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Chronic hepatitis B remains a worldwide health concern. Presently, many drugs, such as Clevudine and Telbivudine, are recommended for the treatment of chronic hepatitis B disease. For this purpose, the quantum chemical analysis of E LUMO-HOMO (E gap ), ionization potential (IP), electron affinity (EA), electronegativity (EN), chemical hardness (η), chemical potential (μ), chemical softness (S), electrophilicity index (ω), electron accepting capability (ω + ), electron-donating capability (ω - ), Nucleophilicity index (N), additional electronic charge (∆N max ), Optical softness (σ 0 ) and Dipole Moment, IR and UV-Vis spectra, molecular electrostatic potential (MEP) profile, Mulliken charge analysis, natural bond orbital (NBO) were examined in this study. The dipole moment of the compounds suggests their binding pose and predicted binding affinity. The electrophilic and nucleophilic regions were identified, and techniques such as NBO, UV-Vis, and IR were used to gain insights into the molecular structure, electronic transitions, and potential drug design for Hepatitis B treatment. Calculations for this study were carried out using the Gaussian 09 program package coupled with the DFT/TDDFT technique. The hybrid B3LYP functional method and the 6-311++G(d, p) basis set were used for the calculations., (© 2024. The Author(s).)

Purpose: Imaging is limited in the evaluation of bacterial infection. Direct imaging of in situ bacteria holds promise for noninvasive diagnosis. We investigated the ability of a bacterial thymidine kinase inhibitor ([ 124 I]FIAU) to image pulmonary and musculoskeletal infections., Methods: Thirty-three patients were prospectively accrued: 16 with suspected musculoskeletal infection, 14 with suspected pulmonary infection, and 3 with known rheumatoid arthritis without infection. Thirty-one patients were imaged with [ 124 I]FIAU PET/CT and 28 with [ 18 F]FDG PET/CT. Patient histories were reviewed by an experienced clinician with subspecialty training in infectious diseases and were determined to be positive, equivocal, or negative for infection., Results: Sensitivity, specificity, positive-predictive value, negative-predictive value, and accuracy of [ 124 I]FIAU PET/CT for diagnosing infection were estimated as 7.7% to 25.0%, 0.0%, 50%, 0.0%, and 20.0% to 71.4% for musculoskeletal infections and incalculable-100.0%, 51.7% to 72.7%, 0.0% to 50.0%, 100.0%, and 57.1% to 78.6% for pulmonary infections, respectively. The parameters for [ 18 F]FDG PET/CT were 75.0% to 92.3%, 0.0%, 23.1% to 92.3%, 0.0%, and 21.4% to 85.7%, respectively, for musculoskeletal infections and incalculable to 100.0%, 0.0%, 0.0% to 18.2%, incalculable, and 0.0% to 18.2% for pulmonary infections, respectively., Conclusions: The high number of patients with equivocal clinical findings prevented definitive conclusions from being made regarding the diagnostic efficacy of [ 124 I]FIAU. Future studies using microbiology to rigorously define infection in patients and PET radiotracers optimized for image quality are needed.

Critical bacterial pathogens of public health and biodefense concerns were engineered to constitutively express Escherichia coli enzyme thymidine kinase (TK) that allows for noninvasive nuclear imaging via phosphorylation and entrapment of radiolabeled nucleoside analog 1-(2'deoxy-2'-fluoro-β-D-arabinofuranosyl)-5-iodouracil (FIAU). Expression of functional TK was established using a nucleoside analog Zidovudine that impeded the growth of tk-engineered bacteria. Significantly, no observable growth differences were detected for FIAU. High resolution mass spectrometry with Pseudomonas aeruginosa PAO1 and its tk variant (PAO1TK) confirmed FIAU phosphorylation and retention only in PAO1TK. In vitro gamma counting with wild-type PAO1, Acinetobacter baumannii and Burkholderia pseudomallei Bp82 and their tk derivatives with [ 18 F]FIAU further confirmed that tk variants selectively incorporated the radiotracer, albeit with varying efficiencies. In vitro [ 18 F]FIAU labeling coupled with in vivo Positron Emission Tomography/Computed Tomography (PET/CT) imaging of PAO1 and PAO1TK confirmed that only PAO1TK can be imaged in mice at sensitivities ≥10 7 bacteria per infection site. This was further verified by administering [ 18 F]FIAU to animals infected with PAO1 and PAO1TK. Utility of tk-engineered P. aeruginosa in noninvasive PET/CT imaging for bacterial therapeutic evaluation in animals was demonstrated employing antibiotic ciprofloxacin, underscoring the immediate use of PAO1TK and potentially other engineered pathogens for evaluating experimental therapeutics.

Clevudine was approved as an antiviral agent for hepatitis B virus, which showed marked, rapid inhibition of virus replication without significant toxicity. However, several studies have reported myopathy associated with clevudine therapy. Also, we experienced seven patients who suffered from myopathy during clevudine therapy. To characterize clevudine-induced myopathy, we collected previously reported cases of clevudine myopathy and analyzed all the cases including our cases. We searched electronic databases that were published in English or Korean using PubMed and KoreaMed. Ninety-five cases with clevudine myopathy, including our seven cases, were selected and analyzed for the demographic data, clinical features, and pathologic findings. The 95 patients with clevudine-induced myopathy comprised 52 women and 43 men aged 48.9 years (27-76 years). The patients received clevudine therapy for about 14.2 months (5-24 months) before the development of symptoms. Weakness mainly involved proximal extremities, especially in the lower extremities, and bulbar and neck weakness were observed in some cases (13.7%). Creatine kinase was elevated in the majority of patients (97.9%). Myopathic patterns on electromyography were observed in most patients examined (98.1%). Muscle biopsy presented patterns compatible with mitochondrial myopathy in the majority (90.2%). The weakness usually improved within about 3 months after the discontinuation of clevudine. Though clevudine has been known to be safe in a 6-month clinical trial, longer clevudine therapy for about 14 months may cause reversible mitochondrial myopathy. Careful clinical attention should be paid to patients with long-term clevudine therapy., Competing Interests: The authors have no potential conflicts of interest to disclose., (© 2017 The Korean Academy of Medical Sciences.)

Aim: We assessed the association between the presence and absence of androgen on the normal biodistribution of the positron emission tomography (PET) cellular proliferation imaging biomarker, [ 18 F]-2'-Fluoro-5-methyl-1-beta-D-arabinofuranosyluracil ( 18 F-FMAU), in mice., Materials and Methods: Non-castrated (n=4) and castrated (n=4) athymic non-tumor-bearing male mice served as models for presence and absence, respectively, of androgen. MicroPET-CT scans were performed 1 h following tail vein administration of 200 uCi of 18 F-FMAU. Imaging was performed at baseline and then at 7-day intervals longitudinally for 35 days only in castrated mice following subcutaneous introduction of a 12.5 mg, 21-day release, dihydrotestosterone pellet. Mean standardized uptake values (SUV mean ) were obtained for liver, heart, and muscle. Several two-group comparisons of average of SUV mean were performed., Results: Pre-pellet baseline average SUV mean (±s.d.) values in castrated mice were significantly lower than baseline non-castrated values, increased on day 15 and reached peak values on day 28, at which time they were significantly higher than corresponding baseline levels in both non-castrated and pre-pellet castrated mice. The peak values decreased significantly following dihydrotestosterone withdrawal., Conclusion: There is a significant modulatory effect of androgen on normal 18 F-FMAU uptake levels in mice liver, heart and muscle tissues., (Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background/aims: Data are lacking regarding the management of chronic hepatitis B (CHB) with resistance to clevudine (CLV). This study evaluated the efficacy of different rescue therapies for CLV-resistant CHB., Methods: Patients with CLV-resistant CHB were enrolled in the cohort, and all patients developed virologic breakthrough during CLV therapy and had confirmed-genotypic resistance to CLV (rtM204I mutation) before enrollment., Results: Of the 107 patients, 12 received adefovir (ADV), 21 received a CLV plus ADV combination (CLV+ADV), 34 received a lamivudine plus ADV combination (LAM+ADV), and 40 received entecavir (ETV) therapy for 48 weeks. The CLV+ADV group had the lowest hepatitis B virus (HBV) DNA level (p<0.0001) and showed the greatest reduction of HBV DNA levels from baseline compared to all other groups (p=0.004) at week 48. HBV DNA was undetectable (<70 IU/mL) in 0%, 57.1%, 21.2%, and 27.5% (p=0.003) of the patients in each group, respectively, at week 48. At the end of the study, the mean alanine transaminase (ALT) level, rate of ALT normalization, and rate of hepatitis B envelope antigen loss or seroconversion did not differ between groups., Conclusions: CLV+ADV combination therapy in patients with CLV-resistant CHB more effectively suppresses HBV replication than ETV, ADV, or LAM+ADV therapy.

Aim . To determine whether nucleot(s)ide analogs therapy has survival benefit for patients with HBV-related HCC after unresectable treatment. Method . A systematic search was conducted through seven electronic databases including PubMed, OVID, EMBASE, Cochrane Databases, Elsevier, Wiley Online Library, and BMJ Best Practice. All studies comparing NA combined with unresectable treatment versus unresectable treatment alone were considered for inclusion. The primary outcome was the overall survival (OS) after unresectable treatment for patients with HBV-related HCC. The secondary outcome was the progression-free survival (PFS). Results were expressed as hazard ratio (HR) for survival with 95% confidence intervals. Results . We included six studies with 994 patients: 409 patients in nucleot(s)ide analogs therapy group and 585 patients without antiviral therapy in control group. There were significant improvements for the overall survival (HR = 0.57; 95% CI = 0.47-0.70; p < 0.001) and progression-free survival (HR = 0.84; 95% CI = 0.71-0.99; p = 0.034) in the NA-treated group compared with the control group. Funnel plot showed that there was no significant publication bias in these studies. When it comes to antiviral drugs and operation method, it also showed benefit in NA-treated group. At the same time, overall mortality as well as mortality secondary to liver failure in NA-treated group was obviously lesser. Sensitivity analyses confirmed the robustness of the results. Conclusions . Nucleot(s)ide analogs therapy after unresectable treatment has potential beneficial effects in terms of overall survival and progression-free survival. NA therapy should be considered in clinical practice., Competing Interests: The authors deny any conflict of interests.

Background: A principal goal for the use of positron emission tomography (PET) in oncology is for real-time evaluation of tumor response to chemotherapy. Given that many contemporary anti-neoplastic agents function by impairing cellular proliferation, it is of interest to develop imaging modalities to monitor these pathways. Here we examined the effect of capecitabine on the uptake of thymidine analogs used with PET: 3'-deoxy-3'-[ 18 F]fluorothymidine ( 18 F-FLT), 1-(2'-deoxy-2'-[ 18 F]fluoro-β-D-arabinofuranosyl) thymidine ( 18 F-FMAU), and 1-(2'-deoxy-2'-[ 18 F]fluoro-β-D-arabinofuranosyl) uracil ( 18 F-FAU) in patients with advanced cancer., Methods: Fifteen patients were imaged, five with each imaging agent. Patients had been previously diagnosed with breast, colorectal, gastric, and esophageal cancers and had not received therapy for at least 4 weeks prior to the first scan, and had not been treated with any prior fluoropyrimidines. Subjects were imaged within a week before the start of capecitabine and on the second day of treatment, after the third dose of capecitabine. Tracer uptake was quantified by mean standard uptake value (SUV mean ) and using kinetic analysis., Results: Patients imaged with 18 F-FLT showed variable changes in retention and two patients exhibited an increase in SUV mean of 172.3 and 89.9 %, while the other patients had changes ranging from +19.4 to -25.4 %. The average change in 18 F-FMAU retention was 0.2 % (range -24.4 to 23.1) and 18 F-FAU was -10.2 % (range -40.3 to 19.2). Observed changes correlated strongly with SUV max but not kinetic measurements., Conclusions: This pilot study demonstrates that patients treated with capecitabine can produce a marked increase in 18 F-FLT retention in some patients, which will require further study to determine if this flare is predictive of therapeutic response. 18 F-FAU and 18 F-FMAU showed little change, on average, after treatment.

Liver biology and function, drug-induced liver injury (DILI) and liver diseases are difficult to study using current in vitro models such as primary human hepatocyte (PHH) monolayer cultures, as their rapid de-differentiation restricts their usefulness substantially. Thus, we have developed and extensively characterized an easily scalable 3D PHH spheroid system in chemically-defined, serum-free conditions. Using whole proteome analyses, we found that PHH spheroids cultured this way were similar to the liver in vivo and even retained their inter-individual variability. Furthermore, PHH spheroids remained phenotypically stable and retained morphology, viability, and hepatocyte-specific functions for culture periods of at least 5 weeks. We show that under chronic exposure, the sensitivity of the hepatocytes drastically increased and toxicity of a set of hepatotoxins was detected at clinically relevant concentrations. An interesting example was the chronic toxicity of fialuridine for which hepatotoxicity was mimicked after repeated-dosing in the PHH spheroid model, not possible to detect using previous in vitro systems. Additionally, we provide proof-of-principle that PHH spheroids can reflect liver pathologies such as cholestasis, steatosis and viral hepatitis. Combined, our results demonstrate that the PHH spheroid system presented here constitutes a versatile and promising in vitro system to study liver function, liver diseases, drug targets and long-term DILI.

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.

The emergence of compensatory mutations in the polymerase gene of drug resistant hepatitis B virus (HBV) is associated with treatment failure. We previously identified a multi-drug resistant HBV mutant, which displayed resistance towards lamivudine (LMV), clevudine (CLV), and entecavir (ETV), along with a strong replication capacity. The aim of this study was to identify the previously unknown compensatory mutations, and to determine the clinical relevance of this mutation during antiviral therapy. In vitro mutagenesis, drug susceptibility assay, and molecular modeling studies were performed. The rtL269I substitution conferred 2- to 7-fold higher replication capacity in the wild-type (WT) or YMDD mutation backbone, regardless of drug treatment. The rtL269I substitution alone did not confer resistance to LMV, ETV, adefovir (ADV), or tenofovir (TDF). However, upon combination with YMDD mutation, the replication capacity under LMV or ETV treatment was enhanced by several folds. Molecular modeling studies suggested that the rtL269I substitution affects template binding, which may eventually lead to the enhanced activity of rtI269-HBV polymerase in both WT virus and YMDD mutant. The clinical relevance of the rtL269I substitution was validated by its emergence in association with YMDD mutation in chronic hepatitis B (CHB) patients with sub-optimal response or treatment failure to LMV or CLV. Our study suggests that substitution at rt269 in HBV polymerase is associated with multi-drug resistance, which may serve as a novel compensatory mutation for replication-defective multi-drug resistant HBV.

Unlabelled: Monitoring genetically altered T cells is an important component of adoptive T cell therapy in patients, and the ability to visualize their trafficking/targeting, proliferation/expansion, and retention/death using highly sensitive reporter systems that do not induce an immunologic response would provide useful information. Therefore, we focused on human reporter gene systems that have the potential for translation to clinical studies. The objective of the in vivo imaging studies was to determine the minimum number of T cells that could be visualized with the different nuclear reporter systems. We determined the imaging sensitivity (lower limit of T cell detection) of each reporter using appropriate radiolabeled probes for PET or SPECT imaging., Methods: Human T cells were transduced with retroviral vectors encoding for the human norepinephrine transporter (hNET), human sodium-iodide symporter (hNIS), a human deoxycytidine kinase double mutant (hdCKDM), and herpes simplex virus type 1 thymidine kinase (hsvTK) reporter genes. After viability and growth were assessed, 10(5) to 3 × 10(6) reporter T cells were injected subcutaneously on the shoulder area. The corresponding radiolabeled probe was injected intravenously 30 min later, followed by sequential PET or SPECT imaging. Radioactivity at the T cell injection sites and in the thigh (background) was measured., Results: The viability and growth of experimental cells were unaffected by transduction. The hNET/meta-(18)F-fluorobenzylguanidine ((18)F-MFBG) reporter system could detect less than 1 × 10(5) T cells because of its high uptake in the transduced T cells and low background activity. The hNIS/(124)I-iodide reporter system could detect approximately 1 × 10(6) T cells; (124)I-iodide uptake at the T cell injection site was time-dependent and associated with high background. The hdCKDM/2'-(18)F-fluoro-5-ethyl-1-β-d-arabinofuranosyluracil ((18)F-FEAU) and hsvTK/(18)F-FEAU reporter systems detected approximately 3 × 10(5) T cells, respectively. (18)F-FEAU was a more efficient probe (higher uptake, lower background) than (124)I-1-(2-deoxy-2-fluoro-1-d-arabinofuranosyl)-5-iodouracil for both hdCKDM and hsvTK., Conclusion: A comparison of different reporter gene-reporter probe systems for imaging of T cell number was performed, and the hNET/(18)F-MFBG PET reporter system was found to be the most sensitive and capable of detecting approximately 35-40 × 10(3) T cells at the site of T cell injection in the animal model., (© 2015 by the Society of Nuclear Medicine and Molecular Imaging, Inc.)

The susceptibilities of gammaherpesviruses, including Epstein-Barr virus (EBV), Kaposi's sarcoma-associated herpesvirus (KSHV), and animal rhadinoviruses, to various nucleoside analogs was investigated in this work. Besides examining the antiviral activities and modes of action of antivirals currently marketed for the treatment of alpha- and/or betaherpesvirus infections (including acyclovir, ganciclovir, penciclovir, foscarnet, and brivudin), we also investigated the structure-activity relationship of various 5-substituted uridine and cytidine molecules. The antiviral efficacy of nucleoside derivatives bearing substitutions at the 5 position was decreased if the bromovinyl was replaced by chlorovinyl. 1-β-D-Arabinofuranosyl-(E)-5-(2-bromovinyl)uracil (BVaraU), a nucleoside with an arabinose configuration of the sugar ring, exhibited no inhibitory effect against rhadinoviruses but was active against EBV. On the other hand, the fluoroarabinose cytidine analog 2'-fluoro-5-iodo-aracytosine (FIAC) showed high selectivity indices against gammaherpesviruses that were comparable to those of brivudin. Additionally, we selected brivudin- and acyclovir-resistant rhadinoviruses in vitro and characterized them by phenotypic and genotypic (i.e., sequencing of the viral thymidine kinase, protein kinase, and DNA polymerase) analysis. Here, we reveal key amino acids in these enzymes that play an important role in substrate recognition. Our data on drug susceptibility profiles of the different animal gammaherpesvirus mutants highlighted cross-resistance patterns and indicated that pyrimidine nucleoside derivatives are phosphorylated by the viral thymidine kinase and purine nucleosides are preferentially activated by the gammaherpesvirus protein kinase., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Background: The durability of off-treatment virologic responses has not been fully elucidated in chronic hepatitis B (CHB) patients who have previously achieved complete virologic suppression with nucleos(t)ide analog (NA) therapy. This study aimed to assess off-treatment virologic relapse rates and to characterize the outcomes of subsequent re-treatment in CHB patients who have discontinued oral NA following complete virologic suppression., Methods: Ninety-five CHB patients who showed complete virologic suppression were withdrawn from NAs: entecavir, lamivudine, and clevudine in 67, 15, and 13 patients, respectively. Consolidation therapy was given for 6 and 12 months for HBeAg-positive and -negative CHB, respectively, before cessation. Virologic relapse was managed with the same NA that had induced complete virologic response before discontinuation., Results: The cumulative rates of virologic relapse at 12 and 24 months were 73.8% and 87.1%, respectively. The relapse rates were independent of HBeAg positivity, HBeAg seroconversion, and type of oral NA. In a multivariate analysis, duration of oral NA therapy was the only significant predicting factor associated with off-treatment virologic relapse. Although the majority of patients regained complete virologic suppression, some patients did not respond to re-treatment with the initial NA and developed genotypic resistance., Conclusions: NA consolidation therapy for 6 and 12 months is associated with high off-treatment virologic relapse in HBeAg-positive and -negative CHB patients, respectively. Drugs with high genetic barriers to resistance should be considered as a rescue therapy for off-treatment relapse in CHB.

Background: 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., Conclusions: 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.

All currently approved antiviral drugs for the treatment of chronic hepatitis B virus (HBV) infection are nucleos(t)ide reverse transcriptase inhibitors (NRTI), which inhibit the DNA synthesis activity of the HBV polymerase. The polymerase is a unique reverse transcriptase (RT) that has a novel protein priming activity in which HP initiates viral DNA synthesis using itself as a protein primer. We have determined the ability of NRTI-triphosphates (TP) to inhibit HBV protein priming and their mechanisms of action. While entecavir-TP (a dGTP analog) inhibited protein priming initiated specifically with dGTP, clevudine-TP (a TTP analog) was able to inhibit protein priming independently of the deoxynucleoside triphosphate (dNTP) substrate and without being incorporated into DNA. We next investigated the effect of NRTIs on the second stage of protein priming, wherein two dAMP nucleotides are added to the initial deoxyguanosine nucleotide. The obtained results indicated that clevudine-TP as well as tenofovir DF-DP strongly inhibited the second stage of protein priming. Tenofovir DF-DP was incorporated into the viral DNA primer, whereas clevudine-TP inhibited the second stage of priming without being incorporated. Finally, kinetic analyses using the HBV endogenous polymerase assay revealed that clevudine-TP inhibited DNA chain elongation by HP in a noncompetitive manner. Thus, clevudine-TP appears to have the unique ability to inhibit HBV RT via binding to and distorting the HP active site, sharing properties with both NRTIs and nonnucleoside RT inhibitors.

Unlabelled: This study was designed to investigate the feasibility of imaging bone marrow stem cells (BMSCs) in experimental middle cerebral artery occlusion (MCAO) rat models with a reporter gene-probe system, HSV1-tk-(131)I-2'-fluoro-2'-deoxy-1-β-d-arabinofuranosyl-5-iodouracil ((131)I-FIAU), and to choose the best strategies for stem cell injection, image acquisition, and imaging in vivo., Methods: A recombinant adenovirus (Ad5-TIBE) carrying the herpes simplex virus type 1 thymidine kinase (TK) reporter gene (HSV1-tk) linked via the internal ribosome entry site to the brain-derived neurotrophic factor therapeutic gene was prepared. After transfection with Ad5-TIBE, BMSCs were introduced into MCAO rat models via local injection into the brain or via injection into the lateral ventricle, carotid artery, and tail vein. Normal rats were used as controls. Twenty-four hours after (131)I-FIAU injection, rats were sacrificed for biodistribution analysis. The expression of the TK gene was evaluated by real-time quantitative polymerase chain reaction and Western blot analysis. Autoradiography was used for ex vivo imaging. SPECT images were obtained in MCAO rat models., Results: The percentage injected dose per gram (%ID/g) in infarcted brain tissue in rats receiving the injection into the brain was 0.124 ± 0.013; this value was significantly higher than those in rats receiving the injection into the ventricle (0.052 ± 0.004), carotid artery (0.061 ± 0.002), and tail vein (0.059 ± 0.005) as well as normal rats (0.005 ± 0.001). No differences were seen in the other cell transplantation groups. The %ID/g in infarcted brain tissue was higher than that in the contralateral brain tissue in all experimental rats but not in normal rats. The expression of the TK gene in rats receiving a local injection into the brain was superior to that in all of the other groups. TK messenger RNA and protein expression showed a positive correlation with the %ID/g in brain tissue. Greater radioactivity at the injection site than in the surrounding and contralateral brain tissues in all experimental rats was indicated through autoradiography. The ratio of counts in bilateral brain tissues reached its peak (6.63) 24 h after (131)I-FIAU injection. SPECT images showed that radioactivity accumulation in the brain was low but increased gradually over time., Conclusion: The HSV1-tk-(131)I-FIAU reporter gene-probe system may be used to monitor BMSC activity in experimental MCAO rat models. Local injection of stem cells may provide an optimal means for cell transplantation, and imaging with (131)I-FIAU 24 h after injection provides peak target-to-nontarget count ratios.

2,2'-Anhydro-1-(3',5'-di-O-acetyl-β-D-arabinofuranosyl)uracil, C13H14N2O7, was obtained by refluxing 2',3'-O-(methoxymethylene)uridine in acetic anhydride. The structure exhibits a nearly perfect C4'-endo ((4)E) conformation. The best four-atom plane of the five-membered furanose ring is O-C-C-C, involving the C atoms of the fused five-membered oxazolidine ring, and the torsion angle is only -0.4 (2)°. The oxazolidine ring is essentially coplanar with the six-membered uracil ring [r.m.s. deviation = 0.012 (5) Å and dihedral angle = -3.2 (3)°]. The conformation at the exocyclic C-C bond is gauche-trans which is stabilized by various C-H...π and C-O...π interactions.

Positron emission tomography (PET) reporter genes allow noninvasive whole-body imaging of transplanted cells by detection with radiolabeled probes. We used a human deoxycytidine kinase containing three amino acid substitutions within the active site (hdCK3mut) as a reporter gene in combination with the PET probe [(18)F]-L-FMAU (1-(2-deoxy-2-(18)fluoro-β-L-arabinofuranosyl)-5-methyluracil) to monitor models of mouse and human hematopoietic stem cell (HSC) transplantation. These mutations in hdCK3mut expanded the substrate capacity allowing for reporter-specific detection with a thymidine analog probe. Measurements of long-term engrafted cells (up to 32 wk) demonstrated that hdCK3mut expression is maintained in vivo with no counter selection against reporter-labeled cells. Reporter cells retained equivalent engraftment and differentiation capacity being detected in all major hematopoietic lineages and tissues. This reporter gene and probe should be applicable to noninvasively monitor therapeutic cell transplants in multiple tissues.

Background/aims: Clevudine is a potent antiviral agent against HBV. However, long-term clevudine therapy may cause myopathy. This study was carried out to identify the efficacy of entecavir switching therapy in chronic hepatitis B patients experiencing clevudine-induced myopathy., Methods: One hundred forty six patients with chronic hepatitis B treated with 30 mg of clevudine per day for 73 weeks (range, 36-132 weeks) were enrolled. Among them, clevudine-induced myopathy occurred in 21 patients (14.4%) which was diagnosed if the patients had symptoms related to myopathy with concurrent CK and AST elevation. All the patients who were diagnosed as clevudine-induced myopathy stopped the therapy, and 17 patients (81%) were switched to entecavir 0.5 mg., Results: The patients with clevudine-induced myopathy were switched to entecavir 0.5 mg for median 68 weeks, and all of them showed disappearance of clinical myopathic symptoms and normalization of CK and AST level within median 2.2 months. Eight patients (47%) were HBeAg positive before entecavir treatment, and HBeAg seroconversion was achieved in 2 patients (25%). HBV DNA level was elevated in 3 patients (17.6%) at the time when the patients were diagnosed as myopathy, all of them achieved virological response with entecavir switching therapy. ALT level was elevated in 3 patients (17.6%) before entecavir treatment, all of them showed normalization of ALT level. During entecavir therapy, genotypic resistance to entecavir or virological breakthrough was not noted., Conclusions: In chronic hepatitis B patients experiencing clevudine-induced myopathy, switching to entecavir 0.5 mg per day showed a resolution of myopathy and adequate viral suppression.

Aim: To evaluate the treatment outcomes of clevudine compared with entecavir in antiviral-naive patients with chronic hepatitis B (CHB)., Methods: We retrospectively analyzed the clinical data of CHB patients treated with clevudine 30 mg/d and compared their clinical outcomes with patients treated with entecavir 0.5 mg/d. The biochemical response, as assessed by serum alanine aminotransferase (ALT) activity, virologic response, as assessed by serum hepatitis B virus DNA (HBV DNA) titer, serologic response, as assessed by hepatitis B e antigen (HBeAg) status, and virologic breakthrough with genotypic mutations were assessed., Results: Two-hundred and fifty-four patients [clevudine (n = 118) vs entecavir (n = 136)] were enrolled. In clevudine-treated patients, the cumulative rates of serum ALT normalization were 83.9% at week 48 and 91.5% at week 96 (80.9% and 91.2% in the entecavir group, respectively), the mean titer changes in serum HBV DNA were -6.03 and -6.55 log(10) copies/mL (-6.35 and -6.86 log(10) copies/mL, respectively, in the entecavir group), and the cumulative non-detection rates of serum HBV DNA were 72.6% and 83.1% (74.4% and 83.8%, respectively, in the entecavir group). These results were similar to those of entecavir-treated patients. The cumulative rates of HBeAg seroconversion were 21.8% at week 48 and 25.0% at week 96 in patients treated with clevudine, which was similar to patients treated with entecavir (22.8% and 27.7%, respectively). The virologic breakthrough in the clevudine group occurred in 9 (7.6%) patients at weeks 48 and 15 (12.7%) patients at week 96, which primarily corresponded to genotypic mutations of rtM204I and/or rtL180M. There was no virologic breakthrough in the entecavir group., Conclusion: In antiviral-naive CHB patients, long-term treatment outcomes of clevudine were not inferior to those of entecavir, except for virologic breakthrough.

Background/aims: Chronic hepatitis B infection is a common cause of secondary membranous nephropathy (MN) in endemic areas. Lamivudine treatment improves renal outcome in patients with hepatitis B virus-associated MN (HBV-MN), but prolonged use leads to the emergence of lamivudine-resistant variants. We describe our experience treating lamivudine-resistant and other strains of HBV-MN with new antiviral drugs., Methods: Of the 89 patients biopsied and diagnosed with MN from 1996 to 2011, 10 positive for hepatitis B surface antigen were recruited for this study. We investigated the clinical courses, therapeutic responses, and prognoses of patients with HBV-MN., Results: The incidence of HBV-MN among the original 89 patients was 11.2%. Of these patients, four were treated with supportive care and six with antiviral drugs. One of the four patients treated with supportive care had a spontaneous remission. Four of the six patients treated with antiviral drugs were given lamivudine, and the other two were given entecavir. Two of the four patients treated with lamivudine achieved complete remission with seroconversion (i.e., development of anti-hepatitis B e antigen antibodies), whereas the other two had lamivudine-resistant strains, which were detected at 22 and 23 months after lamivudine treatment, respectively. We added adefovir to the treatment regimen for one of these patients, and for the other patient we substituted clevudine for lamivudine. Both of these patients experienced complete remission, as did the two patients initially treated with entecavir, neither of whom showed resistance to the drug., Conclusions: New nucleoside analogues, such as entecavir, adefovir, and clevudine, can be effective for treatment of HBV-MN, including lamivudine-resistant strains.

We hypothesized that imaging-based assessment of cellular proliferation in prostate cancer may improve tumor characterization. We therefore evaluated the biodistribution and effect of androgen on tumor uptake of the cellular proliferation imaging marker [(18)F]-2'-fluoro-5-methyl-1-beta-D-arabinofuranosyluracil ((18)F-FMAU) in xenograft mouse models of human prostate cancer. Castrated and noncastrated athymic male mice were implanted with androgen-independent PC3 and androgen-sensitive CWR22 human prostate cancer cells. Dynamic micro-positron emission tomography (PET)/computed tomography was performed for 1 hour followed by 10-minute static scans at 2 and 3 hours. Animals were sacrificed after imaging for biodistribution studies and immunohistochemical staining of tumors for androgen receptor and Ki-67/MIB expression. (18)F-FMAU uptake was significantly higher in all major organs of the castrated animals in comparison with noncastrated mice, with the highest uptake in liver and the lowest uptake in muscle and bone. When compared to PC3 tumors, CWR22 xenografts showed significantly higher tumor to muscle (2.56 ± 0.30 vs 1.99 ± 0.30, p  =  .008) and tumor to liver (1.72 ± 0.12 vs 1.26 ± 0.17, p  =  .0003) uptake ratios in the noncastrated animal at the 3-hour time point. Androgen receptor and Ki-67/MIB expressions were higher in CWR22 than in PC3 xenografts. Our initial preclinical observations suggest that there may be an association between androgen signaling and thymidine metabolism and that (18)F-FMAU PET may be useful in prostate tumor characterization.

Background: Clevudine is a nucleoside analog reverse transcriptase inhibitor that exhibits potent antiviral activity against hepatitis B virus (HBV) without serious side effects. However, mitochondrial myopathy has been observed in patients with chronic HBV infection taking clevudine. Moreover, the development of diabetes was recently reported in patients receiving long-term treatment with clevudine. In this study, we investigated the effects of clevudine on mitochondrial function and insulin release in a rat clonal β-cell line, INS-1E., Methods: The mitochondrial DNA (mtDNA) copy number and the mRNA levels were measured by using quantitative PCR. MTT analysis, ATP/lactate measurements, and insulin assay were performed., Results: Both INS-1E cells and HepG2 cells, which originated from human hepatoma, showed dose-dependent decreases in mtDNA copy number and cytochrome c oxidase-1 (Cox-1) mRNA level following culture with clevudine (10 μM-1 mM) for 4 weeks. INS-1E cells treated with clevudine had reduced total mitochondrial activities, lower cytosolic ATP contents, enhanced lactate production, and more lipid accumulation. Insulin release in response to glucose application was markedly decreased in clevudine-treated INS-1E cells, which might be a consequence of mitochondrial dysfunction., Conclusions: Our data suggest that high-dose treatment with clevudine induces mitochondrial defects associated with mtDNA depletion and impairs glucose-stimulated insulin secretion in insulin-releasing cells. These findings partly explain the development of diabetes in patients receiving clevudine who might have a high susceptibility to mitochondrial toxicity.

Positron emission tomography (PET) reporter gene imaging can be used to non-invasively monitor cell-based therapies. Therapeutic cells engineered to express a PET reporter gene (PRG) specifically accumulate a PET reporter probe (PRP) and can be detected by PET imaging. Expanding the utility of this technology requires the development of new non-immunogenic PRGs. Here we describe a new PRG-PRP system that employs, as the PRG, a mutated form of human thymidine kinase 2 (TK2) and 2'-deoxy-2'-18F-5-methyl-1-β-L-arabinofuranosyluracil (L-18F-FMAU) as the PRP. We identified L-18F-FMAU as a candidate PRP and determined its biodistribution in mice and humans. Using structure-guided enzyme engineering, we generated a TK2 double mutant (TK2-N93D/L109F) that efficiently phosphorylates L-18F-FMAU. The N93D/L109F TK2 mutant has lower activity for the endogenous nucleosides thymidine and deoxycytidine than wild type TK2, and its ectopic expression in therapeutic cells is not expected to alter nucleotide metabolism. Imaging studies in mice indicate that the sensitivity of the new human TK2-N93D/L109F PRG is comparable with that of a widely used PRG based on the herpes simplex virus 1 thymidine kinase. These findings suggest that the TK2-N93D/L109F/L-18F-FMAU PRG-PRP system warrants further evaluation in preclinical and clinical applications of cell-based therapies.

Background: Modeling of short-term viral dynamics of hepatitis B with traditional biphasic model might be insufficient to explain long-term viral dynamics. The aim was to develop a novel method of mathematical modeling to shed light on the dissociation between early and long-term dynamics in previous studies., Methods: We investigated the viral decay pattern in 50 patients from the phase III clinical trial of 24-week clevudine therapy, who showed virological response and HBsAg decline. Immune effectors were added as a new compartment in the model equations. We determined some parameter values in the model using the non-linear least square minimization method., Results: Median baseline viral load was 8.526 Log(10)copies/mL, and on-treatment viral load decline was 5.683 Log(10)copies/mL. The median half-life of free virus was 24.89 hours. The median half-life of infected hepatocytes was 7.39 days. The viral decay patterns were visualized as triphasic curves with decreasing slopes over time: fastest decay in the first phase; slowest in the third phase; the second phase in between., Conclusions: In the present study, mathematical modeling of hepatitis B in patients with virological response and HBsAg decline during 24-week antiviral therapy showed triphasic viral dynamics with direct introduction of immune effectors as a new compartment, which was thought to reflect the reduction of clearance rate of infected cells over time. This modeling method seems more appropriate to describe long-term viral dynamics compared to the biphasic model, and needs further validation.

The long-term fate of stem cells after intramyocardial delivery is unknown. We used noninvasive, repetitive PET/CT imaging with [(18)F]FEAU to monitor the long-term (up to 5 months) spatial-temporal dynamics of MSCs retrovirally transduced with the sr39HSV1-tk gene (sr39HSV1-tk-MSC) and implanted intramyocardially in pigs with induced acute myocardial infarction. Repetitive [(18)F]FEAU PET/CT revealed a biphasic pattern of sr39HSV1-tk-MSC dynamics; cell proliferation peaked at 33-35 days after injection, in periinfarct regions and the major cardiac lymphatic vessels and lymph nodes. The sr39HSV1-tk-MSC-associated [(18)F]FEAU signals gradually decreased thereafter. Cardiac lymphography studies using PG-Gd-NIRF813 contrast for MRI and near-infrared fluorescence imaging showed rapid clearance of the contrast from the site of intramyocardial injection through the subepicardial lymphatic network into the lymphatic vessels and periaortic lymph nodes. Immunohistochemical analysis of cardiac tissue obtained at 35 and 150 days demonstrated several types of sr39HSV1-tk expressing cells, including fibro-myoblasts, lymphovascular cells, and microvascular and arterial endothelium. In summary, this study demonstrated the feasibility and sensitivity of [(18)F]FEAU PET/CT imaging for long-term, in-vivo monitoring (up to 5 months) of the fate of intramyocardially injected sr39HSV1-tk-MSC cells. Intramyocardially transplanted MSCs appear to integrate into the lymphatic endothelium and may help improve myocardial lymphatic system function after MI.

Background: Coccidioidomycosis or Valley fever is caused by a highly virulent fungal pathogen: Coccidioides posadasii or immitis. Vaccine development against Coccidioides is of contemporary interest because a large number of relapses and clinical failures are reported with antifungal agents. An efficient Th1 response engenders protection. Thus, we have focused on developing a dendritic cell (DC)-based vaccine for coccidioidomycosis. In this study, we investigated the immunostimulatory characteristics of an intranasal primary DC-vaccine in BALB/c mouse strain that is most susceptible to coccidioidomycosis. The DCs were transfected nonvirally with Coccidioides-Ag2/PRA-cDNA. Expression of DC-markers, Ag2/PRA and cytokines were studied by flow cytometry, dot-immunoblotting and cytometric bead array methods, respectively. The T cell activation was studied by assessing the upregulation of activation markers in a DC-T cell co-culture assay. For trafficking, the DCs were co-transfected with a plasmid DNA encoding HSV1 thymidine kinase (TK) and administered intranasally into syngeneic mice. The trafficking and homing of TK-expressing DCs were monitored with positron emission tomography (PET) using 18F-FIAU probe. Based on the PET-probe accumulation in vaccinated mice, selected tissues were studied for antigen-specific response and T cell phenotypes using ELISPOT and flow cytometry, respectively., Results: We found that the primary DCs transfected with Coccidioides-Ag2/PRA-cDNA were of immature immunophenotype, expressed Ag2/PRA and activated naïve T cells. In PET images and subsequent biodistribution, intranasally-administered DCs were found to migrate in blood, lung and thymus; lymphocytes showed generation of T effector memory cell population (T(EM)) and IFN-γ release., Conclusions: In conclusion, our results demonstrate that the intranasally-administered primary DC vaccine is capable of inducing Ag2/PRA-specific T cell response. Unique approaches utilized in our study represent an attractive and novel means of producing and evaluating an autologous DC-based vaccine.

Background/aims: clevudine is a potent antiviral agent that has demonstrated efficacy in patients with chronic hepatitis B. This study compared the efficacy of clevudine (C), entecavir (E) and lamivudine (L) in treatment-naive patient with HBeAg-positive chronic hepatitis B., Methods: a total of 146 treatment-naive patients with HBeAg-positive chronic hepatitis B received clevudine, entecavir or lamivudine. C group (n=39) received 30 mg of clevudine, E group (n=39) received 0.5 mg of entecavir and L group (n=68) received 100 mg of lamivudine once a day for more than 48 weeks. The efficacy analysis estimated the mean changes of the HBV DNA levels as a virologic response, the normalization of the ALT levels (less than 35 IU/L) as a biochemical response and loss of HBeAg or seroconversion as a serologic response. The serum HBV DNA level was quantified by hybrid capture and real-time PCR assay., Results: before the administration of clevudine, entecavir and lamivudine, the mean HBV DNA and ALT levels and the gender and age were well balanced among the three groups (p>0.05). For the virologic response at 48 weeks, the mean changes of the HBV DNA levels from baseline of the C, E and L groups were -3.8+/-2.2, -4.5+/-1.9 and -2.5+/-2.1 log copies/mL. C and E group showed superior antiviral activity compared to that of L group (p<0.0001), but no significant differences in antiviral response were noted between C and E groups. For the biochemical response at 48 weeks, the normalization of the ALT levels (less than 35 IU/L) among the C, E and L groups was 82%, 74% and 71%, respectively (p=0.46). The rates of undetectable serum HBV DNA (less than 300 copies/mL) of the C, E and L groups were 39%, 69% and 27%, respectively (p<0.0001). For the serologic response at 48 weeks, the loss of HBeAg was 13%, 31% and 24% and the seroconversion was 10%, 23% and 17%, respectively. There was no difference of efficacy among the three groups regarding ALT normalization or serologic response (p>0.05). Viral breakthrough in C group was noted at 24 weeks (5%) and 48 weeks (21%), but no biochemical breakthrough was noted. The elevation of the serum CK level was noted in only 1 patient of group C at 48 weeks (2.56%) after therapy. For the patients without or with liver cirrhosis (LC), C and E group showed superior antiviral activity compared to that of the L group, but the antiviral activity was more effective in non- LC group than LC group (p<0.0001 vs p=0.036)., Conclusions: clevudine therapy compared with lamivudine for 48 weeks showed significantly potent antiviral efficacy in treatment-naive patients with HBeAg-positive chronic hepatitis B, and especially in the non-LC patients. However, the antiviral efficacy of clevudine was similar to that of entecavir even though taking into account relatively short follow up period and retrospective study.

Background/aims: Clevudine, a pyrimidine nucleoside analogue, has potent antiviral effects in patients with chronic viral hepatitis B (CHB). We report the efficacy of initial treatment with clevudine in naïve patients with CHB living in Daejeon and Chungcheong Province, South Korea., Methods: One hundred five adults with CHB were administered 30 mg of clevudine per day for an average of 51 weeks. We evaluated viral markers and liver biochemistry retrospectively every 3 months., Results: Alanine aminotransferase (ALT), aspartate aminotransferase (AST), and hepatitis B virus (HBV) DNA before the treatment were 184 ± 188 IU/L, 150 ± 138 IU/L, and 7.1 ± 1.2 log copies/mL, respectively. Undetectable rates (< 60 IU/mL) of DNA were 36.2%, 68.9%, 83.6%, 76.2%, and 75.8% at 12, 24, 36, 48, and 60 weeks, respectively. Seroconversion rates were 9.1%, 13.6%, 24.6%, 26.5%, and 26.1% and ALT normalization rates were 64.5%, 78.1%, 87.9%, 90.0% at 12, 24, 36, and 48 weeks, respectively. Six patients (5.7%) had a viral breakthrough., Conclusions: Clevudine is a useful drug in the initial treatment of patients with CHB, with a potent antiviral effect and low incidence of viral breakthrough.

Unlabelled: The basis for the use of nucleoside tracers in PET is that activity of the cell-growth-dependent enzyme thymidine kinase 1 is the rate-limiting factor driving tracer retention in tumors. Recent publications suggest that nucleoside transporters might influence uptake and thereby affect the tracer signal in vivo. Understanding transport mechanisms for different nucleoside PET tracers is important for evaluating clinical results. This study examined the relative role of different nucleoside transport mechanisms in uptake and retention of [methyl-(3)H]-3'-deoxy-3'-fluorothymidine ((3)H-FLT), [methyl-(3)H]-thymidine ((3)H-thymidine), and (3)H-1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-methyluracil ((3)H-FMAU)., Methods: Transport of (3)H-FLT, (3)H-thymidine, and (3)H-FMAU was examined in a single human adenocarcinoma cell line, A549, under both nongrowth and exponential-growth conditions., Results: (3)H-Thymidine transport was dominated by human equilibrative nucleoside transporter 1 (hENT1) under both growth conditions. (3)H-FLT was also transported by hENT1, but passive diffusion dominated its transport. (3)H-FMAU transport was dominated by human equilibrative nucleoside transporter 2. Cell membrane levels of hENT1 increased in cells under exponential growth, and this increase was associated with a more rapid rate of uptake for both (3)H-thymidine and (3)H-FLT. (3)H-FMAU transport was not affected by changes in growth conditions. All 3 tracers concentrated in the plateau phase, nonproliferating cells at levels many-fold greater than their concentration in buffer, in part because of low levels of nucleoside metabolism, which inhibited tracer efflux., Conclusion: Transport mechanisms are not the same for (3)H-thymidine, (3)H-FLT, and (3)H-FMAU. Levels of hENT1, an important transporter of (3)H-FLT and (3)H-thymidine, increase as proliferating cells enter the cell cycle.

Background/aims: Clevudine is a pyrimidine analogue with potent activity against hepatitis B virus (HBV) replication in vitro. In a previous pivotal phase III clinical study, 24 weeks treatment with clevudine 30 mg has been shown to profoundly suppress HBV replication and normalize serum alanine aminotransferase level., Methods: In this study, we compare the efficacy and safety of clevudine (30 mg daily) versus lamivudine (100 mg daily) for 48 weeks in treatment-naive chronic hepatitis B e antigen (HBeAg) positive patients., Results: Ninety-two chronic HBeAg positive patients were randomized to receive clevudine 30 mg daily or lamivudine 100 mg daily in a 1:1 ratio. The clevudine group demonstrated greater viral suppression at week 48 when compared with the lamivudine group (median reduction: 4.27 vs. 3.17 log(10) copies/ml at week 48, p<0.0001). At week 48, serum HBV DNA level was below 300 copies/mL in 73% and 40% in the clevudine and lamivudine groups, respectively (p=0.001). HBeAg seroconversion occurred in 18% of patients in the clevudine group versus 12% in the lamivudine group at week 48. Lamivudine-resistant mutations were detected in 11 (24%) patients in the lamivudine group, who showed viral rebound during lamivudine therapy but no resistance was found in the clevudine group during 48-week treatment period., Conclusions: A 48-week dosing with clevudine 30 mg daily was superior to lamivudine 100 mg daily in suppressing HBV replication, with no emergence of viral breakthrough in patients with HBeAg positive chronic hepatits B.

Unlabelled: In this article, we describe a series of new human-derived reporter genes based on human deoxycytidine kinase (dCK) suitable for clinical PET., Methods: Native dCK and its mutant reporter genes were tested in vitro and in vivo for their phosphorylation of pyrimidine- and acycloguanosine-based radiotracers including 2'-deoxy-2'-fluoroarabinofuranosylcytosine, 2'-fluoro-2'-deoxyarabinofuranosyl-5-ethyluracil (FEAU), penciclovir, and 9-[4-fluoro-3-(hydroxymethyl)butyl]guanine (FHBG) and clinically applied antiviral and anticancer drugs., Results: Cells transduced with dCK mutant reporter genes showed high in vitro and in vivo uptake of pyrimidine-based radiopharmaceuticals ((18)F-FEAU) comparable to that of herpes simplex virus type-1 thymidine kinase (HSV1-tk)-transduced cells. These mutants did not phosphorylate acycloguanosine-based radiotracers ((18)F-FHBG) or antiviral drugs (ganciclovir). Furthermore, the mutants displayed suicidal activation of clinically used pyrimidine-based prodrugs (cytarabine, gemcitabine)., Conclusion: The mutants of human dCK can be used as pyrimidine-specific PET reporter genes for imaging with (18)F-FEAU during treatment with acycloguanosine-based antiviral drugs. Additionally, the prosuicidal activity of these reporters with pyrimidine-based analogs will allow for the safe elimination of transduced cells.

The authors assessed the efficacy and antiviral resistance of 48-week clevudine therapy versus lamivudine in treatment of naïve patients with HBeAg positive chronic hepatitis B. In this retrospective study, a total of 116 HBeAg positive patients, who received 30 mg of clevudine once daily (n=53) or 100 mg of lamivudine once daily (n=63) for 48 weeks, were included. At week 48, clevudine therapy produced a significantly greater mean reductions in serum HBV DNA levels from baseline than lamivudine therapy (-5.2 vs. -4.2 log(10)IU/mL; P=0.005). Furthermore, a significantly higher proportion of patients on clevudine achieved negative serum HBV DNA by PCR (<13 IU/mL) at week 48 (60.4% vs. 38.1%; P=0.025). The incidence of virologic breakthrough in the clevudine group was significantly lower than in the lamivudine group (9.4% vs. 25.4%; P=0.031). However, rates of alanine aminotransferase normalization and HBeAg loss or seroconversion were similar in the two groups (83.0% vs. 81.0%, 11.3% vs. 11.1%; P=0.813, 1.000, respectively). In conclusion, clevudine is more potent for viral suppression and lower for antiviral resistance at week 48 than lamivudine in treatment of naïve patients with HBeAg positive chronic hepatitis B.

Clevudine (CLV) is a nucleoside analog with potent antiviral activity against chronic hepatitis B virus (HBV) infection. Viral resistance to CLV in patients receiving CLV therapy has not been reported. The aim of this study was to characterize CLV-resistant HBV in patients with viral breakthrough (BT) during long-term CLV therapy. The gene encoding HBV reverse transcriptase (RT) was analyzed from chronic hepatitis B patients with viral BT during CLV therapy. Sera collected from the patients at baseline and at the time of viral BT were studied. To characterize the mutations of HBV isolated from the patients, we subjected the HBV mutants to in vitro drug susceptibility assays. Several conserved mutations were identified in the RT domain during viral BT, with M204I being the most common. In vitro phenotypic analysis showed that the mutation M204I was predominantly associated with CLV resistance, whereas L229V was a compensatory mutation for the impaired replication of the M204I mutant. A quadruple mutant (L129M, V173L, M204I, and H337N) was identified that conferred greater replicative ability and strong resistance to both CLV and lamivudine. All of the CLV-resistant clones were lamivudine resistant. They were susceptible to adefovir, entecavir, and tenofovir, except for one mutant clone. In conclusion, the mutation M204I in HBV RT plays a major role in CLV resistance and leads to viral BT during long-term CLV treatment. Several conserved mutations may have a compensatory role in replication. Drug susceptibility assays reveal that adefovir and tenofovir are the most effective compounds against CLV-resistant mutants. These data may provide additional therapeutic options for CLV-resistant patients.

One limitation of HSV1-tk reporter positron emission tomography (PET) 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 analogues. The combination of Peptamen and Nulytely increased radiotracer elimination by increasing bowel motility without inducing dehydration.

Background/aims: Clevudine is a nucleoside analogue that exhibits potent and sustained antiviral effects as a 24-week therapy for chronic hepatitis B (CHB). This study evaluated the efficacy and viral resistance of a 48-week course of clevudine treatment for CHB., Methods: Data on patients with CHB and detectable serum hepatitis B virus (HBV) DNA who were treated with clevudine for 48 weeks or longer were collected retrospectively for this study. Patients who had taken lamivudine within the 3 years prior to this study were excluded. Serum HBV DNA was measured by polymerase chain reaction hybridization (lower detection limit=316 copies/mL). Serum HBV DNA and biochemical data were analyzed at weeks 24 and 48. Developments of viral breakthrough and resistance to the antiviral drug were also monitored., Results: Data from 74 patients (mean age 44 years; M:F=54:20; HBeAg-positive, 47; HBeAg-negative, 27) were included in this study. Ten patients had experienced previous lamivudine treatment. Median HBV DNA at baseline was 6.49 log(10) copies/mL. Median serum HBV DNA reductions from baseline at week 48 were -4.34 log(10) copies/mL (HBeAg-positive, -4.84 log(10) copies/mL; HBeAg-negative, -3.74 log(10) copies/mL). At week 48, serum HBV DNA was not detected in 83.8% of the patients (HBeAg-positive, 76.6%; HBeAg-negative, 96.3%). Normalization of serum alanine aminotransferase levels was achieved in 84.7% of the patients. Viral breakthrough and antiviral resistance developed in two patients at week 48. The development of antiviral resistance was associated with the presence of previous lamivudine treatment and cirrhosis., Conclusion: A 48-week course of clevudine therapy was highly effective in patients with CHB. The risk of development of resistance to clevudine was increased in patients with previous exposure to lamivudine and cirrhosis.

Background: Bacteria can be selectively imaged in experimentally-infected animals using exogenously administered 1-(2'deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5-[(125)I]-iodouracil ([(125)I]-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. [(125)I]FIAU uptake, antimicrobial susceptibilities and in vivo growth characteristics were evaluated for this strain. Using single photon emission computed tomography (SPECT), M. tuberculosis P(hsp60) 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 P(hsp60) TK strain actively accumulated [(125)I]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 P(hsp60) TK strain was stable in vivo and SPECT imaging could detect and localize this strain in both animal models tested., Conclusion: 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.

Adoptive transfer of antigen-specific cytotoxic T lymphocytes (CTLs) has been successfully used to treat patients with different types of cancer. However, the long-term spatial-temporal dynamics of the distribution of systemically infused CTLs remains largely unknown. Noninvasive imaging of adoptively transferred CTLs using molecular-genetic reporter imaging with positron emission tomography and computed tomography (PET-CT) represents an innovative approach to understanding the long-term migratory patterns and therapeutic potential of adoptively transferred T cells. Here we report the application of repetitive PET-CT imaging with [18F]fluoro-5-ethyl-1-beta-D-arabinofuranosyluracil (18F-FEAU) in two nonhuman primates demonstrating that autologous polyclonal macaque T lymphocytes activated and transduced with a retroviral vector encoding for the sr39 mutant herpes simplex virus 1 thymidine kinase (sr39HSV1-tk) reporter gene can be detected after intravenous infusion in discrete lymphoid organs and in sites of inflammation. This study represents a proof of principle and supports the application of 18F-FEAU PET-CT imaging for monitoring the distribution of intravenously administered sr39HSV1-tk gene-transduced CTLs in humans.

Backgrounds/aims: Clevudine is an effective antiviral nucleoside analogue, but there are few data regarding its long-term effects, resistance, and safety. The aim of this study was to evaluate the long-term clinical efficacy of clevudine over a 1-year treatment period in nucleos(t)ide-naive and lamivudine-experienced chronic hepatitis B patients., Methods: Nucleos(t)ide-naive (group A, n=196) and lamivudine-experienced (serum hepatitis B virus, HBV DNA >2,000 copies/mL without resistant mutants at the start of clevudine therapy, group B, n=75) patients were included in this study. Basic clinical characteristics including age, sex, the presence of cirrhosis, laboratory data, and hepatitis B surface antigen (HBeAg) positivity were similar between the two groups. Pretreatment serum levels of HBV DNA were 7.4 and 6.6 log(10) copies/mL (P<0.001). The mean treatment duration was 8 months for both groups (range for group A: 3-21 months; range for group B: 3-20 months). Genotypic analysis for resistant mutations in the reverse transcriptase of HBV was performed after viral breakthrough., Results: After 1 year of therapy, 75.0% and 51.9% of groups A and B, respectively, had HBV DNA levels of <2,000 copies/mL (P=0.032), and HBeAg seroconversion rates were 16.9% and 16.7%, respectively. The rates of viral breakthrough at 1 year were 10.0% (8/80) and 44.4% (12/27), respectively (P<0.001). Proven sites of mutation of HBV DNA polymerase in naive patients were, for example, L80I, L180M, A181V/T, M204I and V207I. Ten patients complained of prominent fatigue and revealed elevated serum levels of aspartate aminotransferase (AST) and creatine phosphokinase (CPK). Two of these patients presented with severe myopathy from which they recovered completely after quitting clevudine., Conclusions: Clevudine is one of the recommended first-line medicines for the treatment of chronic hepatitis B, but it is not free from resistance, particularly in patients with a history of previous lamivudine treatment, but also in naive patients. Clevudine should be avoided in previously lamivudine-exposed patients. In addition, reelevation of serum AST and CPK levels is not a rare occurrence, and close observation and follow-up tests are essential.

Unlabelled: Imaging 2 different molecular-genetic events in a single subject by PET is essential in a variety of in vivo applications. Using herpes simplex virus-1 thymidine kinase (HSV1-tk) mutants with narrower substrate specificities in combination with wild-type HSV1-tk (wtHSV1-tk) would enable differential imaging with corresponding radiotracers, namely 2'-deoxy-2'-(18)F-fluoro-5-ethyl-1-beta-d-arabinofuranosyl-uracil ((18)F-FEAU) and the acycloguanosine derivative 9-(4-(18)F-fluoro-3-[hydroxymethyl]butyl)guanine ((18)F-FHBG). In this study, we evaluated wtHSV1-tk and the A168H mutant, which has been reported to exhibit enhanced acycloguanosine substrate catalytic activity and diminished pyrimidine phosphorylating activity, as PET reporter genes., Methods: Computational analysis was performed to assess the binding mode of FHBG and FEAU to wtHSV1-tk and the A168H variant. U87 cells were stably transduced with wtHSV1-tk or HSV1-tk(A168H) fused with green fluorescent protein and sorted to obtain equivalent transgene expression. In vitro uptake studies were performed to determine rates of substrate accumulation and retention. Nude mice bearing tumors expressing HSV1-tk variants were subsequently imaged using (18)F-FHBG and (18)F-FEAU., Results: Docking results indicate that binding of FHBG to the A168H variant is unaffected whereas the binding of FEAU is hindered because of a steric clash with the bulkier mutant residues. U87 cells expressing HSV1-tk(A168H) accumulated (18)F-FHBG in in vitro uptake studies at a 3-fold higher rate than did cells expressing wtHSV1-tk without any detectable accumulation of (3)H-FEAU. Furthermore, HSV1-tk(A168H) demonstrated no thymidine phosphorylation activity. In contrast, U87 cells expressing wtHSV1-tk preferentially accumulated (3)H-FEAU at an 18-fold higher rate than they did (18)F-FHBG. Tumors expressing wtHSV1-tk or HSV1-tk(A168H) were distinctly imaged with (18)F-FEAU or (18)F-FHBG, respectively. Hence, tumors expressing HSV1-tk(A168H) accumulated 8.4-fold more (18)F-FHBG than did tumors expressing wtHSV1-tk. In addition, wtHSV1-tk tumors, compared with HSV1-tk(A168H)-expressing tumors (which retained baseline levels of the radiotracer), preferentially accumulated (18)F-FEAU., Conclusion: The FEAU and FHBG substrate discrimination capacity of the wtHSV1-tk and HSV1-tk(A168H) reporter enzymes was validated in vivo by PET of mice with tumor xenografts established from U87 cells expressing these different reporters. Thus, HSV1-tk(A168H) may potentially be used as a second reporter gene in combination with wtHSV1-tk to achieve differential PET.

Background: In patients with melanoma, knowledge of regional lymph node status provides important information on outlook. Since lymph node status can influence treatment, surgery for sentinel lymph node (SLN) biopsy became a standard staging procedure for these patients. Current imaging modalities have a limited sensitivity for detection of micrometastases in lymph nodes and, therefore, there is a need for a better technique that can accurately identify occult SLN metastases., Methodology/principal Findings: B16-F10 murine melanoma cells were infected with replication-competent herpes simplex virus (HSV) NV1023. The presence of tumor-targeting and reporter-expressing virus was assessed by [(18)F]-2'-fluoro-2'-deoxy-1-beta-D-beta-arabinofuranosyl-5-ethyluracil ([(18)F]FEAU) positron emission tomography (PET) and confirmed by histochemical assays. An animal foot pad model of melanoma lymph node metastasis was established. Mice received intratumoral injections of NV1023, and 48 hours later were imaged after i.v. injection of [(18)F]FEAU. NV1023 successfully infected and provided high levels of lacZ transgene expression in melanoma cells. Intratumoral injection of NV1023 resulted in viral trafficking to melanoma cells that had metastasized to popliteal and inguinal lymph nodes. Presence of virus-infected tumor cells was successfully imaged with [(18)F]FEAU-PET, that identified 8 out of 8 tumor-positive nodes. There was no overlap between radioactivity levels (lymph node to surrounding tissue ratio) of tumor-positive and tumor-negative lymph nodes., Conclusion/significance: A new approach for imaging SLN metastases using NV1023 and [(18)F]FEAU-PET was successful in a murine model. Similar studies could be translated to the clinic and improve the staging and management of patients with melanoma.

Clevudine is a nucleoside analog of the unnatural beta-L configuration which has potent antiviral activity against hepatitis B virus (HBV). Clevudine is expected to have similar pattern of resistance profile as lamivudine. However, there was no report on the mutation associated with clevudine resistance in patients with chronic hepatitis B. We report a case of young male patient with chronic hepatitis B who presented with clevudine resistance. The patient had received lamivudine therapy for 5 months with reduced serum HBV DNA levels. Then, lamivudine was switched to clevudine monotherapy. After the 6 months of clevudine therapy, the patient developed virologic breakthrough (>1.0chi10(8) copies/mL) as well as biochemical breakthrough, which was associated with the presence of rtM204I plus rtL80I mutant. After switching from clevudine to adefovir, the viral load decreased with biochemical improvement.

Tumor hypoxia is important in the development and treatment of human cancers. We have developed a novel xenograft model for studying and imaging of hypoxia-induced gene expression. A hypoxia-inducible dual reporter herpes simplex virus type 1 thymidine kinase and enhanced green fluorescence protein (HSV1-TKeGFP), under the control of hypoxia response element (9HRE), was stably transfected into human colorectal HT29 cancer cells. Selected clones were further enriched by repeated live cell sorting gated for hypoxia-induced eGFP expression. Fluorescent microscopy, fluorescence-activated cell sorting, and radioactive substrate trapping assays showed strong hypoxia-induced expression of eGFP and HSV1-tk enzyme in the HT29-9HRE cells in vitro. Sequential micropositron emission tomography (PET) imaging of tumor-bearing animals, using the hypoxic cell tracer (18)F-FMISO and the reporter substrate (124)I-FIAU, yielded similar tumor hypoxia images for the HT29-9HRE xenograft but not in the parental HT29 tumor. Using autoradiography and IHC, detailed spatial distributions in tumor sections were obtained and compared for the following hypoxia-associated biomarkers in the HT29-9HRE xenograft: (124)I-FIAU, (18)F-FMISO, Hoechst (perfusion), lectin-TRITC (functional blood vessels), eGFP, pimonidazole, EF5, and CA9. Intratumoral distributions of (124)I-FIAU and (18)F-FMISO were similar, and eGFP, pimonidazole, EF5, and CA9 colocalized in the same areas but not in well-perfused regions that were positive for Hoechst and lectin-TRITC. In enabling the detection of hypoxia-induced molecular events and mapping their distribution in vivo with serial noninvasive positron emission tomography imaging, and multiple variable analysis with immunohistochemistry and fluorescence microscopy, this human xenograft model provides a valuable tool for studying tumor hypoxia and in validating existing and future exogenous markers for tumor hypoxia.

We investigated the possibility of using a pharmacologic agent to modulate viral gene expression to target radiotherapy to tumor tissue. In a mouse xenograft model, we had previously shown targeting of [(125)I]2'-fluoro-2'-deoxy-beta-D-5-iodouracil-arabinofuranoside ([(125)I]FIAU) to tumors engineered to express the Epstein-Barr virus thymidine kinase (EBV-TK). Here we extend those results to targeting of a therapeutic radiopharmaceutical [(131)I]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. With naturally infected EBV tumor cell lines (Burkitt's lymphoma and gastric carcinoma), activation of viral gene expression by pretreatment with bortezomib was required. Marked changes in tumor growth could also be achieved in naturally infected Kaposi's sarcoma herpesvirus tumors after pretreatment with bortezomib. Bortezomib-induced enzyme-targeted radiation therapy illustrates the possibility of pharmacologically modulating tumor gene expression to result in targeted radiotherapy.

We previously reported that both the concentrative (hCNT) and equilibrative (hENT) nucleoside transporters are expressed in the human liver (21). Here we report a study that investigated the expression of these transporters (transcripts and proteins) and their role in the hepatobiliary transport of nucleosides/nucleoside drugs using sandwich-cultured human hepatocytes. In the hepatic tissue, the rank order of the mRNA expression of the transporters was hCNT1 approximately hENT1>hENT2 approximately hCNT2>hCNT3. In sandwich-cultured hepatocytes, the mRNA expression of hCNT2 and hENT2 was comparable to that in hepatic tissue, whereas the expression of corresponding transporters in the two-dimensional hepatocyte cultures was lower. Colocalization studies demonstrated predominant localization of these transporters at the sinusoidal membrane and of hENT1, hCNT1, and hCNT2 at the canalicular membrane. In the sandwich-cultured hepatocytes, ENTs were the major contributors to the transport of thymidine (hENT1, 63%; hENT2, 23%) or guanosine (hENT1, 53%; hENT2, 24%) into the hepatocytes followed by hCNT1 (10%) for thymidine or hCNT2 (23%) for guanosine. Although ribavirin was predominately transported (89%) into the hepatocytes by hENT1, fialuridine (FIAU) was transported by both hENT1 (30%) and hCNTs (61%). The extensively metabolized natural nucleosides were not effluxed into the bile, whereas significant biliary-efflux was observed of FIAU (19%), ribavirin (30%), and formycin B (35%). We conclude that the hepatic activity of hENT1 and hCNT1/2 transporters will determine the in vivo hepatic distribution and therefore the efficacy and/or toxicity of nucleoside drugs used to treat hepatic diseases.