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Basic Science Investigations |
1 Department of Neurology, Memorial Sloan-Kettering Cancer Center, New York, New York
2 Department of Radiology, Memorial Sloan-Kettering Cancer Center, New York, New York
3 Nuclear Medicine Service, Memorial Sloan-Kettering Cancer Center, New York, New York
4 Radiochemistry/Cyclotron Core Facility, Memorial Sloan-Kettering Cancer Center, New York, New York
5 Department of Radiology, PET Imaging Science Center, University of Southern California, Los Angeles, California
6 Preparative Core Resource Facility, Memorial Sloan-Kettering Cancer Center, New York, New York
7 Department of Epidemiology and Biostatistics, Memorial Sloan-Kettering Cancer Center, New York, New York
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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10 min) selective localization of FIAU in RG2TK+ xenografts, whereas FHBG and FHPG are being cleared from the HSV1-tk transduced and wild-type xenografts over the initial 2-h imaging period. Conclusion: The in vitro and in vivo results (including the PET images) show that FIAU is a substantially more efficient probe than FHBG or FHPG for imaging HSV1-tk expression, with greater sensitivity and contrast as well as lower levels of abdominal background radioactivity at 2 and 24 h.
Key Words: HSV1-tk • PET • 2'-fluoro-2'-deoxy-1-ß-D-arabinofuranosyl-5-iodouracil • 9-[3-fluoro-1-hydroxy-2-propoxymethyl] • guanine • 9-[4-fluoro-3-(hydroxymethyl)butyl]guanine • 124I • 18F • reporter gene
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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This paradigm and the use of reporter gene technology had been extended into several different realms. Reporter genes can be used to image vector targeting and the level of suicide gene (HSV1-tk) expression (5), to image the regulation of endogenous genes and signal-transduction pathways (6–8), and to monitor and quantitatively assess the expression of a second transgene that is cis-linked to the reporter gene by an internal ribosome entry site sequence (4,9).
Other radiolabeled probes for imaging HSV1-tk expression and other reporter genes using radiolabeled probes for noninvasive PET imaging have been developed. For example, 9-[(2-hydroxy-1-(hydroxymethyl)ethoxy)methyl]guanine (GCV) and 9-[4-hydroxy-3-(hydroxymethyl)butyl]guanine (PCV) (10–15) and other 18F-labeled acycloguanosine analogs, such as 8-fluoro-9-[(2-hydroxy-1-(hydroxymethyl)ethoxy)methyl]guanine (FGCV) (12,13), 8-fluoro-9-[4-hydroxy-3-(hydroxymethyl)butyl]guanine (FPCV) (14,15), 9-[3-fluoro-1-hydroxy-2-propoxymethyl]guanine (FHPG) (16,17), and 9-[4-fluoro-3-(hydroxymethyl)butyl]guanine (FHBG) (18) have been developed as reporter substrates for imaging wild-type and mutant (14) HSV1-tk expression. Recently, imaging, pharmacokinetics, and dosimetry of 18F-FHBG were reported in healthy volunteers as a preface to imaging HSV1-tk reporter expression in clinical gene therapy trials (19).
The objective of this study was to compare the efficacy of 3 radiolabeled probes (FIAU, FHBG, and FHPG) for in vivo imaging of HSV1-tk expression with PET. The experimental design of the in vitro and in vivo studies provides for paired comparisons between FIAU and FHBG and between FIAU and FHPG. The PET studies and comparisons were also performed on the same animals. Our results show that FIAU is a substantially better probe than either FHBG or FHPG for imaging HSV1-tk expression, with greater sensitivity and lower abdominal background radioactivity at 2 and 24 h.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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18F-FHBG and 18F-FHPG Syntheses
The syntheses of 18F-FHBG and 18F-FHPG were performed with minor modifications according to procedures reported in the literature (16–18). Briefly, 3.7 GBq (100 mCi) of no-carrier-added aqueous H18F from the cyclotron target was added to a solution of potassium carbonate (2–3 mg) and Kryptofix 2.2.2. (12–15 mg; Aldrich Chemical Co., Milwaukee, WI) in CH3CN and H2O (88:12). Azeotropic distillation at 115°C with freshly distilled acetonitrile (3 x 1 mL) under a nitrogen stream efficiently removed the target H2O. N2-(p-Anisyldiphenylmethyl)-9-[(4-tosyl)-3-p-anisyldiphenylmethoxymethylbutyl]guanine (16–18) (3–4 mg dissolved in 0.5 mL of freshly distilled acetonitrile) was introduced to the anhydrous potassium fluoride-Kryptofix 2.2.2. complex. The reaction mixture was heated at 120°C for 20 min and was subsequently allowed to cool, at which time the crude product was passed through a silica Sep-Pak cartridge to remove Kryptofix 2.2.2. and nonreacted fluoride. The Sep-Pak was eluted with 15% methanol/dichloromethane (3.5 mL) and evaporated in vacuo. The residue was acidified with 1N HCl (0.6 mL) and heated for 10 min at 120°C. Upon cooling, the contents were neutralized with 6N NaOH (0.1 mL). The crude product was purified by high-performance liquid chromatography (HPLC) on a semipreparative column (Luna C-18, 10 x 250 mm; Alltech Associates, Inc., Deerfield, IL). The mobile phase consisted of 5% EtOH/H2O and the flow rate was set at 5 mL/min. 18F-FHBG eluted at 12 min under these conditions. 18F-FHPG was prepared similarly from N2-(p-anisyldiphenylmethyl)-9-[(1-anisyldiphenyl)-3-tosyl-2-propoxymethyl]guanine (16–18) and was purified under the same conditions as described above; 18F-FHPG eluted at 7 min.
The radiochemical purities of 18F-FHBG and 18F-FHPG were >97% as measured by radio-HPLC using an analytic Luna C-18 column (4.5 x 250 mm) and 6% acetonitrile/H2O as the mobile phase. For specific activity calculations, standard solutions of each compound were injected to the HPLC column, and the area under ultraviolet (UV) was measured. The radioactive product with known activity was injected to the HPLC column under the same condition, and the area under UV was measured. This area under the UV peak was converted to mass, and activity was divided by the mass to yield specific activity for that preparation (16). In our previous studies we reported the estimated lower limits of specific activities of 18F-FHBG and 18F-FHPG to be 11.8 GBq/µmol (320 Ci/mmol) (18) and 19.5 GBq/µmol (526 Ci/mmol) (16), respectively. In more recent experiments, we found that a small amount of UV active impurity elutes close to the product. We initially thought this peak represented unlabeled product; however, this was subsequently determined to be incorrect. Therefore, a more likely estimate of the specific activity for 18F-FHBG and 18F-FHPG used in these experiments is in the range of 44.4–74.0 GBq/µmol (1,200–2,000 Ci/mmol) (although this was not determined at the time the syntheses were performed for the studies reported in this article). The final product was tested for sterility and pyrogenicity by standard techniques and found to be sterile and free from pyrogens.
In Vitro Assays
RG2TK+ and RG2 cells in culture were used to compare the accumulation of 14C-FIAU (2.07 GBq/mmol [56 mCi/mmol], >99.7% radiochemical purity; Moravek Biochemicals, Brea, CA) and each of the guanosine analogs, 18F-FHBG and 18F-FHPG, as described (1). Paired time-course radiotracer accumulation experiments were run over 180 min and the data were normalized to 3H-thymidine ([TdR] 2,220 GBq/mmol [60 Ci/mmol], >99.5% radiochemical purity; Moravek Biochemicals) accumulation. The incubation medium contained 14C-FIAU (0.37 kBq/mL [0.01 µCi/mL], 0.18 µmol/L), 18F-FHBG (148 kBq/mL [4 µCi/mL], 7.6 pmol/L), or 18F-FHPG (148 kBq/mL [4 µCi/mL], 12.5 pmol/L) and 3H-TdR (3.7 kBq/mL [0.1 µCi/mL], 0.16 pmol/L). The radioactivity assay for the in vitro tissue culture studies involved 18F 
-counting (AutoGamma 5550 spectrometer; Packard Instruments, Meriden, CT) immediately after the experiment, followed 24–36 h later by 3H and 14C ß-isotope counting (Tri-Carb Liquid Scintillation Analyzer, model 1600TR; Packard Instruments) using external standard quench correction and standard dual-counting techniques. All data were expressed as disintegrations per minute (dpm)/g cells (or dpm/mL medium). The net accumulation rate (K) was calculated from the slope of the probe accumulation versustime plot (dpm/g cells ÷ dpm/mL medium vs. time; units = mL medium/g cells/min - a clearance constant).
124I-FIAU Synthesis
124I-FIAU was synthesized as described (6) with some modifications. Briefly, 124I was produced on the Memorial Sloan-Kettering Cancer Center (MSKCC) CS-15 cyclotron using the 124Te(p,n)124I nuclear reaction on an enriched 124TeO2/Al2O3 solid target (20). The 2'-fluoro-2'-deoxy-1-ß-D-arabinofuranosyl-5- (tri-n-butyltin)-uracil (FTBSnAU) precursor was prepared by an adaptation of a procedure by Vaidyanathan et al. (21) in 69% yield and was identified by 1H and 19F nuclear magnetic resonance spectroscopy and mass spectrometry. Na124I was added to a solution of FTBSnAU (125 µg) dissolved in 50 µL methanol, followed by the addition of a 20-µL mixture of 30% hydrogen peroxide/acetic acid (1:3, v/v). Aqueous saturated sodium metabisulfate (0.1 mL) was added to quench the reaction; then the reaction mixture was loaded onto a C18 Sep-Pak cartridge system (Waters, Milford, MA). The C18 cartridge system was eluted with water (35 mL), followed by methanol (3–4 mL) to isolate the 124I-FIAU. The methanol was evaporated and the 124I-FIAU was formulated in physiologic saline (with 5% ethanol added), which was passed through a sterile 0.22-µm Millipore filter (Millipore, Inc., Bedford, MA).
The radiochemical purity was determined by radio-thin-layer chromatography (silica gel plates [Sigma-Aldrich, St. Louis, MO]; eluent, ethyl acetate/acetone/H2O, 14:8:1). 124I-FIAU comigrated with authentic FIAU standard. The radiolabeled product was isolated in 51%–54% radiochemical yield (3 labelings), with radiochemical purities of 
97%. The specific activity of the initial Na124I was found to be >1,110 GBq/µmol (>30 Ci/µmol) (20), so that the specific activity of the 124I-FIAU was estimated to be similar to this value. The structure of FIAU was confirmed by single-crystal x-ray analysis.
131I-FIAU Synthesis
No-carrier-added 131I-FIAU was prepared by the same iododestannylation method (21) as was used for 124I-FIAU. Commercially produced Na131I was obtained from New England Nuclear (North Billerica, MA). Radiochemical yields obtained with 131I averaged 93% (3 labelings), and radiochemical purities were found to be 99%. The specific activities of the 131I-FIAU produced were estimated to be 48–59 GBq/µmol (1.3–1.6 Ci/µmol) on the basis of the manufacturer’s specifications accompanying the Na131I lots used.
Subcutaneous Xenografts and Animal Studies
The experimental protocol involving animals was approved by the Institutional Animal Care and Use Committee of MSKCC. Subcutaneous xenografts were produced in rnu/rnu rats (Frederick Cancer Center, Frederick, MD) weighing 200–250 g by subcutaneous injection of 5 x 106 tumor cells in 200 µL of serum-free cell culture medium under anesthesia (ketamine [87 mg/kglrsqb] and xylazine [13 mg/kg], intraperitoneally). Transduced RG2TK+ cells were placed in the left shoulder (test) and wild-type RG2 cells were placed in the right shoulder (negative control). The animals were monitored for tumor growth by daily measurements of the tumor’s size and the animal’s weight. Animals were studied when the subcutaneous tumor xenografts reached a diameter of 15 mm, 7–14 d after subcutaneous implantation of the RG2 and RG2TK+ cells. The in vivo growth rate, macroscopic and microscopic appearances, and the degree of vascularization were similar in the HSV1-tk transduced and wild-type subcutaneous xenografts.
Three days before study, all animals received 1.0-mL intravenous injections of a 0.9% NaI solution (30 mg/kg NaI) daily for 3 consecutive days to block thyroid uptake of radioactive iodide. For the paired 2-h FHBG-24-h FIAU imaging studies (n = 6), the animals were first injected with 18F-FHBG (27.8 MBq [0.75 mCi] per animal intravenously), imaged over 2 h, and then injected with 124I-FIAU (8.9 MBq [0.24 mCi] per animal intravenously) and reimaged 24 h later. For the paired 2-h FHPG-24-h FIAU imaging studies (n = 6), the animals were first injected with 18F-FHPG (61.1 MBq [1.65 mCi] per animal intravenously), imaged over 2 h, and then injected with 124I-FIAU (13.0 MBq [0.35 mCi] per animal intravenously) and reimaged 24 h later. For the paired 2-h FHBG-2-h FIAU imaging studies (n = 6), the animals were first injected with 18F-FHBG (50.0 MBq [1.35 mCi] per animal intravenously) and imaged over 2 h; 24 h later the same animals were injected with 124I-FIAU (7.0 MBq [0.19 mCi] per animal intravenously) and reimaged over 2 h. Immediately after the 124I-FIAU imaging sessions, animals were euthanized and tissue samples were processed for 124I radioactivity using an AutoGamma 5550 spectrometer (Packard Instruments). Radioactivity is expressed as percentage dose/g tissue (%dose/g tissue).
For the paired 2-h 131I-FIAU-18F-FHBG and the 2-h 131I-FIAU-18F-FHPG tissue sampling studies, 2 groups of animals were studied. Simultaneous intravenous injection of 131I-FIAU (1.81 MBq [0.049 mCi] per animal) and 18F-FHPG (14.8–25.9 MBq [0.40–0.70 mCi] per animal) was performed in 1 group (n = 6), and injection of 131I-FIAU (3.18 MBq [0.086 mCi] per animal) and 18F-FHBG (5.55–13.0 MBq [0.15–0.35 mCi] per animal) was performed in the other group (n = 6). All animals were euthanized at 2 h after injection, and the tissue samples were processed for 131I and 18F radioactivity assay using an AutoGamma 5550 spectrometer. The upward cross-talk of 131I into the 18F channel was 2.7% of the counts in the 131I channel. Corrections for 18F and 131I decay and 131I upward cross-talk were applied to all samples. Radioactivity in the animal studies is expressed as %dose/g tissue.
PET
PET imaging was performed using the Advance Tomograph (General Electric Medical Systems, Milwaukee, WI), with a spatial resolution of 5-mm full width at half maximum (FWHM) at the center of the field of view. This camera was cross-calibrated previously with the AutoGamma 5550 spectrometer. All animals were anesthetized with ketamine (87 mg/kg) and xylazine (13 mg/kg) administered intraperitoneally. Measured attenuation correction was performed using a 7-min-duration transmission scan with two 107.3-MBq (9 mCi) germanium transmission sources. Two-dimensional emission scans were obtained for all studies.
Four studies (n = 6 animals per study) acquired dynamic image data from 10 or 20 min to 130 min after injection; acquisition frames were either 10 or 20 min in duration, depending on the dose of radiopharmaceutical administered. Two studies (n = 6 animals per study) involved imaging at 24 h after injection of 124I-FIAU (20-min acquisition).
Emission counts were corrected for random coincidences, dead time, and scatter. Emission scans were reconstructed using an iterative reconstruction method with measured attenuation correction, smoothed with an 8-mm gaussian filter. The reconstruction parameters were 28 subsets, 2 iterations in a 256 x 256 matrix using a loop filter of 2.15-mm FWHM and a postfilter of 3.0-mm FWHM. Regional tumor radioactivity concentrations (%dose/mL) were estimated from the maximum pixels within regions of interest drawn around the tumor on transaxial slices of the reconstructed image sets.
Statistical Analysis
The statistical significance of comparisons between 2-h FIAU and either 2-h FHBG or 2-h FHPG values for RG2-TK+ and the ratios of RG2-TK+ to RG2 or muscle values was based on 2-sided paired Student t tests for matched values in the same rat. The statistical significance of comparisons between 2-h FIAU and 24-h FIAU values was based on 2-sided 2-sample t tests. Results are reported as mean ± SD. Logarithmic transformations brought the data into closer agreement with implicit assumptions of normal statistical distributions with constant variance.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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To address this issue, some investigators have performed sequential studies comparing 2 or more probes using the same transduced cell line or transduced xenograft. Alternatively, multilabel paired studies have been performed involving the simultaneous administration of 2 or more different radiolabeled probes to the same culture dish (1,12,14,15,24) or to the same xenograft-bearing animal (14,15,24). These studies provide the best direct in vitro and in vivo comparisons of different probes. On the basis of paired probe accumulation studies in HSV1-tk transduced cells performed in vitro (12,14,15,24), a comparison of values normalized to that of GCV (set at 1.0) yields the following nucleoside/GCV values: FGCV, 0.5 (9,11); PCV, 2.0 (12,14); FPCV, 0.2 (14); FPCV, 1.0 (15); FIAU, 23 (1). Normalization of the in vitro data to acyclovir (which is accumulated in HSV1-tk transduced cells less avidly than GCV (11)) yields the following nucleoside/ACV values: FHPG, 1–8, and FIAU, 40–100; the FIAU/FHPG values varied between 5- and 100-fold (24). A direct comparison of the 2 probes in vivo showed that the FIAU/FHPG ratio in HSV1-tk transduced tumors increased from 21 at 30 min to 119 at 4 h (24). Also note that FHPG cleared rapidly from the transduced tumor; a maximum of 2.2 ± 0.2 %dose/g was observed at 1 h, which fell to 0.2 ± 0.03 %dose/g at 4 h after injection. In contrast, FIAU remained essentially constant during this interval (12.7 ± 2.4 and 10.5 ± 3.9 %dose/g, respectively). Our results confirm this observation.
The objective of this study was to compare the efficacy of 3 radiolabeled probes of current interest (FIAU, FHBG, and FHPG) for in vivo imaging of HSV1-tk expression with PET. The in vitro and in vivo experimental design provided for paired comparisons between FIAU and FHBG and between FIAU and FHPG. The PET imaging comparisons were performed on the same animals on sequential days (within 24 h). The 2-h nucleoside accumulation comparisons were performed on the same tissue samples (animals) using double-label radiotracer techniques. These paired comparisons clearly show a 20-fold greater sensitivity of FIAU compared with that of FHBG for imaging HSV1-tk expression and a >50-fold sensitivity advantage for FIAU compared with that of FHPG.
The PET images revealed high levels of radioactivity in the viscera of the animals (Fig. 2), whereas comparatively low levels of radioactivity were measured in the tissue sampling experiments (Table 1). This apparent inconsistency is explained by the fact that almost all visceral radioactivity observed on the PET images of FIAU, FHBG, and FHPG was intraluminal, not in the wall of the viscera that were sampled for counting. The time course of FIAU- and FHPG-derived radioactivity in stomach and intestine was assessed (data not presented). 124I radioactivity remained largely in the stomach, whereas 18F radioactivity was observed to rapidly move through the intestine. The hepatobiliary route of excretion of 18F-acycloguanosine derivatives has been well documented (12–19,24,25,32).
The issue of metabolic degradation and presence of radiolabeled metabolites was not addressed in this study. It has been shown that FIAU is resistant to metabolic degradation (29); pharmacologic doses of FIAU are excreted largely unchanged through the kidney and urine in patients (30). However, the intraluminal localization of radioactivity to stomach and to thyroid in animals not receiving a blocking dose of iodide indicates some deiodination of tracer 124I-FIAU in vivo. GCV and other acycloguanosine analogs are also resistant to metabolic degradation (48). 18F-FGCV, 18F-FPCV, and 18F-FHPG have been shown to be metabolically stable in blood and liver (17,19,20,22), and studies on healthy volunteers and primates indicate that 18F-FHBG is excreted largely unchanged in the urine (19,32). Further studies specifically addressing the issue of radiolabeled metabolites in blood and tissue need to be performed.
Another issue that has not been addressed in this study or earlier HSV1-tk imaging studies is whether probe transport across cell and tissue membranes is limiting and affects the images. Nucleosides are polar, and several classes of equilibrative (33) and concentrative (34) nucleoside transporters have been identified in cell membranes. The Na+-dependent concentrative transporters are expressed primarily in epithelial cells and proliferating tissues; they have been characterized as purine selective (N1, SPNT, or CNT2), pyrimidine selective (N2, CNT1), and broadly selective (N3, N4, and N5) (34). Although the data are indirect, there appears to be a wide range of nucleoside transporters expressed in different cell lines and tissue (organs) (35,36) as well as depressed levels of expression in some tumors (36). Transport limitations may affect the acycloguanosine compounds more than the pyrimidine nucleoside derivatives (10,24,25). If nucleoside (probe) transport is a rate-determining step for probe accumulation in HSV1-tk transduced cells and tissue, nucleoside transporter expression could affect the images and account for some of the variability reported in the literature (10,24). Detailed transport studies comparing different acycloguanosine and pyrimidine nucleoside probes have not been reported, although modification of the 3'-position, loss of the 3'-hydroxyl, loss of a portion of the sugar ring, and lack of conformational flexibility are factors that significantly decrease the affinity of some nucleoside analogs to nucleoside transporters, which results in a decreased transport into the cells (10,37).
It has not been resolved whether nucleoside (probe) transport across cell membranes significantly affects net accumulation of radioactivity (and intensity of the images) in HSV1-tk-expressing tissue. Rapid accumulation of FIAU in RG2TK+ xenografts is revealed in the early (10–20 min) image frames (Figs. 3 and 4), and this is encouraging. Also note that initial 2-min-duration emission scans were obtained as scout views (5–10 min after intravenous injection of the radiolabeled probes), and they provided good quality images of the RG2TK+ xenograft in animals injected with FIAU but not with FHBG or FHPG. This suggests that cell membrane transport may not be limiting for FIAU; further in vivo and in vitro kinetic studies are needed to establish this point.
The results of the paired 2-h studies clearly show the sensitivity and dynamic range advantages of FIAU over FHBG or FHPG for imaging HSV1-tk expression. The ability to image late, 24 h after FIAU administration, also provides the opportunity to achieve high image specificity because of physiologic washout of background radioactivity and the retention of the radioactivity in HSV1-tk transduced tissue (1–3). Although FIAU is usually labeled with 124I for PET studies (or with 123I or 131I for gamma-camera scintigraphy) and late imaging is usually performed, our results show the feasibility of early FIAU imaging. Good quality images can be obtained within the first 30 min to 2 h after FIAU administration (Figs. 2–4). Thus, radiolabeling FIAU with 18F or 11C is an alternative strategy to using FHBG or FHPG; it would provide additional positron-emitting FIAU probes with high HSV1-tk sensitivity and dynamic range. In addition, the relatively short half-life of these radionuclides would facilitate repeated or sequential PET imaging of HSV1-tk expression.
To enhance the HSV1-tk imaging sensitivity of radiolabeled acycloguanosine probes, the use of site-mutated variants of wild-type HSV1-tk as the reporter gene has been proposed recently (14). This proposal developed from earlier work that showed enhanced ganciclovir and acyclovir cytotoxicity and enhanced drug phosphorylation with site-mutated HSV1-tk variants compared with wild-type HSV1-tk (38,39). Initial results obtained with the HSV1-sr39tk mutant (generated by semirandom sequence mutagenesis) revealed enhanced sensitivity for PCV and FPCV, but this enhancement was modest (2-fold) (14). Furthermore, the 2-fold enhancement in probe accumulation contrasts with the 300-fold difference in GCV 50% inhibitory concentration (IC50) between HSV1-sr39tk and wild-type HSV1-tk C6 transfectants (39). This disparity raises the question of whether cell membrane transport of PCV and FPCV was limiting in the radiolabeled probe accumulation studies run over a period of 15–240-min (14); in contrast, the GCV IC50 studies were run over a 3-d period, providing a longer period for GCV to enter the cell and exert a cytotoxic effect (38–40). Our previous GCV uptake studies in tissue culture were run over a 24-h period and the cell/medium concentration ratios were initially low in RG2 and RG2TK+ cells (1): at 1 h, the GCV ratios (uncorrected for cell-adherent medium) were 0.68 ± 0.12 and 0.64 ± 0.21 mL/g for RG2 and RG2TK+ cells, respectively; at 4 h, the ratios were 0.70 ± 0.16 and 0.97 ± 0.35 mL/g, respectively; at 8 h, the ratios were 0.69 ± 0.10 and 1.17 ± 0.08 mL/g, respectively; and at 24 h, the ratios were 0.94 ± 0.09 and 2.8 ± 1.0 mL/g, respectively. The K values for GCV were 0.00020 ± 0.00004 mL/min/g cells in RG2 cells and 0.00156 ± 0.00009 mL/min/g cells in RG2TK+ cells (values determined from data presented in (1)) and are consistent with slow GCV equilibration across RG2 and RG2TK+ cell membranes. An approximation of the probe-cell equilibration times in vitro can be obtained from the reciprocal of the K values; for FIAU, FHBG, FHPG, and GCV flux into RG2TK+ cells, the calculated values are 2.6, 43, 130, and 641 min, respectively. Thus, cell membrane transport could be a factor in limiting FHBG, FHPG, and GCV accumulation in transduced cells.
The sensitivity and dynamic range of a PET reporter probe are important considerations that will impact on animal and patient studies. Our data clearly show the advantages of FIAU compared with FHBG and FHPG. Greater sensitivity and dynamic range provide the ability to image lower levels of reporter gene expression over a wider range of expression levels. This advantage translates into several practical benefits: Lower quantities of vector are required for successful transduction of target tissue with the reporter system, a wider range of image intensity is provided for more accurate quantitation of reporter expression, and a more sensitive measure of weakly activated reporter systems is provided. For example, the higher sensitivity and wider dynamic range of PET imaging with FIAU allows more accurate monitoring of small changes in the activity of various signal-transduction pathways, when expression of the HSV1-tk reporter gene is controlled by a signal-transduction specific cis-acting promoter/enhancer element. Our recent studies showed that the p53 signal-transduction pathway activity in tumors could be imaged successfully with 124I-FIAU and PET when the HSV1-tk gene was placed under control of an artificial cis-acting p53-specific promoter (6). In another study, we showed that highly specific images of T-cell activation could be obtained when the HSV1-tk gene was placed under control of an artificial cis-acting promoter that is specific to the nuclear factor of activated T-cells (7).
Looking ahead to the clinical application of these reporter systems, it is necessary to consider the differences in maximum allowable injected dose of the reporter substrate, the differences in the rates of decay of the radiolabels, and, for PET imaging, the differences in positron abundance. Except for FHBG (19,32), detailed dosimetry studies in humans or primates have not been reported for these tracers. The biodistribution and dosimetry in healthy subjects after intravenous administration of 70–229 MBq (1.9–6.2 mCi) 18F-FHBG showed that the urinary bladder wall was the dose-limiting organ: 0.094 mGy/MBq (0.35 rad/mCi) (19). If the dose limitations are similar to those of other tracers labeled with the same radioisotopes (namely, an 5-mCi maximum for 124I and an 10 mCi maximum for 18F), the following comparisons could be made. The 2:1 advantage of the 18F-labeled probes over the 124I- probes is further extended to 8:1 when positron abundance (23% for 124I and 98% for 18F) is considered but is back to 4:1 for imaging at 2 h because of decay. However, even when this 4:1 advantage in counts for 18F over 124I is considered, the positron signal from 124I-FIAU is still expected to be significantly higher than that from either 18F-FHBG or 18F-FHPG. Furthermore, note that the ratios of transduced to nontransduced tissues are not affected by these considerations, and the image contrast would be expected to be similar to that observed in this study.
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CONCLUSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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2 h, whereas FHPG clearance is exponential. Abdominal background activity of FHBG was greater than that of FHPG because of the more rapid hepatobiliary clearance of FHBG. Transport across cell membranes may be a rate-limiting process for imaging HSV1-tk expression with radiolabeled acycloguanosine probes, but this does not appear to be the case for radiolabeled FIAU.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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For correspondence or reprints contact: Ronald G. Blasberg, MD, Department of Neurology, Room K923, Memorial Sloan-Kettering Cancer Center, 1275 York Ave., Box 52, New York, NY 10021.
E-mail: Blasberg{at}neuro1.mskcc.org
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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-particle-emitting eudoradiotherapeutic agent undergoing DNA incorporation. Cancer Res. 1996;56:1204–1209.This article has been cited by other articles:
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L. H. Wei, T. Olafsen, C. Radu, I. J. Hildebrandt, M. R. McCoy, M. E. Phelps, C. Meares, A. M. Wu, J. Czernin, and W. A. Weber Engineered Antibody Fragments with Infinite Affinity as Reporter Genes for PET Imaging J. Nucl. Med., November 1, 2008; 49(11): 1828 - 1835. [Abstract] [Full Text] [PDF]
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 P. Brader, J. Stritzker, C. C. Riedl, P. Zanzonico, S. Cai, E. M. Burnazi, E.R. Ghani, H. Hricak, A. A. Szalay, Y. Fong, et al. Escherichia coli Nissle 1917 Facilitates Tumor Detection by Positron Emission Tomography and Optical Imaging Clin. Cancer Res., April 15, 2008; 14(8): 2295 - 2302. [Abstract] [Full Text] [PDF]
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T. Miyagawa, G. Gogiberidze, I. Serganova, S. Cai, J. A. Balatoni, H. T. Thaler, L. Ageyeva, N. Pillarsetty, R. D. Finn, and R. G. Blasberg Imaging of HSV-tk Reporter Gene Expression: Comparison Between [18F]FEAU, [18F]FFEAU, and Other Imaging Probes J. Nucl. Med., April 1, 2008; 49(4): 637 - 648. [Abstract] [Full Text] [PDF]
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J. Culver, W. Akers, and S. Achilefu Multimodality Molecular Imaging with Combined Optical and SPECT/PET Modalities J. Nucl. Med., February 1, 2008; 49(2): 169 - 172. [Abstract] [Full Text] [PDF]
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V. Ponomarev, M. Doubrovin, A. Shavrin, I. Serganova, T. Beresten, L. Ageyeva, C. Cai, J. Balatoni, M. Alauddin, and J. Gelovani A Human-Derived Reporter Gene for Noninvasive Imaging in Humans: Mitochondrial Thymidine Kinase Type 2 J. Nucl. Med., May 1, 2007; 48(5): 819 - 826. [Abstract] [Full Text] [PDF]
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D.-X. Fu, Y. C. Tanhehco, J. Chen, C. A. Foss, J. J. Fox, V. Lemas, J.-M. Chong, R. F. Ambinder, and M. G. Pomper Virus-Associated Tumor Imaging by Induction of Viral Gene Expression Clin. Cancer Res., March 1, 2007; 13(5): 1453 - 1458. [Abstract] [Full Text] [PDF]
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J.-C. Tseng, P. B. Zanzonico, B. Levin, R. Finn, S. M. Larson, and D. Meruelo Tumor-Specific In Vivo Transfection with HSV-1 Thymidine Kinase Gene Using a Sindbis Viral Vector as a Basis for Prodrug Ganciclovir Activation and PET J. Nucl. Med., July 1, 2006; 47(7): 1136 - 1143. [Abstract] [Full Text] [PDF]
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H.-E. Wang, H.-M. Yu, R.-S. Liu, M. Lin, J. G. Gelovani, J.-J. Hwang, H.-J. Wei, and W.-P. Deng Molecular Imaging with 123I-FIAU, 18F-FUdR, 18F-FET, and 18F-FDG for Monitoring Herpes Simplex Virus Type 1 Thymidine Kinase and Ganciclovir Prodrug Activation Gene Therapy of Cancer J. Nucl. Med., July 1, 2006; 47(7): 1161 - 1171. [Abstract] [Full Text] [PDF]
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S. S. Yaghoubi, M. A. Couto, C.-C. Chen, L. Polavaram, G. Cui, L. Sen, and S. S. Gambhir Preclinical Safety Evaluation of 18F-FHBG: A PET Reporter Probe for Imaging Herpes Simplex Virus Type 1 Thymidine Kinase (HSV1-tk) or Mutant HSV1-sr39tk's Expression J. Nucl. Med., April 1, 2006; 47(4): 706 - 715. [Abstract] [Full Text] [PDF]
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Z. Xiong, Z. Cheng, X. Zhang, M. Patel, J. C. Wu, S. S. Gambhir, and X. Chen Imaging Chemically Modified Adenovirus for Targeting Tumors Expressing Integrin {alpha}v{beta}3 in Living Mice with Mutant Herpes Simplex Virus Type 1 Thymidine Kinase PET Reporter Gene J. Nucl. Med., January 1, 2006; 47(1): 130 - 139. [Abstract] [Full Text] [PDF]
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A. R. Buursma, A. M.J. Beerens, E. F.J. de Vries, A. van Waarde, M. G. Rots, G. A.P. Hospers, W. Vaalburg, and H. J. Haisma The Human Norepinephrine Transporter in Combination with 11C-m-Hydroxyephedrine as a Reporter Gene/Reporter Probe for PET of Gene Therapy J. Nucl. Med., December 1, 2005; 46(12): 2068 - 2075. [Abstract] [Full Text] [PDF]
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S. Y. Cho, L. Ravasi, L. P. Szajek, J. Seidel, M. V. Green, H. A. Fine, and W. C. Eckelman Evaluation of 76Br-FBAU as a PET Reporter Probe for HSV1-tk Gene Expression Imaging Using Mouse Models of Human Glioma J. Nucl. Med., November 1, 2005; 46(11): 1923 - 1930. [Abstract] [Full Text] [PDF]
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S.-C. Hung, W.-P. Deng, W. K. Yang, R.-S. Liu, C.-C. Lee, T.-C. Su, R.-J. Lin, D.-M. Yang, C.-W. Chang, W.-H. Chen, et al. Mesenchymal Stem Cell Targeting of Microscopic Tumors and Tumor Stroma Development Monitored by Noninvasive In vivo Positron Emission Tomography Imaging Clin. Cancer Res., November 1, 2005; 11(21): 7749 - 7756. [Abstract] [Full Text] [PDF]
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M. V. Simoes, M. Miyagawa, S. Reder, C. Stadele, R. Haubner, W. Linke, T. Lehner, P. Epple, M. Anton, M. Schwaiger, et al. Myocardial Kinetics of Reporter Probe 124I-FIAU in Isolated Perfused Rat Hearts After In Vivo Adenoviral Transfer of Herpes Simplex Virus Type 1 Thymidine Kinase Reporter Gene J. Nucl. Med., January 1, 2005; 46(1): 98 - 105. [Abstract] [Full Text] [PDF]
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M. M. Alauddin, A. Shahinian, R. Park, M. Tohme, J. D. Fissekis, and P. S. Conti Synthesis and Evaluation of 2'-Deoxy-2'-18F-Fluoro-5-Fluoro-1-{beta}-D-Arabinofuranosyluracil as a Potential PET Imaging Agent for Suicide Gene Expression J. Nucl. Med., December 1, 2004; 45(12): 2063 - 2069. [Abstract] [Full Text] [PDF]
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M. Miyagawa, M. Anton, R. Haubner, M. V. Simoes, C. Stadele, W. Erhardt, S. Reder, T. Lehner, B. Wagner, S. Noll, et al. PET of Cardiac Transgene Expression: Comparison of 2 Approaches Based on Herpesviral Thymidine Kinase Reporter Gene J. Nucl. Med., November 1, 2004; 45(11): 1917 - 1923. [Abstract] [Full Text] [PDF]
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J.-C. Richard, Z. Zhou, D. L. Chen, M. A. Mintun, D. Piwnica-Worms, P. Factor, D. E. Ponde, and D. P. Schuster Quantitation of Pulmonary Transgene Expression with PET Imaging J. Nucl. Med., April 1, 2004; 45(4): 644 - 654. [Abstract] [Full Text] [PDF]
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P. Ray, A. De, J.-J. Min, R. Y. Tsien, and S. S. Gambhir Imaging Tri-Fusion Multimodality Reporter Gene Expression in Living Subjects Cancer Res., February 15, 2004; 64(4): 1323 - 1330. [Abstract] [Full Text] [PDF]
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M. F. Kircher, J. R. Allport, E. E. Graves, V. Love, L. Josephson, A. H. Lichtman, and R. Weissleder In Vivo High Resolution Three-Dimensional Imaging of Antigen-Specific Cytotoxic T-Lymphocyte Trafficking to Tumors Cancer Res., October 15, 2003; 63(20): 6838 - 6846. [Abstract] [Full Text] [PDF]
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 J.-C. Richard, Z. Zhou, D. E. Ponde, C. S. Dence, P. Factor, P. N. Reynolds, G. D. Luker, V. Sharma, T. Ferkol, D. Piwnica-Worms, et al. Imaging Pulmonary Gene Expression with Positron Emission Tomography Am. J. Respir. Crit. Care Med., May 1, 2003; 167(9): 1257 - 1263. [Abstract] [Full Text] [PDF]
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| JOURNAL OF NUCLEAR MEDICINE TECHNOLOGY | THE JOURNAL OF NUCLEAR MEDICINE |
) and 18F-FHPG-14C-FIAU (
) accumulation data (mL medium/g cells) are shown. (B) 18F-FHBG-14C-FIAU and 18F-FHPG-14C-FIAU comparisons with 3H-TdR are shown: 14C-FIAU (•), 18F-FHBG (
). Each point represents paired comparison of uptake results obtained from single triple-label experiment; experimental times varied between 10 and 180 min.
