculated relative to water (n = 3). *P < 0.05. dC = deoxycytidine.

  • FIGURE 5. 
    FIGURE 5. 

    Biodistribution of 18F-labeled unnatural nucleosides in mice. (A) Small-animal PET/CT images of C57BL/6J mice. (B) Quantification of PET data. Probe uptake was normalized to muscle background (absolute uptake values are shown in Supplemental Fig. 6). (C) l-18F-FAC small-animal PET/CT scan of dCK knockout mouse. P values were calculated relative to FAC for each specific tissue. *P < 0.05; n = 5 (18F-FAC), n = 3 (l-18F-FAC), n = 3 (l-18F-FMAC), n = 2 (l-18F-FFAC), and n = 2 (l-18F-FCAC). %ID/g = percentage injected dose per gram of tissue; Bl = urinary bladder; B. Marrow = bone marrow; BM = bone marrow; GI = gastrointestinal tract; KO = knockout; L = liver; S = spleen; SG = salivary gland; Thy = thymus.

  • FIGURE 6. 
    FIGURE 6. 

    l-18F-FAC and l-18F-FMAC small-animal PET/CT images of malignant and autoimmune lymphoproliferative disorders. (A) l-18F-FAC and l-18F-FMAC small-animal PET/CT of L1210 lymphoma tumors. L1210 parental cell line (WT, solid-lined circle) and dCK-deficient variant L1210-10K (10K, dash-lined circle) were injected subcutaneously under the left and right shoulders of the mouse, respectively. Only the parental cell line accumulated both probes. (B) l-18F-FAC and l-18F-FMAC small-animal PET/CT of autoimmune B6.MRL-Faslpr/J mice. Both probes detected cervical, axillary, and brachial lymphadenopathy in these mice. %ID/g = percentage injected dose per gram of tissue; L = liver; LN = lymph nodes; S = spleen; Thy = thymus; WT = wild-type. Number of mice per probe ≥ 3.

Tables

  • TABLE 1

    Kinetic Parameters for FAC and Its Analogs with l-Chirality

    ProbeKM (μM)Relative kcatRelative kcat/KM
    FAC0.76 ± 0.151.01.0
    l-FMAC1.02 ± 0.151.00.96
    l-FCAC0.61 ± 0.090.961.20
    l-FBAC6.54 ± 0.992.700.31

    • kcat and specificity constant (kcat/KM) were determined using purified recombinant human dCK, and values are given relative to FAC.

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