Substitution at rt269 in Hepatitis B Virus Polymerase Is a Compensatory Mutation Associated with Multi-Drug Resistance.

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.

Fialuridine induces acute liver failure in chimeric TK-NOG mice: a model for detecting hepatic drug toxicity prior to human testing.

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.

High-dose clevudine impairs mitochondrial function and glucose-stimulated insulin secretion in INS-1E cells.

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 ß-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.

Identification and characterization of clevudine-resistant mutants of hepatitis B virus isolated from chronic hepatitis B patients.

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.

Clevudine: a promising therapy for the treatment of chronic hepatitis B.

Chronic hepatitis B virus (HBV) infection, affecting approximately 350 million people worldwide, is associated with significant morbidity and mortality. In the past 10 years, hepatitis B therapy research has led to a multitude of available antiviral therapies: IFN-alpha, pegylated IFN-alpha(2a), lamivudine, adefovir, entecavir, telbivudine and tenofovir. To further improve reductions in viral load and resistance profiles, development of new HBV therapeutic strategies has been an important focus. One such therapy is clevudine, an analogue of the beta-L configuration. Clevudine is already licensed in Korea for anti-HBV therapy (Bukwang Pharmaceuticals, Seoul, Korea). Unique to clevudine is its ability to maintain antiviral activity following discontinuation of therapy. Typically, hepatitis B treatment requires continuous therapy to prevent reactivation. Sustained response is uncommon except in hepatitis B antigen (HBeAg)-positive patients who developed HBeAg seroconversion. This article reviews chronic HBV and its therapy options. Specifically, it describes clevudine's potent and sustained antiviral activity as observed in vitro and in vivo.

Clevudine for the treatment of chronic hepatitis B virus infection.

Chronic hepatitis B virus (HBV) infection is a major health problem that is responsible for < or = 1 million deaths and 500,000 cases of hepatocellular carcinoma worldwide each year. Drugs that are currently approved by the FDA for the treatment of chronic HBV consist of two groups: the immunomodulators, such as conventional IFN-alpha and pegylated IFN-alpha2a; and nucleoside/nucleotide analogues, such as lamivudine, adefovir dipivoxil and entecavir. However, due to the limitations of these agents, newer agents with improved efficacy are currently being developed. One nucleoside/nucleotide analogue that is drawing a wide range of interest is clevudine, which is an analogue of the unnatural beta-L configuration. In the woodchuck hepatitis virus (WHV), clevudine 10 mg/kg has proven to be effective in suppressing viral replication with < or = 9 log10 decreases in WHV. At this dose, a significant reduction of intrahepatic WHV RNA and covalently closed circular WHV DNA levels can also be observed. Treatment with clevudine 10 mg/kg can confer additional antiviral benefit in the form of a more sustained reduction in WHV replication, serum woodchuck hepatitis surface antigen and intrahepatic woodchuck hepatitis core antigen expression following the withdrawal of clevudine. In humans, clevudine 10, 50, 100 or 200 mg/day for 28 days can reduce the median HBV DNA by -2.5, -2.7, -3 and -2.6 log10, respectively. More importantly, this suppression of antiviral activity is maintained at 12 and 24 weeks post treatment. Based on the early results of clevudine, more large-scale human studies with clevudine monotherapy or combination therapy is eagerly awaited.

Imaging DNA synthesis in vivo with 18F-FMAU and PET.

UNLABELLED: We imaged DNA synthesis in vivo with PET and (18)F-1-(2'-deoxy-2'-fluoro-beta-d-arabinofuranosyl)thymine (FMAU), which is phosphorylated by thymidine kinases and incorporated into DNA. METHODS: We produced (18)F-FMAU and injected the tracer into 5 normal dogs and studied them by imaging or biodistribution for up to 2.5 h. The pharmacokinetics of FMAU in blood and urine were determined using high-performance liquid chromatography analysis. At the end of each study, selected tissues were removed to measure the total activity retained in these tissues. In addition, the selected tissues were extracted by acid precipitation, by which the macromolecules can be precipitated to determine the radioactivity of (18)F-FMAU incorporated into DNA. RESULTS: Imaging and tissue analysis showed increased activity in the lymph nodes, stomach, small intestine, and bone marrow, with mean standardized uptake values of 1.4, 1.6, 2.3, and 3.9, respectively, because of varying degrees of increased cell proliferation. In contrast, (18)F-FMAU was distributed with tissue-to-muscle ratios of approximately 1.0 in nonproliferative organs such as lung, liver, and kidneys. Analysis of the tissue extracts using acid precipitation demonstrated that 88% of activity in marrow and 65% of activity in small intestine was acid precipitated. However, more than 90% of activity in the nonproliferating tissues such as heart and lungs was in the supernatant. Increased activity was seen in the heart because of a high level of thymidine kinase 2 and in the gallbladder because of excretion. Analysis of blood and urine demonstrated that more than 95% of activity was present as intact (18)F-FMAU at the end of the studies. CONCLUSION: The results showed that (18)F-FMAU was selectively retained in DNA of the proliferating tissues and was resistant to degradation. These features indicate that (18)F-FMAU might be an alternative to (11)C-thymidine for imaging DNA synthesis in normal tissues and tumors.

A preclinical model for noninvasive imaging of hypoxia-induced gene expression; comparison with an exogenous marker of tumor hypoxia.

PURPOSE: Hypoxia is associated with tumor aggressiveness and is an important cause of resistance to radiation therapy and chemotherapy. Assays of tumor hypoxia could provide selection tools for hypoxia-modifying treatments. The purpose of this study was to develop and characterize a rodent tumor model with a reporter gene construct that would be transactivated by the hypoxia-inducible molecular switch, i.e., the upregulation of HIF-1. METHODS: The reporter gene construct is the herpes simplex virus 1-thymidine kinase (HSV1-tk) fused with the enhanced green fluorescent protein (eGFP) under the regulation of an artificial hypoxia-responsive enhancer/promoter. In this model, tumor hypoxia would up-regulate HIF-1, and through the hypoxia-responsive promoter transactivate the HSV1-tkeGFP fusion gene. The expression of this reporter gene can be assessed with the 124I-labeled reporter substrate 2'-fluoro-2'-deoxy-1-beta-D-arabinofuranosyl-5-iodouracil (124I-FIAU), which is phosphorylated by the HSV1-tk enzyme and trapped in the hypoxic cells. Animal positron emission tomography (microPET) and phosphor plate imaging (PPI) were used in this study to visualize the trapped 124I-FIAU, providing a distribution of the hypoxia-induced molecular events. The distribution of 124I-FIAU was also compared with that of an exogenous hypoxic cell marker, 18F-fluoromisonidazole (FMISO). RESULTS: Our results showed that 124I-FIAU microPET imaging of the hypoxia-induced reporter gene expression is feasible, and that the intratumoral distributions of 124I-FIAU and 18F-FMISO are similar. In tumor sections, detailed radioactivity distributions were obtained with PPI which also showed similarity between 124I-FIAU and 18F-FMISO. CONCLUSION: This reporter system is sufficiently sensitive to detect hypoxia-induced transcriptional activation by noninvasive imaging and might provide a valuable tool in studying tumor hypoxia and in validating existing and future exogenous markers for tumor hypoxia.

Positron emission tomography imaging for herpes virus infection: Implications for oncolytic viral treatments of cancer.

Molecular therapy using viruses would benefit greatly from a non-invasive modality for assessing dissemination of viruses. Here we investigated whether positron emission tomography (PET) scanning using [(124)I]-5-iodo-2'-fluoro-1-beta-d-arabinofuranosyl-uracil (FIAU) could image cells infected with herpes simplex viruses (HSV). Using replication-competent HSV-1 oncolytic viruses with thymidine kinase (TK) under control of different promoters, we demonstrate that viral infection, proliferation and promoter characteristics all interact to influence FIAU accumulation and imaging. In vivo, as few as 1 x 107 viral particles injected into a 0.5-cm human colorectal tumor can be detected by [(124)I]FIAU PET imaging. PET signal intensity is significantly greater at 48 hours compared with that at 8 hours after viral injection, demonstrating that PET scanning can detect changes in TK activity resulting from local viral proliferation. We also show the ability of FIAU-PET scanning to detect differences in viral infectivity at 0.5 log increments. Non-invasive imaging might be useful in assessing biologically relevant distribution of virus in therapies using replication-competent HSV.

Preclinical investigation of L-FMAU as an anti-hepatitis B virus agent.

Preclinical aspects of a potent anti-hepatitis B virus (HBV) L-nucleoside, 1-(2-fluoro-5-methyl-beta-L-arabino-furanosyl)uracil (L-FMAU) are described. L-FMAU was prepared from L-ribose derivatives via either L-xylose or L-arabinose. L-FMAU shows potent antiviral activity against hepatitis B virus (EC50 5.0 microM in H1 cells) with high selectivity in vitro. L-FMAU is not incorporated into mitochondrial DNA and no significant lactic acid production was observed in vitro. L-FMAU is phosphorylated by thymidine kinase as well as deoxycytidine kinase, ultimately to the triphosphate, which inhibits HBV DNA polymerase as the mechanism of antiviral action. Preliminary in vivo toxological studies suggest no apparent toxicity for 30 days at 50 mg/kg/day in mice and for 3 months in woodchucks (10 mg/kg/day). L-FMAU also has respectable bioavailability in rats. L-FMAU shows potent anti-HBV activity in vivo against woodchuck hepatitis virus in chronically infected woodchucks and there is no significant virus rebound after cessation of the drug treatment.

Herpesviruses.

AIDS: The signs, symptoms, diagnoses, and treatment of human herpesviruses are discussed, including advances and refinements in treatment options. Various treatment drugs, such as Zovirax, Famvir, Cidofovir, Foscarnet, Valtrex, and Virend, are examined. Genital herpes vaccines and possible alternative therapies are reviewed. In particular, varicella zoster virus, the virus that produces chicken pox and shingles, is examined, including its signs, symptoms, and intervention strategies using famciclovir and sorivudine. Final comments discuss the Epstein-Barr virus, the cause of mononucleosis, the newly discovered human herpesviruses, and the future prospects for identifying and appropriately treating herpesviruses.^ieng

Construction of a herpes simplex virus/varicella-zoster virus (HSV/VZV) thymidine kinase recombinant with the pathogenic potential of HSV and a drug sensitivity profile resembling that of VZV.

A recombinant of herpes simplex virus (HSV) was constructed in which the HSV thymidine kinase (TK) gene was deleted and the varicella-zoster virus (VZV) TK gene was introduced into the US5 region under the control of the human cytomegalovirus IE promoter. Infection with the recombinant (R18) led to the induction of TK although the kinetics of synthesis resembled those of a 'late' gene product. The recombinant was virulent in the zosteriform mouse model with the pattern of pathogenesis similar to that of wild-type HSV-1. The sensitivity of the recombinant to several nucleoside analogues was assessed and in most cases (BVaraU, ACV and GCV) it resembled VZV rather than HSV. The enhanced sensitivity of the recombinant to BVaraU compared with wild-type HSV resulted in a far greater response to treatment with BVaraU as assessed in the mouse model.

New nucleoside analogues for chronic hepatitis B.

In recent years, an in vitro system for screening nucleoside analogues against hepatitis B virus has yielded several compounds with a high therapeutic index. Phase I and II studies have also shown a potent in vivo antiviral effect of fialuridine and lamivudine in patients with chronic hepatitis B. The use of fialuridine was associated with unexpectedly severe mitochondrial dysfunction; in contrast, lamivudine had virtually no side-effects. Experience in liver transplant patients with recurrent hepatitis B shows that famciclovir may be another effective antivirotic drug with good tolerance.

Effects of 2'-fluorinated arabinosyl-pyrimidine nucleosides on duck hepatitis B virus DNA level in serum and liver of chronically infected ducks.

The 2'-fluorinated arabinosyl-pyrimidine nucleosides, 1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5-iodocytosine (FIAC) and 1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5-methyluracil (FMAU), are new antiviral compounds with in vitro inhibitory activity against the DNA polymerase of hepadnaviruses. Those compounds also induced permanent inhibition of viral replication in woodchucks chronically infected by woodchuck hepatitis virus. The effects of these antiviral compounds were assessed in ducks chronically infected by duck hepatitis B virus (DHBV). Following intraperitoneal administration for 5 days, FMAU (2 mg/kg/day) and FIAC (10 mg/kg/day) induced a transient decrease in DHBV replication, as shown by the decrease in both the serum and liver DHBV DNA level. After stopping therapy, DHBV replication rebounded immediately to the pretreatment level. The supercoiled form of liver viral DNA was found to be less affected by the therapy. By contrast, no obvious antiviral effect was observed with vidarabine monophosphate (ara-AMP) (80 mg/kg/day) therapy. No sign of toxicity was observed during the course of the treatment. These preliminary results confirmed in the DHBV model the higher efficacy of FIAC and FMAU as compared to ara-AMP. Pharmacokinetic studies are needed to explain the differences observed in viral replication in these 2 models of HBV infection.

Efficacy of oral treatment with BV-araU against cutaneous infection with herpes simplex type 1 in shaved mice.

The effect of oral BV-araU was tested in cutaneous model infections of shaved Balb/c mice with herpes simplex virus type 1 (HSV-1). Progression of cutaneous symptoms associated with cutaneous infection with HSV-1 F strain was inhibited by BV-araU at doses of 20 and 50 mg/kg twice daily, beginning one day post-infection, resulting in significant increase in the survival rate. Onset of disease was suppressed in most animals receiving 100 mg of BV-araU per kg. BV-araU (20 mg/kg or more) also significantly increased the survival rate of mice infected with HSV-1 WT-51 strain. The efficacy of BV-araU was not affected by gender or age (6-9 weeks) of the mice. BV-araU was effective even when the treatment was started 2.5 days post-infection. The efficacy of BV-araU against F strain infection was comparable to that of acyclovir, but acyclovir showed therapeutic effects at lower doses compared with BV-araU against WT-51 strain infection. Against infection of cyclophosphamide-treated immunosuppressed mice with HSV-1 KOS(S) strain, BV-araU decreased the morbidity rate and severity of symptoms at doses of 200 and 100 mg/kg, respectively, and all mice given 50 mg of BV-araU or more per kg survived, suggesting oral efficacy can be achieved against HSV-1 infections in immunosuppressed individuals.

Evaluation of 1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5-ethyluracil in a rabbit model of herpetic keratitis.

The nucleoside analog 1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5- ethyluracil (FEAU) was tested in a rabbit model of acute herpetic keratitis and its effectiveness compared with that of acyclovir (ACV). FEAU or ACV was applied topically 3 times daily, beginning 3 days post-HSV-1 inoculation and continued for a period of 7 days. FEAU at a concentration of 1% (w/v) or 3% ACV resulted in significant lessening of the severity of corneal lesions, conjunctivitis, iritis, and corneal clouding at 24 to 48 h after beginning chemotherapy. No toxic reaction was observed in any rabbit eyes treated with either FEAU or ACV. The duration of virus shedding into tear film and colonization of the trigeminal ganglia, however, were not reduced by either FEAU or ACV treatment begun 3 days post-inoculation. Fifty percent effective dose (ED50) of FEAU determinations performed on isolates from tear film and on the virus inoculum in secondary rabbit kidney cultures yielded a range of 4.6-7 microM, with two in vitro resistant isolates having ED50S of greater than or equal to 1500 microM of FEAU. Fifty percent cell growth inhibition for FEAU was 3000 microM at 72 h.

Evaluation of (E)-5-(2-bromovinyl)- and 5-vinyl-1-beta-D-arabinofuranosyluracil (BrVaraU, VaraU) in the treatment of experimental herpes simplex virus type 1 keratitis in rabbits: comparison with (E)-5-(2-bromovinyl)-2'-deoxyuridine (BrVUdR).

The 5-substituted 1-beta-D-arabinofuranosyl (araU) analogues, (E)-5-(2-bromovinyl)-araU (BrVaraU) and 5-vinyl-araU (VaraU), which can be considered as structural analogues of (E)-5-(2-bromovinyl)-2'-deoxyuridine (BrVUdR), are potent and selective inhibitors of herpes simplex virus type 1 (HSV-1) replication in vitro. BrVaraU and VaraU have been compared with BrVUdR for their therapeutic effect on acute HSV-1 keratitis in rabbits. Both araU derivatives applied as 0.1% eyedrops suppressed the development of keratitis as monitored by the reduced number of herpes efflorescences. The healing effect of BrVaraU and VaraU was less pronounced than that of 0.1% BrVUdR eyedrops, the difference between BrVUdR and VaraU being statistically significant at the 10th day of treatment. As a further indication of the healing effect the number of cornea with opacities seen after cessation of drug treatment were 3.3, 7.4, 27.6 and 46.9% for the BrVUdR-BrVaraU-, VaraU- and placebo-treated eyes, respectively.