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Basic Science Investigations |
Department of Radiology, PET Imaging Science Center, University of Southern California, Los Angeles; and Gene Therapy Laboratory, Norris Comprehensive Cancer Center, University of Southern California, Los Angeles, California
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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Key Words: PET • gene therapy • [18F]FHBG • virus • colon cancer
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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The selective monophosphorylation of antiviral nucleoside analogs by virus-encoded thymidine kinase has been exploited in gene therapy of cancer (13–15). Gene therapy for treatment of human disease is currently being explored, and several approaches have been tested in patients (16). In this process, murine cells that are engineered to produce retroviral vectors that carry the gene for HSV thymidine kinase (HSV-tk) are injected into a tumor. Drug sensitivity is conferred upon tumor cells after expression of the HSV-tk gene. Ganciclovir is converted by the resulting HSV-tk to the monophosphorylated product, which is then converted to di- and triphosphates by host kinases, leading to tumor cell death (15). This approach is effective in the treatment of experimental glioma (13,15), hepatoma (17), and melanoma (18).
Treatment planning and individualization of gene therapy could be aided by a noninvasive method for measuring monophosphorylation induced in tumors to assess whether a therapeutically sufficient enzyme level exists before application of the prodrug ganciclovir (19,20). Moreover, the measurement of therapy effects on tumor metabolism of nucleoside analogs may be useful for predicting therapeutic outcome at an early stage of treatment (21).
PET using tracers of tumor metabolism has been used in the diagnosis and evaluation of treatment response in a variety of tumors (22). Development of PET for monitoring gene therapy with the suicide gene HSV-tk is underway (19–21,23–31). Many of these studies are designed to use a fluoro analog of ganciclovir, 9-([3-fluoro-1-hydroxy-2-propoxy]methyl)guanine (FHPG) (24,25,27–31). FHPG is selectively phosphorylated by HSV-tk at two thirds the rate of thymidine (9). Absence of viral kinase in noninfected cells precludes phosphorylation and, hence, trapping within these cells. [18F]FHPG has been shown to be potentially useful as a PET imaging agent of gene expression and incorporation in transduced tumors (24,27–31).
Penciclovir, a carba analog of ganciclovir, is active against HSV types 1 and 2, varicella-zoster virus, and Epstein–Barr virus (8,12). Preliminary work from our group showed that the fluoro analog of penciclovir, 9-(4-fluoro-3-hydroxymethylbutyl)guanine (FHBG), is selectively phosphorylated by HSV-tk and has higher accumulation in transduced cells than does FHPG (26). This finding suggests that [18F]FHBG might be better than [18F]FHPG in PET imaging of gene expression in tumors. Recently, [18F]FHBG has shown considerable promise relative to other indicators of HSV-tk expression in preliminary imaging studies in small animals (32–35). In this study, we report a detailed evaluation of this tracer in cell culture and in tumor-bearing nude mice using transduced and nontransduced HT-29 human colon cancer cells. We also report a preliminary assessment of the in vivo pharmacology of [18F]FHBG in a nonhuman primate model.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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Radiotracer
Sterile, pyrogen-free [18F]FHBG was synthesized following the method developed in our laboratory (26). The product was isolated by high-performance liquid chromatography (HPLC) using 15% acetonitrile in water. After isolation, solvent was evaporated, and the product was reconstituted with saline solution. The final product was passed through a 0.22-µm Millipore filter (Millipore, Bedford, MA) and collected into a sterile apyrogenic vial. Sterility and pyrogenicity tests were performed on the final preparations after the radioactivity had decayed. Pyrogenicity was tested using LAL reagent and E. coli endotoxin type 055:B5. Sterility was tested using thioglycollate and trypticase media.
Cell Line and Transduction
HT-29 human colon cancer cells were obtained from the American Type Culture Collection (Rockville, MD). Cells were transduced with the retroviral vector G1Tk1SvNa following a literature method (36). The construction of the retroviral vector G1Tk1SvNa was described elsewhere (37). Briefly, the HSV-tk was inserted into the backbone of pG1XSvNa vector (Gene Therapy Inc., Los Angeles, CA) and was under the control of viral long terminal repeat promoter. The amphotropic packaging cell line PA317 was used to make the viral supernatant, and viral titering was done in NIH3T3 cells (36). The HT-29 cells were grown in 100-mm dishes (1 x 107 cells per dish) in the presence of Dulbecco’s modified Eagle’s medium (DMEM) with a high glucose concentration (4.5 g/L) and supplemented with 2 mmol/L glutamine and 10% fetal bovine serum ([FBS] HyClone Laboratory, Logan, UT). Cells were transduced with the viral supernatants supplemented with 8 µg/mL (1,5-dimethyl-1,5-diazaundecamethylene)polymethobromide (Polybrene; Sigma, St. Louis, MO) at a multiplicity of infection of 1 viral particle per cell. Cells were selected with 0.6 mg/mL G-418 (Geneticin; GIBCO Life Technology, Grand Island, NY) for 10 d and used for RNA extraction. The transduced HT-29 cells displayed >99% transduction by methylene blue staining after selection. Cells in culture were reselected monthly (every 4 or 5 passages) to maintain >99% transduction for experimental work. Cells stored at -80°C in liquid nitrogen maintain >99% transduction indefinitely. The expression of proviral transcript was analyzed by northern hybridization with thymidine kinase probe as well as neomycin complementary DNA probe to verify the expression of the genes. In vivo gene expression in this HT-29 tumor line has been verified by immunocytochemical staining using a polyclonal rabbit antibody against the HSV-tk protein (36) and, therefore, was not assayed directly in the tumors grown in this study. The doubling time for transduced cells was 48 h, whereas that for wild-type cells was 24 h.
In Vitro Studies
Transduced and nontransduced control cells (approximately 5 million) were plated in triplicate to a Petri dish (100 mm) and cultured in 10 mL media for 24 h. Logarithmically growing cells were incubated with fresh media (7 mL) and 370 kBq [18F]FHBG for 1 and 5 h. After incubation and removal of media, the cells were washed with phosphate-buffered saline ([PBS] 3 x 10 mL) and exposed to trypsin. After neutralization with 10% FBS in DMEM, cells were centrifuged for 5 min at 1,200 rpm and supernatants were discarded. Cells were resuspended in PBS (4 mL), and the number of viable cells was counted using a hemocytometer. The cell suspensions were further centrifuged and the cell pellets were separated. Cellular radioactivity was measured in a Cobra II Auto Gamma Counter (Packard Instrument Co., Meriden, CT). The ratio of activity uptake in transduced and nontransduced cells was obtained for each of 3 experiments per time point.
To determine the molecular distribution of radiolabel, cells incubated for 5 h with radiotracer were treated with 1N perchloric acid ([PCA] 100 µL per cell pellet), and the acidic mixtures were cooled to 4°C for 10 min. The acidic cell suspensions were combined and centrifuged for 1 min at 14,000 rpm. The supernatant was separated, neutralized with 2.5N KOH, and analyzed by HPLC after removing the perchlorate salt by centrifugation. HPLC was performed using a Waters Associates system (Milford, MA) equipped with model 510 pumps, a variable-wavelength ultraviolet (UV) detector operated at 254 nm, and a C18 reversed-phase analytic column (Rainin, Walnut Creek, CA). Elution was performed at a flow of 1 mL/min with a gradient from 95% A (1% acetonitrile [MeCN], 1.5 mmol/L tetrabutylammonium hydroxide, and 1 mmol/L potassium dihydrogen phosphate in water, pH 3.3) and 5% B (30% MeCN, 1.5 mmol/L tetrabutylammonium hydroxide, and 25 mmol/L potassium dihydrogen phosphate in water, pH 3.3) to 50% A and 50% B over a 10-min period, followed by a gradient to 0% A and 100% B over an additional 3 min. Fractions were collected at 30-s intervals using a fraction collector (LKB Pharmacia, Uppsala, Sweden). Radioactivity in each fraction was measured in a 
-counter and plotted.
In Vivo Studies on Mice
In vivo studies, including blood clearance and biodistribution, were conducted on tumor-bearing nude mice at 2 and 5 h after injection. Tumors were grown in 6-wk-old athymic nude mice (Harlan–Sprague–Dawley; Harlan, Indianapolis, IN) by inoculation of 10 million cells (either transduced or nontransduced) under the skin in the left thigh region. When the tumor was approximately 1 cm in size, animals were used for the experiment as described below. Animal studies were performed under an approved institutional animal care and use committee (IACUC) protocol.
Four groups of mice (n = 5 per group), 2 with transduced tumors and 2 with nontransduced tumors, were injected intravenously through a tail vein with the radiotracer [18F]FHBG (approximately 2 MBq; 200 µL). Activity injected into each mouse was measured by placing the mouse in a dose calibrator (Capintec, Ramsey, NJ). In 3 randomly selected mice bearing transduced tumors, blood was collected in capillary tubes (1.33 µL) from the contralateral tail vein after rupture with a needle; samples were obtained 1, 2, 5, 10, 20, 40, 60, and 120 min after injection. Activity in each blood sample was measured with the -counter, and the percentage injected dose per gram of body weight (%ID/g) was calculated. Animals were anesthetized with sodium pentobarbital (40 mg/kg) and sacrificed at 2 or 5 h after injection. Organs and tumors were excised and weighed. Tissue radioactivity was measured with the -counter. For each mouse, [18F]FHBG uptake was expressed as the %ID/g and as the ratio of tumor to organ uptake.
In Vivo Studies on Monkeys
Two male cynomolgus monkeys (5.2 and 7.0 kg) (Macaca fascicularis; Biomedical Resources Foundation, Inc., Houston, TX) were anesthetized with sodium pentobarbital and prepared for arterial blood sampling by catheterization of a femoral artery. The animals were positioned supine in a CTI/Siemens 953A PET scanner (Knoxville, TN) and injected intravenously with 23–28 MBq/kg [18F]FHBG. Five sequential whole-body, attenuation-corrected PET scans of increasing duration were obtained between 0 and 2 h after injection. Blood samples were taken at increasing intervals from 30 s to 120 min after injection. A urine sample was also obtained from 1 monkey by transabdominal puncture. Blood samples were immediately placed in ice and then centrifuged. An aliquot was removed from each sample, weighed, and counted for radioactivity. In samples drawn for metabolite analysis, the remaining plasma was prepared for HPLC analysis by acid extraction with PCA (1N), and neutralization with KOH (4 mol/L). The acid-soluble fraction (ASF) was analyzed by ion-pair HPLC using a C8 reversed-phase analytic column (Radial Pak; Waters) and isocratic elution. The mobile phase consisted of 5% MeCN in 20 mmol/L phosphate buffer containing 2.5 mmol/L tetrabutylammonium hydroxide (pH 6.5), and the flow was 1.0 mL/min. Eluents were passed through an in-line UV detector (Waters), and eluted fractions were collected and assayed for radioactivity. Metabolites and [18F]FHBG eluted within 12 min. The procedure for analysis of the molecular distribution of radiolabel in urine was the same as that for plasma. Monkeys were studied under an approved IACUC protocol.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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Uptake and Biodistribution Studies
Figure 1 summarizes uptake of [18F]FHBG in vitro in transduced and nontransduced cells. At 1 and 5 h, activity uptake in transduced cells was 31 (P < 0.001) and 71 (P < 0.001) times higher, respectively, than that in the control cells. Figure 2 shows HPLC radiochromatograms of the ASF from transduced and nontransduced cells at 5 h of incubation. Four radioactive peaks were observed for transduced cells, corresponding to the parent compound and its presumed mono-, di-, and triphosphates, estimated as 10%, 6%, 54%, and 30% of recovered activity, respectively. The peak at 11 min is attributed to [18F]FHBG, whereas the peaks at 16, 20.5, and 23.5 min were tentatively assigned as mono-, di-, and triphosphates, respectively. Analysis of the ASF of nontransduced cells showed only 1 radioactive peak at 11 min, which corresponds to the parent compound [18F]FHBG.
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5 times that in normal tissues and organs other than bone at 2 and 5 h.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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In vitro studies revealed a much higher accumulation of [18F]FHBG with time in transduced cells compared with that in nontransduced control cells, consistent with monophosphorylation of the tracer by HSV-tk. Similar patterns of accumulation of [3H]ganciclovir and [18F]FHPG have been observed in virus-infected and transduced cells (2,28). However, uptake of [18F]FHBG at 5 h was 12-fold higher than that of [18F]FHPG in the same cell line (28), suggesting that the rate of phosphorylation of [18F]FHBG by HSV-tk or stability (or both) and intracellular retention of nucleotides thus formed is much higher than that with [18F]FHPG. A similar relationship has been shown for the parent compounds penciclovir and ganciclovir in virus-infected cell lines (5,7,12).
In control cells, no significant uptake of [18F]FHBG was observed over time, with the small amount of activity uptake representing nonspecific accumulation of the parent compound. That cellular accumulation of FHBG is unrelated to cell growth is supported by the observed 2-fold higher cell growth rate in wild-type cells (doubling time, 24 h) compared with that in transduced cells (doubling time, 48 h). Thus, higher uptake in transduced cells supports the hypothesis that the tracer is selectively phosphorylated by HSV-tk only and not by native thymidine kinase.
HPLC analysis of the ASF of the transduced cells after 5 h of incubation with [18F]FHBG showed 4 radioactive peaks. The elution profile of these peaks is consistent with that of [3H]penciclovir and its phosphates in cultured cells (12). In the analysis of a mixture of standard guanosine and its phosphates, the nucleoside and mono-, di-, and triphosphates had similar retention times of 10.5, 15.5, 19.7, and 22.8 min in our HPLC system. By analogy, the second peak in the experiments with [18F]FHBG has been assigned as the monophosphate, the third peak as the diphosphate, and the fourth peak as the triphosphate of [18F]FHBG. The intensity of the triphosphate peak was less compared with that of the diphosphate. Because nucleoside triphosphates are known to be relatively unstable (38), some of the triphosphate may have been hydrolyzed to the more stable diphosphate during the work-up, leading to inaccuracies regarding the relative proportions observed by HPLC. Because standard unlabeled phosphates of FHBG are not available, assignment of these nucleotides is tentative, and further studies on gradient elution for separation and identification of these phosphates are necessary for precise identification of these species. However, presumptive identification of these species suggests that, after monophosphorylation by HSV-tk, FHBG, like its parent compound penciclovir, is converted into its di- and triphosphates.
In vivo blood clearance studies showed that radioactivity from injected [18F]FHBG was cleared rapidly and monotonically in mice and monkeys. This agrees with observations of [18F]FHPG in nude mice (28) and [18F]FHBG in healthy human volunteers (39). At 2 h after injection in nude mice, uptake of [18F]FHBG was greater than that seen previously with [18F]FHPG for most organs and tissues studied (Fig. 7A). 18F from the 2 radiotracers washed out of most normal tissues and organs at approximately the same fractional rates between 2 and 5 h (Fig. 7). The one exception was bone. Earlier in vivo studies with [18F]FHPG in nude mice did not show uptake in bone matrix—that is, nearly all activity within bone samples was accounted for in the marrow and washed out with time (28). However, in the case of [18F]FHBG, activity concentration within bone samples was almost as high at 5 h as at 2 h, indicating that the activity did not wash out with time. Bone uptake could be attributed to the accumulation of the radiotracer in bone marrow or free fluoride in bone (or both). To differentiate between bone and marrow uptake, bones were treated with 0.5% sodium dodecyl sulfate, and marrow was removed using a needle. Bones without marrow were counted again, and no significant change of activity was observed after removal of marrow. At 5 h, 92% ± 3% of the activity was in the bone matrix compared with 89% ± 4% at 2 h. These results support the hypothesis that activity in bone at both times was predominately caused by incorporation of free fluoride by the bone matrix in addition to some nonspecific tracer uptake in the marrow. The close similarity of mean bone uptake among the 4 groups of mice (control and transduced tumors at 2 and 5 h) and the relatively small SDs for bone uptake indicate that the degree of defluorination was similar among individual mice. Finally, as shown in Figure 6, the high uptake of 18F in bone detected in mice did not occur in monkeys. Thus, there appears to be a significant difference between mice and monkeys with regard to defluorination of FHBG. As in the cell studies discussed above, higher in vivo uptake in transduced tumors compared with nontransduced tumors supports the hypothesis that [18F]FHBG is selectively phosphorylated by HSV-tk. By 5 h, the tumor-to-blood ratio in transduced tumors was approximately 21, whereas for nontransduced tumors it remained low, at approximately 1.6 (Table 2). The %ID/g in transduced tumors was approximately 13-fold higher than that in the nontransduced tumors at this time point. This finding is likely related to washout of nonspecifically bound drug and clearance of the background activity along with continued trapping of the remaining circulating parent compound, which presumably accounts for the increasing retention of drug in transduced cells over time.
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Direct assay of circulating metabolites of [18F]FHBG was not done in mice but was addressed in the nonhuman primate model. When coupled with the rapid disappearance of radiolabel from the blood (Fig. 5), the data indicate that the contribution of labeled metabolites to integral plasma activity remains small during the first 2 h after injection of [18F]FHBG (Table 3). This finding implies that the exposure of tumors and normal tissues to circulating metabolites of the injected compound is small, as reflected in the observation that 97% of the activity seen in urine at 2 h was on [18F]FHBG. Similar results were reported for the urine of normal human volunteers at 2 h, although the presence of plasma metabolites was not addressed (39).
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CONCLUSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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FOOTNOTES |
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For correspondence or reprints contact: Peter S. Conti, MD, PhD, PET Imaging Science Center, University of Southern California, 2250 Alcazar St., Suite 101, Los Angeles, CA 90033-4609.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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