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1.  Bacterial Thymidine Kinase as a Non-Invasive Imaging Reporter for Mycobacterium tuberculosis in Live Animals 
PLoS ONE  2009;4(7):e6297.
Bacteria can be selectively imaged in experimentally-infected animals using exogenously administered 1-(2′deoxy-2′-fluoro-β-D-arabinofuranosyl)-5-[125I]-iodouracil ([125I]-FIAU), a nucleoside analog substrate for bacterial thymidine kinase (TK). Our goal was to use this reporter and develop non-invasive methods to detect and localize Mycobacterium tuberculosis.
Methodology/Principal Findings
We engineered a M. tuberculosis strain with chromosomally integrated bacterial TK under the control of hsp60 - a strong constitutive mycobacterial promoter. [125I]FIAU uptake, antimicrobial susceptibilities and in vivo growth characteristics were evaluated for this strain. Using single photon emission computed tomography (SPECT), M. tuberculosis Phsp60 TK strain was evaluated in experimentally-infected BALB/c and C3HeB/FeJ mice using the thigh inoculation or low-dose aerosol infection models. M. tuberculosis Phsp60 TK strain actively accumulated [125I]FIAU in vitro. Growth characteristics of the TK strain and susceptibility to common anti-tuberculous drugs were similar to the wild-type parent strain. M. tuberculosis Phsp60 TK strain was stable in vivo and SPECT imaging could detect and localize this strain in both animal models tested.
We have developed a novel tool for non-invasive assessment of M. tuberculosis in live experimentally-infected animals. This tool will allow real-time pathogenesis studies in animal models of TB and has the potential to simplify preclinical studies and accelerate TB research.
PMCID: PMC2706987  PMID: 19606217
2.  Herpes Simplex Virus Thymidine Kinase Imaging in Mice with (1-(2’-deoxy-2’-[18F] fluoro-1-β-D-arabinofuranosyl)-5-iodouracil) and metabolite (1-(2’-deoxy-2’-[18F] fluoro-1-β-D-arabinofuranosyl)-5-uracil) 
FIAU, (1-(2’-deoxy-2’-fluoro-1-β-D-arabinofuranosyl)-5-iodouracil) has been used as a substrate for herpes simplex virus thymidine kinases (HSV-TK and HSV-tk, for protein and gene expression respectively) and other bacterial and viral thymidine kinases for noninvasive imaging applications. Previous studies have reported the formation of a de-iodinated metabolite of 18F-FIAU. This study reports the dynamic tumor uptake, biodistribution and metabolite contribution to the activity of 18F-FIAU seen in HSV-tk gene expressing tumors and compares the distribution properties with its de-iodinated metabolite 18F-FAU.
CD-1 nu/nu mice with subcutaneous MH3924A and MH3924A-stb-tk+ xenografts on opposite flanks were used for the biodistribution and imaging studies. Mice were injected IV with either 18F-FIAU or 18F-FAU. Mice underwent dynamic imaging with each tracer for 65 min followed by additional static imaging up to 150 min post injection for some animals. Animals were sacrificed at 60 or 150 min post injection. Samples of blood and tissue were collected for biodistribution and metabolite analysis. Regions of interest were drawn over the images obtained from both tumors to calculate the time activity curves.
Biodistribution and imaging studies showed the highest uptake of 18F-FIAU in the MH3924A-stb-tk+ tumors. Dynamic imaging studies revealed a continuous accumulation of 18F-FIAU in HSV-TK expressing tumors over 60 min. The mean biodistribution values (SUV±SE) for MH3924A-stb-tk+ were 2.07±0.40, 6.15±1.58, and that of MH3924A tumors were 0.19±0.07, 0.47±0.06 at 60 and 150 min respectively. In 18F-FIAU injected mice, at 60 min nearly 63% of blood activity was present as its metabolite 18F-FAU. Imaging and biodistribution studies with 18F-FAU demonstrated no specific accumulation in MH3924A-stb-tk+ tumors and SUVs for both the tumors were similar to those observed with muscle.
18F-FIAU shows a continuous accumulation of activity in HSV-TK expressing tumors. 18F-FAU does not show any preferential accumulation in HSV-TK expressing tumors. In the 18F-FIAU treated mice, the 18F-FAU contribution to the total uptake seen in HSV-TK positive tumors is minimal.
PMCID: PMC3107601  PMID: 19506865
Fluorine-18; FIAU; HSV-TK; Gene Expression; Metabolism; PET Imaging
3.  Comparison of Cell-Labeling Methods with 124I-FIAU and 64Cu-PTSM for Cell Tracking Using Chronic Myelogenous Leukemia Cells Expressing HSV1-tk and Firefly Luciferase 
Cell-tracking methods with molecular-imaging modality can monitor the biodistribution of cells. In this study, the direct-labeling method with 64Cu-pyruvaldehyde-bis(N4-methylthiosemicarbazone) (64Cu-PTSM), indirect cell-labeling methods with herpes simplex virus type 1-thymidine kinase (HSV1-tk)-mediated 124I-2′-fluoro-2′-deoxy-1-β-d-arabinofuranosyl-5-iodouracil (124I-FIAU) were comparatively investigated in vitro and in vivo for tracking of human chronic myelogenous leukemia cells. K562-TL was established by retroviral transduction of the HSV1-tk and firefly luciferase gene in the K562 cell. K562-TL cells were labeled with 64Cu-PTSM or 124I-FIAU. Cell labeling efficiency, viability, and radiolabels retention were compared in vitro. The biodistribution of radiolabeled K562-TL cells with each radiolabel and small-animal positron emission tomography imaging were performed. Additionally, in vivo and ex vivo bioluminescence imaging (BLI) and tissue reverse transcriptase–polymerase chain reaction (RT-PCR) analysis were used for confirming those results. K562-TL cells were efficiently labeled with both radiolabels. The radiolabel retention (%) of 124I-FIAU (95.2%±1.1%) was fourfold higher than 64Cu-PTSM (23.6%±0.7%) at 24 hours postlabeling. Viability of radiolabeled cells was statistically nonsignificant between 124I-FIAU and 64Cu-PTSM. The radioactivity of each radiolabeled cells was predominantly accumulated in the lungs and liver at 2 hours. Both the radioactivity of 64Cu-PTSM- and 124I-FIAU-labeled cells was highly accumulated in the liver at 24 hours. However, the radioactivity of 124I-FIAU-labeled cells was markedly decreased from the body at 24 hours. The K562-TL cells were dominantly localized in the lungs and liver, which also verified by BLI and RT-PCR analysis at 2 and 24 hours postinjection. The 64Cu-PTSM-labeled cell-tracking method is more efficient than 124I-FIAU-labeled cell tracking, because of markedly decrease of radioactivity and fast efflux of 124I-FIAU in vivo. In spite of a high labeling yield and radiolabel retention of 124I-FIAU in vitro, the in vivo cell-tracking method using 64Cu-PTSM could be a useful method to evaluate the distribution and targeting of various cell types, especially, stem cells and immune cells.
PMCID: PMC3516418  PMID: 23009582
gene transfer; molecular imaging; PET
4.  The Use of 14C-FIAU to Predict Bacterial Thymidine Kinase Presence: Implications for Radiolabeled FIAU Bacterial Imaging 
Nuclear medicine and biology  2013;40(5):638-642.
Currently available infectious disease imaging techniques cannot differentiate between infection and sterile inflammation or between different types of infections. Recently, radiolabeled FIAU was found to be a substrate for the thymidine kinase (TK) enzyme of multiple pathogenic bacteria, leading to its translational use in the imaging of bacterial infections. Patients with immunodeficiencies, however, are susceptible to a different group of pathogenic bacteria when compared to immunocompetent subjects. In this study, we wanted to predict the usefulness of radiolabeled FIAU in the detection of bacterial infections commonly occurring in patients with immunodeficiencies, in vitro, prior to attempting in vivo imaging with 124I-FIAU-PET.
We obtained representative strains of bacterial pathogens isolated from actual patients with genetic immunodeficiencies. We evaluated the bacterial susceptibility of different strains to the effect of incubation with FIAU, which would implicate the presence of the thymidine kinase (TK) enzyme. We also incubated the bacteria with 14C-FIAU and consequently measured its rate of incorporation in the bacterial DNA using a liquid scintillation counter.
Unlike the other bacterial strains, the growth of Pseudomonas aeruginosa was not halted by FIAU at any concentration. All the tested clinical isolates demonstrated different levels of 14C-FIAU uptake, except for P. aeruginosa.
Radiolabeled FIAU has been successful in delineating bacterial infections, both in preclinical and pilot translational studies. In patients with immunodeficiencies, Pseudomonas infections are commonly encountered and are usually difficult to differentiate from fungal infections. The use of radiolabeled FIAU for in vivo imaging of those patients, however, would not be useful, considering the apparent lack of TK enzyme in Pseudomonas. One has to keep in mind that not all pathogenic bacteria possess the TK enzyme and as such will not all retain FIAU. Our technique is simple, and can be easily used to assess whether a certain bacterial strain of interest can or cannot be visualized using radiolabeled FIAU.
PMCID: PMC3665620  PMID: 23541824
Bacterial imaging; PET; FIAU; thymidine kinase
5.  Activity of 1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5-iodouracil against simian varicella virus infections in African green monkeys. 
The fluorinated pyrimidines 1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5-iodouracil (FIAU) and 1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5-methyluracil (FMAU) are highly effective inhibitors of herpesvirus infections in vitro and in vivo. This report is concerned with an evaluation of their activities in African green monkeys (Cercopithecus aethiops) infected with simian varicella virus, a herpesvirus closely related to human varicella-zoster virus. Oral or intravenous administration of FIAU at 50 mg/kg per day as divided doses beginning 48 h after virus inoculation prevented the development of evidences of clinical infection. Oral treatment with FIAU at 30 mg/kg per day deferred as late as 7 days after virus inoculation modified the course of the disease. When treatment was started 48 h after virus inoculation, daily doses of FIAU as small as 1 mg/kg inhibited development of infections; daily doses of 0.2 mg/kg were ineffective. At the latter dose FMAU prevented development of clinical disease, suggesting that it was more active than FIAU. No signs of FIAU toxicity were observed, with the single exception of an early but transitory elevation in aspartate aminotransferase activity in serum.
PMCID: PMC180356  PMID: 3729332
6.  Biotransformation and elimination of [2-14C]-1-(2-deoxy-2'-fluoro-beta-D -arabinofuranosyl)-5-iodocytosine in immunosuppressed patients with herpesvirus infections. 
The metabolism of the drug [2-14C]-1-(2'-deoxy-2'-fluoro-beta-D -arabinofuranosyl)-5-iodocytosine (FIAC), a potent inhibitor of herpesvirus replication, was studied in immunosuppressed patients with herpesvirus infections. FIAC was administered intravenously by 15-min infusion and by mouth 24 h later to four patients at doses of 50 or 100 mg/m2. FIAC was cleared from the plasma primarily by biotransformation in liver, kidney, and peripheral blood, with a terminal-phase half-life of 0.92 to 1.80 h (mean, 1.36 h) after intravenous administration. The area under the concentration-time curve from zero to infinity (AUC0-infinity) for FIAC was 1.6 to 4.7% (mean, 3.4%) of the AUC0-infinity for total radioactivity. 1-(2'-Deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5-iodouracil (FIAU) was the major metabolite; the AUC0-infinity for FIAU was 54.3 to 72.5% (mean, 63.4%) of the AUC0-infinity for total radioactivity. The terminal-phase half-life for FIAU was 3.32 to 4.49 h (mean, 3.91 h); FIAU was cleared from plasma by renal elimination and further biotransformation. lesser amounts of 1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)uracil, 1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)cytosine, the glucuronide conjugates of these metabolites, and the glucuronide conjugates of FIAC and FIAU were also formed. A comparison of the AUC0-infinity for total radioactivity after intravenous and oral administration suggested that nearly all of the oral dose was absorbed. Plasma levels of FIAU, also a potent inhibitor of herpesvirus replication in vitro, exceeded the 50% effective dose for herpes simplex virus and varicella-zoster virus as late as 12 h after administration of FIAC.
PMCID: PMC180143  PMID: 2990323
7.  In Vivo characterization of a reporter gene system for imaging hypoxia-induced gene expression 
Nuclear medicine and biology  2009;36(7):821-831.
To characterize a tumor model containing a hypoxia-inducible reporter gene and to demonstrate utility by comparison of reporter gene expression to the uptake and distribution of the hypoxia tracer 18F-fluoromisonidazole (18F-FMISO).
I. Three tumors derived from the rat prostate cancer cell line R3327-AT were grown in each of two rats as follows: 1. parental R3327-AT, 2. positive control R3327-AT/PC in which the HSV1-tkeGFP fusion reporter gene was expressed constitutively, 3. R3327-AT/HRE in which the reporter gene was placed under the control of a HIF-responsive promoter sequence (HRE). Animals were co-administered a hypoxia-specific marker (pimonidazole) and the reporter gene probe 124I-2′-fluoro-2′-deoxy-1-ß-d-arabinofuranosyl-5-iodouracil (124I-FIAU) 3hr prior to sacrifice. Statistical analysis of the spatial association between 124I-FIAU uptake and pimonidazole fluorescent staining intensity was then performed on a pixel-by-pixel basis.
II. Utility of this system was demonstrated by assessment of reporter gene expression versus the exogenous hypoxia probe 18F-FMISO. Two rats, each bearing a single R3327-AT/HRE tumor, were injected with 124I-FIAU (3hr before sacrifice) and 18F-FMISO (2hr before sacrifice). Statistical analysis of the spatial association between 18F-FMISO and 124I-FIAU on a pixel-by-pixel basis was performed.
I. Correlation coefficients between 124I-FIAU uptake and pimonidazole staining intensity were: 0.11 in R3327-AT tumors, −0.66 in R3327-AT/PC and 0.76 in R3327-AT/HRE, confirming that only in the R3327-AT/HRE tumor was HSV1-tkeGFP gene expression associated with hypoxia.
II. Correlation coefficients between 18F-FMISO and 124I-FIAU uptakes in R3327-AT/HRE tumors were r= 0.56, demonstrating good spatial correspondence between the two tracers.
We have confirmed hypoxia-specific expression of the HSV1-tkeGFP fusion gene in the R3327-AT/HRE tumor model and demonstrated the utility of this model for the evaluation of radiolabeled hypoxia tracers.
PMCID: PMC2754273  PMID: 19720294
Hypoxia; cancer; reporter gene; nuclear medicine; PET tracer validation
8.  Phosphorylation of the anti-hepatitis B nucleoside analog 1-(2'-deoxy-2'-fluoro-1-beta-D-arabinofuranosyl)-5-iodouracil (FIAU) by human cytosolic and mitochondrial thymidine kinase and implications for cytotoxicity. 
The capacity of recombinant human cytosolic thymidine kinase (TK1) and bovine mitochondrial thymidine kinase (TK2) to phosphorylate the antiviral analogs 1-(2'-deoxy-2'-fluoro-1-beta-D-arabinofuranosyl)-5-iodouracil (FIAU) and 1-(2'-deoxy-2'-fluoro-1-beta-D-arabinofuranosyl)-5-methyluracil (FMAU) has been analyzed. The Vmax/Km ratios for FIAU and FMAU with TK2 are about 30% of that for deoxythymidine, while the corresponding values for TK1 are 2 and 5%, respectively. Thus, these two analogs are more efficient substrates for TK2 than for TK1, which may be part of the explanation for the mitochondrial toxicity associated with FIAU during treatment of hepatitis B infection.
PMCID: PMC163369  PMID: 8726039
9.  Differential effects of the incorporation of 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-iodouracil (FIAU) on the binding of the transcription factors, AP-1 and TFIID, to their cognate target DNA sequences. 
Nucleic Acids Research  1996;24(21):4111-4116.
The thymidine analog, 1-(2-deoxy-2-fluoro-beta-D-arabino-furanosyl)-5-iodouracil (FIAU), is incorporated into DNA in cell culture and in vivo. To investigate the effect of incorporation of FIAU into DNA on the binding of transcription factors, oligonucleotide duplexes which bind specifically to activator protein 1 (AP-1) or to TFIID were synthesized and binding of these oligonucleotides to their respective proteins was studied using gel-shift analysis. When thymidine at position -3, -1, 1 or 7 (relative to the first thymidine of the core binding sequence) was replaced with FIAU, binding to AP-1 was approximately 82, 28, 86 and 51%, respectively, of the binding to the non-substituted oligonucleotide to AP-1. When thymidine at position 3 or 5 (each adjacent to the center of dyad symmetry) was replaced with FIAU, binding to AP-1 was abrogated. Oligonucleotides containing FIAU at positions -1, 3 or 5, were much less able to compete with radiolabeled wild-type oligonucleotides for binding to AP-1. In contrast, the presence of FIAU, depending on its location, resulted in the increased binding of TFIID to its consensus target DNA sequence. These results indicate that incorporation of FIAU into DNA may induce local conformational changes resulting in the altered ability of transcriptional factors to bind to their cognate DNA sequences. Additional studies demonstrated that the presence of FIAU at a position 5' to the cleavage site in the consensus sequence T*TAA (where * is the cleavage site) inhibited restriction of the oligomeric duplex by MseI.
PMCID: PMC146219  PMID: 8932359
10.  FIAU: From reporter gene imaging to imaging of bacterial proliferation 
The radioiodinated thymidine analogue, FIAU, is a tracer that has been developed for reporter gene, for cells that were transfected with herpes simplex virus thymidine kinase, HSV-TK. FIAU is also a specific substrate of bacterial TK due to the homology between viral and bacterial TK. In this issue of AJNMMI (, Pullamb-hatla et al. reported that the accumulation of 125I-FIAU in pulmonary infectious foci correlated with the bacterial burden in the lungs. 125I-FIAU could be used to monitor the efficacy of anti-microbial therapy in mice. Potentially 124I-FIAU PET could be used to discriminate microbial from sterile inflammation in patients with prosthetic implants.
PMCID: PMC3477741  PMID: 23133817
Infection; inflammation; reporter gene imaging; positron emission tomography (PET)
11.  Activities of 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-iodocytosine and its metabolites against herpes simplex virus types 1 and 2 in cell culture and in mice infected intracerebrally with herpes simplex virus type 2. 
As measured by plaque and yield reduction assays, several metabolites of 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-iodocytosine (FIAC) were highly active against herpes simplex virus types 1 and 2. These metabolites included the 2'-deoxy-2'-fluoroarabinosyl derivatives of 5-iodouracil (FIAU), cytosine (FAC), uracil (FAU), and thymine (FMAU). In mice inoculated intracerebrally with herpes simplex virus type 2, the relative order of potency of these compounds and licensed antiviral drugs was as follows: FMAU much greater than FIAC approximately equal to FIAU greater than acyclovir approximately equal to vidarabine much greater than FAC approximately equal to FAU. One of the main metabolites of FMAU, 2'-fluoro-5-hydroxymethyl-arabinosyluracil, was essentially inactive in vivo. FIAC-, FIAU-, FMAU-, FAC-, and FAU-resistant herpes simplex virus variants prepared in cell culture were found to be (i) devoid of viral thymidine kinase, (ii) cross-resistant to one another and resistant to drugs requiring viral thymidine kinase for activation, and (iii) sensitive to vidarabine or phosphonoformate. These results indicate that FIAC, FIAU, and FMAU require the virally encoded thymidine kinase for activation and suggest that the antiviral activity of FAU and FAC in cell cultures is also mediated by this enzyme. The interaction of the fluoroarabinosyl pyrimidine nucleosides with herpes simplex virus thymidine kinase in a cell-free system is also described.
PMCID: PMC180368  PMID: 3015003
12.  Fialuridine Induces Acute Liver Failure in Chimeric TK-NOG Mice: A Model for Detecting Hepatic Drug Toxicity Prior to Human Testing 
PLoS Medicine  2014;11(4):e1001628.
Gary Peltz, Jeffrey Glenn, and colleagues report that a pre-clinical mouse toxicology model can detect liver toxicity of a drug that caused liver failure in several early clinical trial participants in 1993.
Please see later in the article for the Editors' Summary
Seven of 15 clinical trial participants treated with a nucleoside analogue (fialuridine [FIAU]) developed acute liver failure. Five treated participants died, and two required a liver transplant. Preclinical toxicology studies in mice, rats, dogs, and primates did not provide any indication that FIAU would be hepatotoxic in humans. Therefore, we investigated whether FIAU-induced liver toxicity could be detected in chimeric TK-NOG mice with humanized livers.
Methods and Findings
Control and chimeric TK-NOG mice with humanized livers were treated orally with FIAU 400, 100, 25, or 2.5 mg/kg/d. The response to drug treatment was evaluated by measuring plasma lactate and liver enzymes, by assessing liver histology, and by electron microscopy. After treatment with FIAU 400 mg/kg/d for 4 d, chimeric mice developed clinical and serologic evidence of liver failure and lactic acidosis. Analysis of liver tissue revealed steatosis in regions with human, but not mouse, hepatocytes. Electron micrographs revealed lipid and mitochondrial abnormalities in the human hepatocytes in FIAU-treated chimeric mice. Dose-dependent liver toxicity was detected in chimeric mice treated with FIAU 100, 25, or 2.5 mg/kg/d for 14 d. Liver toxicity did not develop in control mice that were treated with the same FIAU doses for 14 d. In contrast, treatment with another nucleotide analogue (sofosbuvir 440 or 44 mg/kg/d po) for 14 d, which did not cause liver toxicity in human trial participants, did not cause liver toxicity in mice with humanized livers.
FIAU-induced liver toxicity could be readily detected using chimeric TK-NOG mice with humanized livers, even when the mice were treated with a FIAU dose that was only 10-fold above the dose used in human participants. The clinical features, laboratory abnormalities, liver histology, and ultra-structural changes observed in FIAU-treated chimeric mice mirrored those of FIAU-treated human participants. The use of chimeric mice in preclinical toxicology studies could improve the safety of candidate medications selected for testing in human participants.
Please see later in the article for the Editors' Summary
Editors' Summary
Before new drugs are approved for clinical use, they undergo extensive preclinical (laboratory-based) and clinical testing. In the preclinical studies, scientists investigate the causes of diseases, identify potential new drugs, and test promising drug candidates in animals. Animal testing is performed to determine whether the new drug is likely to work, and to screen for drug-induced toxicity. In preclinical toxicology studies, new drugs are given to two or more animal species to find out whether the drug has any short- or long-term toxic effects such as damage to the liver (hepatotoxicity). Drugs that pass these animal tests enter clinical trials. Phase I clinical trials test new drugs in a handful of healthy volunteers or patients to evaluate their safety and to identify possible side effects. In phase II trials, a larger group of patients receives the new drug to evaluate its safety further and to get an initial idea of its effectiveness. Finally, in phase III trials, very large groups of patients are randomly assigned to receive the new drug or an established treatment for their disease. These randomized controlled trials provide detailed information about the effectiveness and safety of a candidate drug, and must be completed before a drug can be approved for clinical use.
Why Was This Study Done?
Since animals are not perfect models for people, candidate drugs can cause toxicities in clinical trials that were not predicted by preclinical toxicology testing performed using animal species. For example, in 1993, 15 participants in a phase II trial were given a nucleoside analogue called fialuridine to treat hepatitis B virus infection (nucleoside analogues often have antiviral activity). Seven participants developed liver failure and lactic acidosis (buildup of lactic acid in the blood). Analysis of liver tissue from the affected participants revealed steatosis (fatty degeneration), intracellular fat droplets, and swollen mitochondria (these organelles are the powerhouses of the cell). Five participants subsequently died, and two had to have a liver transplant. In preclinical toxicology testing in mice, rats, dogs, and primates, there had been no indications that fialuridine would be hepatotoxic in people. It now seems that the expression of a nucleoside transporter in the mitochondria of humans but not of other animals may underlie the human-specific mitochondrial toxicity and hepatotoxicity of fialuridine. With several other nucleoside analogues in development, a better screening tool for human-specific mitochondrial toxicity is needed. In this study, the researchers investigate whether fialuridine toxicity can be detected in TK-NOG mice with chimeric (humanized) livers. TK-NOG mice are immunodeficient mice that have been genetically engineered so that human liver cells (hepatocytes) transplanted into these animals establish a long-lived mature “human organ.”
What Did the Researchers Do and Find?
The researchers treated chimeric (with transplanted human liver cells) and control (without transplanted human liver cells) TK-NOG mice with several doses of fialuridine. After treatment with the highest dose (1,600-fold above the dose used in the phase II trial) for four days, the chimeric mice developed liver failure and lactic acidosis. Moreover, steatosis and lipid and mitochondrial abnormalities developed in the regions of their livers that contained human hepatocytes but not in regions that contained mouse hepatocytes. Notably, the control mice had not developed liver toxicity after 14 days of treatment with the highest dose of drug. Liver toxicity was also easily detectable in chimeric mice that had been treated for 14 days with a fialuridine dose only 10-fold above that used in the human trial. Treatment with another nucleoside analogue that does not cause liver toxicity in people did not cause liver toxicity in the chimeric mice.
What Do These Findings Mean?
These findings show that fialuridine-induced liver toxicity can be readily detected using TK-NOG mice that have humanized livers at drug doses only 10-fold higher than those that caused liver failure in the phase II trial. Although the liver toxicity developed much more quickly in these mice than in the human trial participants, the clinical features, laboratory abnormalities, and structural changes seen in the fialuridine-treated chimeric TK-NOG mice closely mirrored those seen in fialuridine-treated people. The use of TK-NOG mice containing humanized livers in toxicology testing will not reveal whether drugs have human-specific toxicities outside the liver. Since they are highly immunocompromised, chimeric TK-NOG mice cannot be used to detect immune-mediated drug toxicities. Nevertheless, these findings suggest that the use of chimeric mice in toxicology studies could help improve the safety of candidate drugs that are tested in humans.
Additional Information
Please access these websites via the online version of this summary at
The US Food and Drug Administration, the body that approves drugs for clinical use in the US, provides an overview for patients about the drug development process from the laboratory to the clinic
The UK Medicines and Healthcare Products Regulatory Agency (MHRA) provides more detailed information for patients and the public about the drug development process, including a section on preclinical research, which includes information on animal testing
The US National Institutes of Health provides information about clinical trials, including personal stories from people who have taken part in clinical trials
The UK National Health Service Choices website has information for patients about clinical trials and medical research, including personal stories about participation in clinical trials
Understanding Animal Research is a UK advocacy group that provides information about the importance of animal research to the public, teachers, scientists, journalists, and policy makers
Wikipedia has a page on animal testing (note that Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
PMCID: PMC3988005  PMID: 24736310
13.  Imaging of Musculoskeletal Bacterial Infections by [124I]FIAU-PET/CT 
PLoS ONE  2007;2(10):e1007.
Traditional imaging techniques for the localization and monitoring of bacterial infections, although reasonably sensitive, suffer from a lack of specificity. This is particularly true for musculoskeletal infections. Bacteria possess a thymidine kinase (TK) whose substrate specificity is distinct from that of the major human TK. The substrate specificity difference has been exploited to develop a new imaging technique that can detect the presence of viable bacteria.
Methodology/Principal Findings
Eight subjects with suspected musculoskeletal infections and one healthy control were studied by a combination of [124I]FIAU-positron emission tomography and CT ([124I]FIAU-PET/CT). All patients with proven musculoskeletal infections demonstrated positive [124I]FIAU-PET/CT signals in the sites of concern at two hours after radiopharmaceutical administration. No adverse reactions with FIAU were observed.
[124I]FIAU-PET/CT is a promising new method for imaging bacterial infections.
PMCID: PMC1994593  PMID: 17925855
14.  Improved synthesis of 2′-deoxy-2′-[18F]-fluoro-1-β-d-arabinofuranosyl-5-iodouracil ([18F]-FIAU) 
Nuclear medicine and biology  2010;37(4):439-442.
An improved synthesis of 2′-[18F]-fluoro-2′-deoxy-1-β-d-arabinofuranosyl-5-iodouracil ([18F]-FIAU) has been developed. The method utilizes trimethylsilyl trifluoromethanesulfonate (TMSOTf) catalyzed coupling of 2-deoxy-2-[18F]-fluoro-1,3,5-tri-O-benzoyl-d-arabinofuranose with 2,4-bis(trimethylsilyloxy)-5-iodouracil to yield the protected dibenzoyl-[18F]-FIAU. Dibenzoyl-[18F]-FIAU was deprotected with sodium methoxide to yield a mixture of α- and β-anomers in a ratio of 1:1, which were purified by HPLC. The procedure described in this article eliminates the need for HBr activation of the sugar prior to coupling with silylated iodouracil and is suitable for automation. The total reaction time was about 110 min, starting from [18F]-fluoride. The average isolated yield of the required β-anomer was 10±6% (decay corrected) with average specific activity of 125 mCi/μmol.
PMCID: PMC4410717  PMID: 20447555
FIAU; PET; Microfluidics; HSV1-tk
15.  Functional Coexpression of HSV-1 Thymidine Kinase and Green Fluorescent Protein: Implications for Noninvasive Imaging of Transgene Expression 
Neoplasia (New York, N.Y.)  1999;1(2):154-161.
Current gene therapy technology is limited by the paucity of methodology for determining the location and magnitude of therapeutic transgene expression in vivo. We describe and validate a paradigm for monitoring therapeutic transgene expression by noninvasive imaging of the herpes simplex virus type 1 thymidine kinase (HSV-1-tk) marker gene expression. To test proportional coexpression of therapeutic and marker genes, a model fusion gene comprising green fluorescent protein (gfp) and HSV-1-tk genes was generated (tkgfp gene) and assessed for the functional coexpression of the gene product, TKGFP fusion protein, in rat 9L gliosarcoma, RG2 glioma, and W256 carcinoma cells. Analysis of the TKGFP protein demonstrated that it can serve as a therapeutic gene by rendering tkgfp transduced cells sensitive to ganciclovir or as a screening marker useful for identifying transduced cells by fluorescence microscopy or fluorescence-activated cell sorting (FACS). TK and GFP activities in the TKGFP fusion protein were similar to corresponding wild-type proteins and accumulation of the HSV-1-tk-specific radiolabeled substrate, 2′-fluoro-2′-deoxy-1β-d-arabino-furanosyl-5-iodo-uracil (FIAU), in stability transduced clones correlated with gfp-fluorescence intensity over a wide range of expression levels. The tkgfp fusion gene itself may be useful in developing novel cancer gene therapy approaches. Valuable information about the efficiency of gene transfer and expression could be obtained by non-invasive imaging of tkgfp expression with FIAU and clinical imaging devices (gamma camera, positron-emission tomography [PET], single photon emission computed tomography [SPECT]), and/or direct visualization of gfp expression in situ by fluorescence microscopy or endoscopy.
PMCID: PMC1508134  PMID: 10933050
thymidine kinase; ganciclovir; FIAU; cancer gene therapy; fusion genes; imaging
16.  Treatment of primary acute genital herpes in guinea pigs by intraperitoneal administration of fluoropyrimidines. 
FIAC [1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5-iodocytosine], FIAU [1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5-iodouracil], and FMAU [1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5-methyluracil] were evaluated for their efficacies in the treatment of genital infections with herpes simplex virus type 2 in guinea pigs. Intraperitoneal administration of these drugs in daily doses of 100 mg/kg of body weight initiated 24 h after virus inoculation and repeated 2 successive days thereafter inhibited development of genital lesions and reduced shedding of virus without evoking untoward reactions. In a comparative study with this 3-day dosage schedule, the efficacy of daily doses of 50 mg of FMAU per kg was greater than that of the same doses of FIAC and FIAU, in that order; all these were more effective than daily doses of 50, 100, or 200 mg of acyclovir or of 500 mg of phosphonoformic acid per kg. These differences in efficacy were enhanced when treatment was delayed for 2 to 3 days after inoculation.
PMCID: PMC176169  PMID: 6239591
17.  Imaging of Viral Thymidine Kinase Gene Expression by Replicating Oncolytic Adenovirus and Prediction of Therapeutic Efficacy 
Yonsei Medical Journal  2008;49(5):811-818.
We have used a genetically attenuated adenoviral vector which expresses HSVtk to assess the possible additive role of suicidal gene therapy for enhanced oncolytic effect of the virus. Expression of TK was measured using a radiotracer-based molecular counting and imaging system.
Materials and Methods
Replication-competent recombinant adenoviral vector (Ad-ΔE1B19/55) was used in this study, whereas replication-incompetent adenovirus (Ad-ΔE1A) was generated as a control. Both Ad-ΔE1B19/55-TK and Ad-ΔE1A-TK comprise the HSVtk gene inserted into the E3 region of the viruses. YCC-2 cells were infected with the viruses and incubated with 2'-deoxy-2'-fluoro-β-D-arabinofuranosyl-5-iodouracil (I-131 FIAU) to measure amount of radioactivity. The cytotoxicity of the viruses was determined, and gamma ray imaging of HSVtk gene was performed. MTT assay was also performed after GCV treatment.
On gamma counter-analyses, counts/minute (cpm)/µg of protein showed MOIs dependency with ΔE1B19/55-TK infection. On MTT assay, Ad-ΔE1B19/55-TK led to more efficient cell killing than Ad-ΔE1A-TK. On plate imaging by gamma camera, both Ad-ΔE1B19/55-TK and Ad-ΔE1A-TK infected cells showed increased I-131 FIAU uptake in a MOI dependent pattern, and with GCV treatment, cell viability of ΔE1B19/55-TK infection was remarkably reduced compared to that of Ad-ΔE1A-TK infection.
Replicating Ad-ΔE1B19/55-TK showed more efficient TK expression even in the presence of higher-cancer cell killing effects compared to non-replicating Ad-ΔE1A-TK. Therefore, GCV treatment still possessed an additive role to oncolytic effect of Ad-ΔE1B19/55-TK. The expression of TK by oncolytic viruses could rapidly be screened using a radiotracer-based counting and imaging technique.
PMCID: PMC2615367  PMID: 18972602
Oncolysis; adenovirus; thymidine kinase; gene therapy; radiotracer
18.  Sensitive and specific radioimmunoassay for fialuridine: initial assessment of pharmacokinetics after single oral doses to healthy volunteers. 
Fialuridine (FIAU) is a halogen-substituted analog of thymidine that was undergoing clinical investigation as a drug for the treatment of chronic hepatitis B viral infection. However, clinical trials of FIAU were terminated after adverse events occurred following chronic oral administration. Prior to the termination of clinical trials, a sensitive assay was needed for the measurement of FIAU because of the anticipated low dose administered to patients. We therefore undertook the development of a radioimmunoassay (RIA). A specific antiserum was raised in rabbits following immunization with a 5'-O-hemisuccinate analog of FIAU coupled to keyhole limpet hemocyanin. Radiolabeled FIAU was synthesized by a destannylation procedure by using sodium [125I]iodide. We developed a competitive-binding procedure and used precipitation with polyethylene glycol as the method for separating the bound and free forms of FIAU. The RIA is sensitive (0.2 ng/ml), specific (negligible interference from known metabolites and endogenous nucleosides), and reproducible (interassay coefficients of variation range from 5 to 19.7% for serum controls). We used the RIA to assess the pharmacokinetics of FIAU in healthy adult volunteers following administration of a single 5-mg oral dose. The sensitivity of the RIA permitted the detection of a prolonged elimination phase for FIAU in healthy volunteers and dogs, with mean elimination half-lives of 29.3 and 35.3 h, respectively. We conclude the RIA is a valid method for the quantification of FIAU in biological fluids.
PMCID: PMC284697  PMID: 7811032
19.  Focused ultrasound enhanced molecular imaging and gene therapy for multifusion reporter gene in glioma-bearing rat model 
Oncotarget  2015;6(34):36260-36268.
The ability to monitor the responses of and inhibit the growth of brain tumors during gene therapy has been severely limited due to the blood-brain barrier (BBB). A previous study has demonstrated the feasibility of noninvasive in vivo imaging with 123I-2′-fluoro-2′-deoxy-5-iodo-1-β-D-arabinofuranosyluracil (123I-FIAU) for monitoring herpes simplex virus type 1 thymidine kinase (HSV1-tk) cancer gene expression in an experimental animal model. Here, we tested the enhancement of SPECT with 123I-FIAU and ganciclovir (GCV) treatment in brain tumors after BBB disruption induced by focused ultrasound (FUS) in the presence of microbubbles. We established an orthotopic F98 glioma-bearing rat model with trifusion reporter genes. The results of this study showed that the rat model of HSV1-tk-expressing glioma cells could be successfully detected by SPECT imaging after FUS-induced BBB disruption on day 10 after implantation. Compared to the control group, animals receiving the GCV with or without sonication exhibited a significant antitumor activity (P < 0.05) of glioma cells on day 16 after implantation. Moreover, combining sonication with GCV significantly inhibited tumor growth compared with GCV alone. This study demonstrated that FUS may be used to deliver a wide variety of theranostic agents to the brain for molecular imaging and gene therapy in brain diseases.
PMCID: PMC4742175  PMID: 26429860
focused ultrasound; molecular imaging; gene therapy; blood-brain barrier; brain tumor
20.  Bortezomib-induced enzyme-targeted radiotherapy in herpesvirus-associated tumors 
Nature medicine  2008;14(10):1118-1122.
We investigated the possibility of using a pharmacologic agent to modulate viral gene expression in order to target radiotherapy to tumor tissue. In a murine xenograft model, we had previously shown targeting of [125I]2'-fluoro-2'-deoxy-beta-D-5-iodouracilarabinofuranoside ([125I]FIAU) to tumors engineered to express the Epstein-Barr virus (EBV)-thymidine kinase (TK). Here we extend those results to targeting of a therapeutic radiopharmaceutical [131I]FIAU to slow or stop tumor growth or to achieve tumor regression. These outcomes were achieved in xenografts with tumors that constitutively expressed the EBV-TK, as well as with naturally-infected EBV tumor cell lines. Burkitt's lymphoma and gastric carcinoma required activation of viral gene expression by pretreatment with bortezomib. Marked changes in tumor growth could also be achieved in naturally-infected Kaposi's sarcoma herpesvirus (KSHV) tumors following bortezomib activation. Bortezomib-induced enzyme-targeted radiation (BETR) therapy illustrates the possibility of pharmacologically modulating tumor gene expression to effect targeted radiotherapy.
PMCID: PMC2709824  PMID: 18776891
21.  Efficacy and selectivity of some nucleoside analogs as anti-human cytomegalovirus agents. 
1-(2'-Deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5-iodocytosine (FIAC), 1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5-methyluridine (FMAU), 1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5-iodouridine (FIAU), and 1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5-ethyluridine (FEAU) were evaluated for antiviral activities against human cytomegalovirus (HCMV) and compared with 9-[(2-hydroxyethoxy)methyl]guanine (acyclovir) and E-5-(2'-bromovinyl)-2'-deoxyuridine (BVDU). The relative anti-HCMV potencies of these compounds, as determined by calculating the dose of drug which inhibited 50% plaque formation, were in order of decreasing potency: FIAC greater than FIAU greater than FMAU greater than acyclovir greater than FEAU greater than BVDU. The antiviral activity of FIAC occurred at levels much lower than those that caused cytotoxic or cytostatic effects in uninfected fibroblasts. Neither thymidine nor deoxycytidine reversed the anti-HCMV activity of FIAC, indicating that this drug was not acting as an analog of the natural nucleosides. FIAC was not phosphorylated by cytosols of HCMV-infected cells to a greater extent that by those of uninfected cells, indicating that, unlike the antiviral activity against herpes simplex virus type 1, the selectivity of this drug is probably not based on a virus-specified pyrimidine kinase.
PMCID: PMC185363  PMID: 6316843
22.  Comparative efficacy and selectivity of some nucleoside analogs against Epstein-Barr virus. 
The effects of (2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-iodocytosine (FIAC), 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-methyluridine (FMAU), 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-iodouridine (FIAU), (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVdU), and 9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG or BW B759U) on the replication of Epstein-Barr virus (EBV) in vitro were evaluated and compared with that of acyclovir (ACV). The relative potencies of these drugs, on the basis of anti-EBV activity, were: FIAC = FIAU greater than FMAU greater than DHPG greater than BVdU greater than ACV; on the basis of the therapeutic index they were: BVdU greater than DHPG greater than FIAC greater than ACV greater than FIAU greater than FMAU. Differential inhibition of EBV-associated polypeptides by these drugs was observed.
PMCID: PMC180200  PMID: 2992367
23.  Different strategies for reducing intestinal background radioactivity associated with imaging HSV1-tk expression using established radionucleoside probes 
Molecular imaging  2010;9(1):47-58.
One limitation of HSV1-tk reporter PET imaging with nucleoside analogues is the high background radioactivity in the intestine. We hypothesized that endogenous expression of thymidine kinase in bacterial flora could phosphorylate and trap such radiotracers, contributing to the high radioactivity levels in the bowel and therefore explored different strategies to increase fecal elimination of radiotracer.
Intestinal radioactivity was assessed by in vivo microPET imaging and ex vivo tissue sampling following intravenous injection of 18F-FEAU, 124I-FIAU or 18F-FHBG in a germ-free mouse strain. We also explored the use of an osmotic laxative agent and/or a 100% enzymatically hydrolyzed liquid diet.
No significant differences in intestinal radioactivity were observed between germ-free and normal mice. 18F-FHBG-derived intestinal radioactivity levels were higher than those of 18F-FEAU and 124I-FIAU; the intestine-to-blood ratio was more than 20-fold higher for 18F-FHBG than for 18F-FEAU and 124I-FIAU. The combination of Peptamen and Nulytely lowered intestinal radioactivity levels and increased (2.2-fold) the HSV1-tk transduced xenograft-to-intestine ratio for 18F-FEAU.
Intestinal bacteria in germ-free mice do not contribute to the high intestinal levels of radioactivity following injection of radionucleoside analogs. The combination of Peptamen and Nulytely increased radiotracer elimination by increasing bowel motility without inducing dehydration.
PMCID: PMC3068838  PMID: 20128998
reporter gene imaging; HSV1-tk; PET imaging; bacteria; background radioactivity
24.  An improved strategy for the synthesis of [18F]-labeled arabinofuranosyl nuclosides 
Nuclear medicine and biology  2012;39(8):1182-1188.
The expression of the herpes simplex virus type-1 thymidine kinase (HSV1-tk) gene can be imaged efficaciously using a variety of 2′-[18F]fluoro-2′-deoxy-1-b-D-arabinofuranosyl-uracil derivatives [[18F]-FXAU, X= I(iodo), E(ethyl), and M(methyl)]. However, the application of these derivatives in clinical and translational studies has been impeded by their complicated and long syntheses (3–5 h). To remedy these issues, in the study at hand we have investigated whether microwave or combined catalysts could facilitate the coupling reaction between sugar and nucleobase and, further, have probed the feasibility of establishing a novel approach for [18F]-FXAU synthesis.
We have demonstrated that the rate of the trimethylsilyl trifluoromethanesulfonate (TMSOTf)-catalyzed coupling reaction between the 2-deoxy-sugar and uracil derivatives at 90°C can be significantly accelerated by microwave-driven heating or by the addition of Lewis acid catalyst (SnCl4). Further, we have observed that the stability of the α- and β-anomers of [18F]-FXAU derivatives differs during the hydrolysis step. Using the microwave-driven heating approach, overall decay-corrected radiochemical yields of 19–27% were achieved for [18F]-FXAU in 120 min at a specific activity of >22 MBq/nmol (595 Ci/mmol). Ultimately, we believe that these high yielding syntheses of [18F]-FIAU, [18F]-FMAU and [18F]-FEAU will facilitate routine production for clinical applications.
PMCID: PMC3517724  PMID: 22819195
25.  Priming of duck hepatitis B virus reverse transcription in vitro: premature termination of primer DNA induced by the 5'-triphosphate of fialuridine. 
Journal of Virology  1994;68(12):8265-8269.
Hepadnaviruses employ a unique mechanism for the initiation of RNA-directed DNA synthesis. Initially, four bases (5'-GTAA-3') are added to a tyrosine residue of the viral polymerase by reverse transcription of a bulge sequence in epsilon, a stem-loop structure which functions as the packaging signal for pregenomic RNA. This protein-DNA complex acts as the primer for minus-strand elongation from the 3' sequence, DR1. To understand this process in greater detail, we investigated whether the protein-mediated priming of viral DNA synthesis is affected by nucleotide analogs. By using cell-free expression of duck hepatitis B virus (DHBV) reverse transcriptase (G.-H. Wang and C. Seeger, Cell 71:663-670, 1992), the 5'-triphosphate of the thymidine analog fialuridine (FIAU) was shown to inhibit the incorporation of radiolabeled TMP into primer DNA in a dose-dependent manner. Inhibition by the 5'-triphosphate of FIAU (FIAU-TP) was nearly complete at a concentration of 10 microM. The dideoxynucleotide analogs ddGTP, ddTTP, and 3'-azidodeoxythymidine triphosphate, known inhibitors of DHBV endogenous DNA polymerase, did not affect substantially the synthesis of primer DNA. Alternate substrate analysis suggested that FIAU is incorporated efficiently into nascent primer DNA as an analog of thymidine. Using site-directed mutagenesis to construct a mutant RNA template yielding a primer with the sequence 5'-GTAC-3', we demonstrated that FIAU-TP inhibited the incorporation of TMP, had no effect on that of dAMP, and decreased markedly the incorporation of dCMP. These results show that the synthesis of full-length DHBV primer DNA is inhibited by FIAU-TP but not by the dideoxynucleotide analogs that we tested. The significance of these findings as they relate to HBV DNA replication is discussed.
PMCID: PMC237293  PMID: 7525986

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