Safety, tolerability, efficacy and pharmacodynamics of the selective JAK1 inhibitor GSK2586184 in patients with systemic lupus erythematosus.

We aimed to evaluate the pharmacodynamics, efficacy, safety and tolerability of the JAK1 inhibitor GSK2586184 in adults with systemic lupus erythematosus (SLE). In this adaptive, randomized, double-blind, placebo-controlled study, patients received oral GSK2586184 50-400 mg, or placebo twice daily for 12 weeks. Primary endpoints included interferon-mediated messenger RNA transcription over time, changes in Safety of Estrogen in Lupus National Assessment-SLE Disease Activity Index score, and number/severity of adverse events. A pre-specified interim analysis was performed when ≥ 5 patients per group completed 2 weeks of treatment. In total, 84-92% of patients were high baseline expressors of the interferon transcriptional biomarkers evaluated. At interim analysis, GSK2586184 showed no significant effect on mean interferon transcriptional biomarker expression (all panels). The study was declared futile and recruitment was halted at 50 patients. Shortly thereafter, significant safety data were identified, including elevated liver enzymes in six patients (one confirmed and one suspected case of Drug Reaction with Eosinophilia and Systemic Symptoms), leading to immediate dosing cessation. Safety of Estrogen in Lupus National Assessment-SLE Disease Activity Index scores were not analysed due to the small number of patients completing the study. The study futility and safety data described for GSK2586184 do not support further evaluation in patients with SLE. Study identifiers: GSK Study JAK115919; ClinicalTrials.gov identifier: NCT01777256.

Efficacy of lamivudine re-treatment in a patient with hepatitis B virus (HBV) recurrence after liver transplantation and HBV-DNA breakthrough during the first treatment.

BACKGROUND: Transplantation for terminal hepatitis B virus (HBV) disease is aggravated by a high rate of reinfection and disease recurrence. Lamivudine, a new nucleoside analog, is a potent inhibitor of HBV synthesis, but its use may lead to the emergence of HBV-DNA polymerase mutants resistant to the drug. METHODS AND RESULTS: We describe the case of a patient who developed an HBV recurrence after liver transplantation and was treated with lamivudine. An HBV-DNA breakthrough occurred 7 months after the start of therapy, and the drug was stopped after 9 months. The molecular state of HBV-DNA was analyzed, and a mutation in the YMDD (tyrosine, methionine, aspartate, aspartate) locus of HBV-DNA polymerase was identified. Nine months after the suspension of lamivudine the patient experienced a new hepatic attack accompanied by high HBV-DNA levels. Lamivudine was given again. Serum HBV-DNA levels normalized after 45 days of re-treatment, but lamivudine-resistant mutants were again the prevalent viral population after 3 months. CONCLUSIONS: The case described suggests that retherapy with lamivudine after a first emergence of YMDD mutants is temporarily effective in recontrolling HBV synthesis but ultimately induces the accelerated reemergence of a prevalently mutant population of HBV. This emphasizes the need for combined antiviral therapy.

Inhibition of duck hepatitis B virus replication by hypericin.

Hypericin was found to be active against a member of the hepatitis B virus family, duck hepatitis B virus (DHBV). After a single 1 h incubation with hypericin, cells stably-transfected with a clone of DHBV stopped producing infectious virus for several days, though virus-like particles continued to be released into the culture medium. Characterization of these virions revealed a buoyant density characteristic of infectious virus preparations and lower than that of virus cores, suggesting that the particles were enveloped. Western blot analysis suggested, however, that the viral preS protein in surface antigen particles and, by inference, in virions, was present in covalently cross-linked aggregates. Evidence of a similar level of aggregation of the core subunit of virion nucleocapsids was not found, nor was there evidence of a similar high level of aggregation of cell-associated core and preS proteins. Hypericin was only slightly virucidal against DHBV and culture medium from treated cultures did not block initiation of infection when added to DHBV susceptible cultures prior to a challenge with infectious DHBV. Thus, the primary antiviral activity of hypericin against DHBV replication appears to be exerted at a late step in viral morphogenesis.

Quantitative analysis of HBV cccDNA from clinical specimens: correlation with clinical and virological response during antiviral therapy.

Attempts to investigate changes in various forms of intrahepatic hepatitis B virus (HBV) DNA during antiviral therapy have been hampered by limitations in technologies and scarcity of adequate tissue for analysis. We used a sensitive, specific assay to detect and quantitate covalently closed circular DNA (cccDNA) from total intrahepatic HBV DNA in clinical liver specimens. Total HBV DNA and cccDNA from 21 needle-biopsy specimens were quantified, with levels ranging from 0.1 to 9.8 copies/cell and 0.3 to 491.0 copies/cell, respectively. Then, we performed the same determinations on baseline and week-52 liver needle-biopsy specimens from eight patients enrolled in a clinical trial and evaluated the association between intrahepatic HBV DNA levels and serological and virological endpoints. In most patients, levels of intrahepatic HBV DNA, including cccDNA, decreased over the 52-week study, regardless of therapy or serological outcome. Higher ratios of cccDNA to total HBV DNA were detected at week 52 than at baseline indicating a shift in predominance of nonreplicating virus in posttreatment specimens. In patients who achieved treatment-related or spontaneous hepatitis B e antigen (HBeAg) responses, including those harbouring tyrosine-methionine-aspartate-aspartate-mutant HBV, levels of intrahepatic and serum HBV DNA suppression were greater than those in patients without HBeAg responses. In conclusion, this pilot study of intrahepatic HBV replicative forms in patients with chronic hepatitis B indicated that total intrahepatic and, specifically, cccDNA levels are not static but change as a reflection of serological and virological events.

Suppression of RNA synthesis by a specific antiviral activity in Sindbis virus-infected Aedes albopictus cells.

An antiviral protein is released by mosquito cells persistently infected with Sindbis virus. Differences in both sensitivity to and production of this virus-specific activity were apparent in three independently produced Aedes albopictus cell lines. This activity inhibits total viral RNA synthesis in a time-dependent manner. The antiviral effect is maximally realized when cells are treated with the activity 48 h before infections. These data suggest that the antiviral activity induces an antiviral state in treated cells which prevents the formation or efficient function of viral RNA-synthesizing complexes.

Exclusion of superinfecting homologous virus by Sindbis virus-infected Aedes albopictus (mosquito) cells.

The infection of tissue-cultured Aedes albopictus (mosquito) cells by an alphavirus ultimately results in a persistently infected cell population which can be maintained in the laboratory for years. One characteristic of this culture is that it will not support the replication of superinfecting homologous virus. We have previously shown that mosquito cells persistently infected with Sindbis virus produce an antiviral agent which when applied to uninfected mosquito cells suppresses Sindbis virus replication. The exclusion of virus replication in the antiviral-agent-treated cells is similar to the phenomenon of homologous interference described in alphavirus-infected vertebrate cells. In this study we examined the expression of homologous interference in three lines of mosquito cells and compared the expression of homologous interference to the effects of the antiviral activity. The cell lines were found to differ in their ability to express homologous interference, and evidence suggests that the mosquito cells may suppress replication by homologous interference or by the action of the antiviral agent.

Effect of actinomycin D and cycloheximide on replication of Sindbis virus in Aedes albopictus (mosquito) cells.

Production of Sindbis virus in the presence of transcription and translation inhibitors was examined in three Aedes albopictus cell lines. Addition of cycloheximide to heat-resistant Sindbis virus (SVHR)-infected mosquito cells arrested viral RNA synthesis completely, in contrast to the effects of this drug on virus-infected vertebrate cells. Production of mature virus by both SVHR (a variant commonly used as a wild-type virus) and SBamr (a mutant which is resistant to the effects of 18 h of pretreatment of vertebrate cells with actinomycin D) in mosquito u4.4, C6-36, and C7-10 cells was inhibited by 2 h of pretreatment with actinomycin D. Pretreatment with this drug for 2 h slightly enhances virus production in vertebrate cells. Treatment of mosquito cells with actinomycin D resulted in shutoff of SVHR RNA synthesis. The mutant SBamr was able to overcome the effects of actinomycin D on viral RNA synthesis and produced both 26S and 49S RNAs, even though no viral structural proteins or mature particles were produced in the presence of the drug. This result suggests that, in the presence of actinomycin, the nonstructural genes of SBamr are translated sufficiently to allow for RNA synthesis but that 26S RNA may not be translated to an extent that allows significant virus production. These data demonstrate that host components are involved in at least two distinct steps in the production of Sindbis virus in mosquito cells: (i) production of viral RNA and (ii) synthesis of viral structural polypeptides.

Lack of effect of antiviral therapy in nondividing hepatocyte cultures on the closed circular DNA of woodchuck hepatitis virus.

The template for synthesis of hepadnaviral RNAs is a covalently closed circular (ccc) DNA located in the nucleus of the infected hepatocyte. Hepatocytes are normally long-lived and nondividing, and antiviral therapies in chronically infected individuals face the problem of eliminating not only the replicative forms of viral DNA found in the cytoplasm but also the cccDNA from the nucleus. Because cccDNA does not replicate semiconservatively, it is not an obvious target for antiviral therapy. However, elimination of cccDNA might be facilitated if its half-life were short in comparison to the generation time of hepatocytes and if new cccDNA formation were effectively blocked. We have therefore measured cccDNA levels in woodchuck hepatocyte cultures following in vitro infection with woodchuck hepatitis virus and treatment with inhibitors of viral DNA synthesis. The viral reverse transcriptase inhibitors lamivudine (3TC) [(-)-beta-L-2',3'-dideoxy-3'-thiacytidine), FTC (5-fluoro-2',3'-dideoxy-3'-thiacytidine) and ddC (2',3'-dideoxycytidine) were added to the cultures beginning at 4 days postinfection. Treatment for up to 36 days with 3TC reduced the amount of cccDNA in the cultures not more than twofold compared to that of an untreated control. Treatment with ddC for 36 days and with FTC for 12 days resulted in effects similar to that of treatment with 3TC. Moreover, the declines in cccDNA appeared to reflect the loss of hepatocytes from the cultures rather than of cccDNA from hepatocytes. These results emphasize the important role of the longevity of the infected hepatocytes in the persistence of an infection.

Characterization of the core promoter and enhancer of duck hepatitis B virus.

The core gene promoter of duck hepatitis B virus (DHBV) has been localized and an enhancer element has been found in a region of the DHBV genome immediately upstream of the core promoter. This enhancer was able to activate expression from both the core promoter and the S promoter of DHBV, as well as from the heterologous thymidine kinase and simian virus 40 early promoters, but not from the DHBV PreS promoter, which was active both in the absence and in the presence of the enhancer. The activity of the enhancer showed a preference for cell cultures of hepatic origin, suggesting a possible tissue preference in vivo.

Efficient duck hepatitis B virus production by an avian liver tumor cell line.

Duck hepatitis B virus (DHBV) is produced in small amounts following transfection of human hepatoma or hepatoblastoma cell lines with cloned viral DNA. In a search for better hosts for DHBV replication, two avian liver cell lines were investigated. One of these cell lines, LMH, produced 5 to 10 times more DNA replicative intermediates and 10 to 20 times more infectious DHBV than did either of the two human cell lines, HuH-7 and Hep G2. Utilization of cell lines in genetic analyses of virus replication is often dependent upon obtaining efficient complementation between cotransfected viral genomes. We assayed transcomplementation of a viral polymerase (pol) gene mutant, which is rather inefficient in transfected human cells, and found that viral DNA synthesis was at least 20 times more efficient following cotransfection of LMH cells than in similarly transfected HuH-7 cells. Recombination, a potential interpretation problem in complementation assays, occurred at low levels in the cotransfected cultures but was substantially reduced or eliminated by creation of an LMH subline stably expressing the viral polymerase. This cell line, pol-7, supported the replication of DHBV pol mutants at ca. 10 to 15% of the level of virus replication obtained following transfection with wild-type viral DNA. By transcomplementation of a pol gene mutant in LMH cells, we were able to produce sufficient virus with the mutant genome to investigate the role of polymerase in covalently closed circular DNA amplification. Our results substantiate the hypothesis that covalently closed circular DNA is synthesized by the viral reverse transcriptase.

Evidence that less-than-full-length pol gene products are functional in hepadnavirus DNA synthesis.

Duck hepatitis B virus mutants containing frameshift or stop codon mutations in a portion of the viral pol gene separating the terminal protein and reverse transcriptase domains had a leaky phenotype and, depending on the location and type of mutation, synthesized up to 10% as much viral DNA as did the wild type. This region of the pol gene had previously been reported to be refractory to missense mutations; in fact, the leakiness of most of our mutants appeared attributable to translational suppression, which would also be expected to introduce amino acid changes. However, at least one mutant (pH1093 + 2), which was ca. 10% as active as the wild type, appeared to use a novel pathway to express the viral pol gene. Our analyses indicated that pH1093 + 2 synthesized the viral reverse transcriptase as a fusion protein with the amino-terminal portion of the pre-S envelope protein. Thus, in this case, the products of the terminal-protein and reverse transcriptase domains of the pol gene would function as separate protein species, though perhaps noncovalently joined in a dimeric structure during assembly of DNA replication complexes. Evidence was also obtained that was consistent with the idea that the wild-type pol gene may, at least in certain instances, be expressed as functional, subgenic polypeptides.

Chronic active hepatitis B exacerbations in human immunodeficiency virus-infected patients following development of resistance to or withdrawal of lamivudine.

Lamivudine is a nucleoside analog with activity against human immunodeficiency virus (HIV) and hepatitis B virus (HBV). Patients coinfected with HIV and HBV may have hepatitis flares when lamivudine therapy is discontinued or when resistance of HBV to lamivudine emerges. This retrospective, descriptive study conducted in three tertiary care medical centers describes patients coinfected with HIV type 1 and HBV who presented with a spectrum of clinical and subclinical hepatitic responses to lamivudine withdrawal or resistance. One patient had fulminant hepatic failure and a second patient had subclinical hepatitis when lamivudine therapy was discontinued and a more efficacious antiretroviral regimen was substituted. Three patients had flares of hepatitis after 13 to 18 months of lamivudine therapy. Lamivudine withdrawal or emergence of lamivudine-resistant mutants in patients coinfected with HIV and HBV may result in severe hepatitis. Clinicians caring for patients with coinfection with HIV and HBV should be aware of the possibility that a hepatitis B flare may occur in previously asymptomatic carrier patients.

Emergence of drug-resistant populations of woodchuck hepatitis virus in woodchucks treated with the antiviral nucleoside lamivudine.

Lamivudine [(-)-beta-L-2',3'-dideoxy-3'-thiacytidine] reduces woodchuck hepatitis virus (WHV) titers in the sera of chronically infected woodchucks by inhibiting viral DNA synthesis. However, after 6 to 12 months, WHV titers begin to increase toward pretreatment levels. Three WHV variants with mutations in the active site of the DNA polymerase gene are present at this time (W. S. Mason et al., Virology 245:18-32, 1998). We have asked if these mutant viruses were responsible for the lamivudine resistance and if their emergence caused an immediate rise in virus titers. Cell cultures studies implied that the mutants were resistant to lamivudine. Emergence of mutant WHV was not always associated, however, with an immediate rise in virus titers in the serum. One of the three types of mutant viruses became prominent in serum up to 7 months before titers in serum actually began to increase, at a time when wild-type virus was still predominant in the liver. The two other mutants did not show this behavior but were detected in serum and liver later, just at the time that virus titers began to rise. A factor linking all three mutants was that a similar duration of drug administration preceded the rise in titers, irrespective of which mutant ultimately prevailed. A simple explanation for these results is that the increase in virus titers following emergence of drug-resistant mutants can occur only as the preexisting wild-type virus is cleared from the hepatocyte population, allowing spread of the mutants. Thus, prolonged suppression of virus titers in the serum may sometimes be a measure of the stability of hepatocyte infection rather than of a successful therapeutic outcome.

(-)-cis-5-fluoro-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]cytosine (524W91) inhibits hepatitis B virus replication in primary human hepatocytes.

The anti-hepatitis B virus (HBV) activity of (-)-cis-5-fluoro-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]cytosine (524W91) in cultures of primary human hepatocytes was examined. 524W91 was anabolized to the active 5'-triphosphate in these cells. HBV replication was equally inhibited in cultures incubated with 524W91 when the drug was added 24 h preinfection, at infection, or 24 h postinfection. 524W91 inhibited HBV replication by 50% at less than 20 nM in human hepatocytes.

Replication of DHBV genomes with mutations at the sites of initiation of minus- and plus-strand DNA synthesis.

We have examined the consequences on duck hepatitis B virus DNA synthesis of deleting the 5' and 3' copies of the 12 base sequence, DR1, from the viral pregenome. With the wild-type virus, reverse transcription initiates at nt 2537 within the 3' copy of DR1. When this sequence was deleted, initiation of reverse transcription was found at two other sites located closer to the 3' end of the pregenome (nt 2576 and nt 2644). The 3-base motif UUA was the only sequence common to these sites as well as the wild-type initiation site in DR1. Deletion of the 5' copy of DR1 did not alter minus strand synthesis, but led to aberrant priming of plus strand synthesis to generate predominantly linear rather than relaxed circular, double-stranded viral DNA, in agreement with the recent report by Loeb et al. (EMBO J. 10, 3533-3540, 1991). A mutant lacking only the 3' copy of DR1 rapidly converted to wild type in transfected cells. This apparently occurred as a consequence of conversion of newly synthesized relaxed circular to covalently closed circular (CCC) DNA, which might then serve as a template for the synthesis of wild-type viral RNAs. A mutant lacking only the 5' copy of DR1 did not exhibit this behavior. These results support the conclusion that amplified CCC DNA serves as transcriptional template.

Identification and characterization of mutations in hepatitis B virus resistant to lamivudine. Lamivudine Clinical Investigation Group.

Cirrhosis and hepatocellular carcinoma occur as long-term complications of chronic hepatitis B virus (HBV) infection. Antiviral therapy is potentially a successful approach for the treatment of patients with HBV infection, which includes the nucleoside analog, lamivudine [(-)2'-deoxy-3'-thiacytidine, 3TC]. Although resistance to lamivudine therapy has been reported in several HBV-infected patients, the pattern of resistance-associated mutations in HBV has not been fully characterized. We report a DNA sequence database that includes a 500-base pair region of the HBV polymerase gene from 20 patients with clinical manifestations of lamivudine resistance. Analysis of the database reveals two patterns of amino acid substitutions in the tyrosine, methionine, aspartate, aspartate (YMDD) nucleotide-binding locus of the HBV polymerase. HBV DNA from the sera of patients in Group I exhibits a substitution of valine for methionine at residue 552, accompanied by a substitution of methionine for leucine at residue 528. Patients in Group II had only an isoleucine-for-methionine substitution at position 552. Reconstruction of these mutations in an HBV replication-competent plasmid was performed in a transient transfection cell assay to determine the function/relevance of these mutations to lamivudine resistance. Both Group I and Group II mutations resulted in a substantial decrease in sensitivity to lamivudine treatment (> 10,000-fold shift in IC50 over wild-type [wt] IC50), strongly indicating that these mutations were involved in resistance to lamivudine. A hypothetical model of the HBV reverse transcriptase has been generated for further study of the role of these mutations in lamivudine resistance.

Efficacy of lamivudine in patients with hepatitis B e antigen-negative/hepatitis B virus DNA-positive (precore mutant) chronic hepatitis B. Lamivudine Precore Mutant Study Group.

This placebo controlled, double-blind study evaluated the efficacy and safety of lamivudine in patients with hepatitis B e antigen (HBeAg)-negative/hepatitis B virus (HBV) DNA-positive chronic hepatitis B. Patients were randomized to receive 100 mg lamivudine orally once daily for 52 weeks (n = 60) or placebo for 26 weeks (n = 65). Patients who were HBV DNA positive at week 24 were withdrawn at week 26. The primary efficacy endpoint was loss of serum HBV DNA plus normalization of alanine transaminase (ALT) at week 24. A significantly higher proportion of patients receiving lamivudine (63%) had a complete response at week 24 compared with patients receiving placebo (6%) (P <.001). Secondary efficacy parameters included histological response from baseline to week 52 in the lamivudine-treated patients. At week 52, 60% of lamivudine-treated patients with liver biopsy specimens available showed histological improvement (>/=2-point reduction in Knodell necro-inflammatory score), 29% showed no change, and 12% worsened. In a ranked assessment of pretreatment and post-treatment biopsy pairs 11% improved, 86% showed no change, and 2% worsened in fibrosis. At week 52, 27% of patients receiving lamivudine had YMDD (tyrosine-methionine-aspartate-aspartate amino acid motif of HBV polymerase) variant HBV. The incidence of adverse events and laboratory abnormalities was similar in both groups. In conclusion, lamivudine treatment results in a significant virological and biochemical improvement compared with placebo, induces an improvement or no change in histology in most patients, and is well tolerated. The response to lamivudine therapy in HBeAg-negative patients is similar to the response reported in previous studies of patients with HBeAg-positive chronic hepatitis B.

Two sensitive PCR-based methods for detection of hepatitis B virus variants associated with reduced susceptibility to lamivudine.

Two novel assays, a restriction fragment length polymorphism (RFLP) assay and an assay based on the 5'-nuclease activity of Taq DNA polymerase, were developed for screening viral variants in lamivudine-treated patients' sera containing <1,000 copies of the hepatitis B virus (HBV) genome per ml. Both assays were designed to detect single-nucleotide changes within the HBV DNA polymerase gene that are associated with lamivudine resistance in vitro and have been used to screen a number of patients' sera for variant virus. Results obtained with these assays and standard sequencing technology were compared with regard to throughput, ability to detect individual virus species present at low concentrations, and ability to detect, distinguish, and quantitate wild-type (wt) and HBV tyrosine methionine(552) aspartate aspartate motif variants in mixed viral populations. Unlike DNA sequencing, both assays are amenable to high-throughput screening and were shown to be able to quantitatively detect variant virus in the presence of a background of wt virus. As with DNA sequencing, both new assays incorporate a PCR amplification step and are able to detect the relatively low amounts of virus found in lamivudine-treated patients' sera. However, these assays are far less labor intensive than the DNA-sequencing techniques presently in use. Overall, the RFLP assay was more sensitive than DNA sequencing in detecting and determining the ratios of wt to variant virus. Furthermore, the RFLP assay and 5'-nuclease assay were equally sensitive in the detection of mixed viral species, but the RFLP assay was superior to the 5'-nuclease assay in the quantitation of mixed viral species. These assays should prove useful for further understanding of virological response to therapy and disease progression.

T-helper cell response to woodchuck hepatitis virus antigens after therapeutic vaccination of chronically-infected animals treated with lamivudine.

BACKGROUND/AIMS: Immunotherapy of patients chronically-infected with hepatitis B virus (HBV) may have the risk of fulminant hepatitis. This risk might be diminished if immunotherapy was carried out under conditions of low viremia. METHODS: Five woodchucks chronically-infected with woodchuck hepatitis virus (WHV), a virus closely related to HBV, were treated with lamivudine for 23 weeks. At week 10, when viremia had decreased by 3-5 logs, three woodchucks were vaccinated with woodchuck hepatitis virus surface antigen (WHsAg) plus the T-helper determinant FISEAIIHVLHSR. RESULTS: It was found that the administration of lamivudine only, had no effect on the T-helper response against WHV antigens. By contrast, vaccination induced T-helper responses against WHV antigens, shifting the cytokine profile from Th2 to Th0/Th1, but was without effect on viremia, WHsAg levels, or anti-WHs antibodies. Analysis of liver biopsies showed that lamivudine administration may have reduced hepatic inflammation. By contrast, vaccination clearly enhanced hepatic inflammation. After lamivudine withdrawal, viremia returned to high levels. CONCLUSIONS: These results suggest that therapeutic vaccination of chronically-infected woodchucks under conditions of low viremia shifts the cytokine profile against viral antigens towards Th0/Th1. This shift may prevent the efficient induction of anti-WHs antibodies.