Treatment of chronic hepadnavirus infection in a woodchuck animal model with an inhibitor of protein folding and trafficking.

A novel strategy for anti-viral intervention of hepatitis B virus (HBV) through the disruption of the proper folding and transport of the hepadnavirus glycoproteins is described. Laboratory reared woodchucks chronically infected with woodchuck hepatitis virus (WHV) were treated with N-nonyl-deoxynojirimycin (N-nonyl-DNJ), an inhibitor of the endoplasmic reticulum (ER) alpha-glucosidases. The woodchucks experienced significant dose dependent decreases in enveloped WHV, resulting in undetectable amounts in some cases. The reduction in viremia correlated with the levels of hyperglucosylated glycan in the serum of treated animals. This correlation supports the mechanism of action associated with the drug and highlights the extreme sensitivity of the virus to this type of glycan inhibitor. At N-nonyl-DNJ concentrations that prevented WHV secretion, the glycosylation of most serum glycoproteins appeared unaffected, suggesting great selectivity for this class of therapeutics. Indeed, this may account for the low toxicity of the compound over the treatment period. We provide the first evidence that glucosidase inhibitors can be used in vivo to alter specific steps in the N-linked glycosylation pathway and that this inhibition has anti-viral effects.

Induction of hepatitis B virus core gene in human cells by cytosine demethylation in the promoter.

A recombinant human cell line constructed by transfection of epithelial cells with a plasmid containing the hepatitis B virus core gene (HBc) was used to study the regulation of HBc gene expression. Methylation of a single Hpa II site 280 base pairs upstream from the structural gene was found to regulate the expression of the core gene. Expression increased in cells treated with 5'-azacytidine as a result of cytosine demethylation at this site, and there was a fivefold increase in the number of HBc gene transcripts in total cellular messenger RNA. The varied life cycle of hepatitis B virus in disease such as viral hepatitis and liver cancer may therefore be attributable to the site-specific regulation of the gene involved in replication of the viral DNA and to the cytophathic effects elicited by this gene in human cells.

Mitogen-induced replication of woodchuck hepatitis virus in cultured peripheral blood lymphocytes.

Peripheral blood lymphocytes (PBLs) isolated from woodchucks chronically infected with the woodchuck hepatitis virus (WHV) carry low levels of nonreplicating WHV DNA. When PBLs from chronic carrier woodchucks were activated in culture with the generalized mitogen lipopolysaccharide (LPS), WHV DNA replication was initiated in cells obtained from one of three animals examined. Intracellular WHV core particles, containing WHV DNA replication intermediates, RNA/DNA hybrid molecules, and an active endogenous DNA polymerase, appeared 3 days after the start of LPS stimulation. After 5 to 7 days of LPS stimulation, WHV DNA-containing particles, which displayed the properties of intact, mature virions, were released into the culture medium. These studies provide evidence for reactivation of a latent WHV infection of circulating lymphoid cells and indicate that the presence of nonreplicating hepadnaviral DNA in lymphoid cells represents a potentially active infection following cellular activation.

High-frequency transfection and cytopathology of the hepatitis B virus core antigen gene in human cells.

A protoplast fusion method was developed to stably transfect human cells with pSV2-derived plasmids at frequencies greater than 10(-3). This procedure made it possible to test the biological effect of a hepatitis B virus (HBV) gene independent of the viral structures required for infection. A pSV2gpt+ plasmid constructed to carry a subgenomic fragment of HBV that contained the core antigen gene (HBc gene) was transfected into human cells. A human epithelial cell line was stably transfected with the HBc+ gene by selecting recipient cells for expression of guanine phosphoribosyl transferase expression. With this gpt+/HBc+ cell line it was shown that growth in serum-free medium or treatment with 5'-azacytidine stimulates the production of the HBV core antigen. A hepatocellular carcinoma carrying the entire HBV genome was stimulated to produce the HBc gene product in response to the same factors that stimulated HBcAg production in the gpt+/HBc+ cell line constructed by transfection. The temporal relation between the cytopathologic response and HBc gene expression was similar for both cell types, indicating a primary role for HBc gene expression in the cytopathology of HBV-infected human liver.

Transformation of human bronchial epithelial cells transfected by Harvey ras oncogene.

Transfection of normal human bronchial epithelial (NHBE) cells with a plasmid carrying the ras oncogene of Harvey murine sarcoma virus (v-Ha ras) changed the growth requirements, terminal differentiation, and tumorigenicity of the recipient cells. One of the cell lines isolated after transfection (TBE-1) was studied extensively and shown to contain v-Ha ras DNA. Total cellular RNA from TBE-1 cells hybridized to v-Ha ras structural gene fragment probes five to eight times more than RNA from parental NHBE cells. The TBE-1 cells expressed phosphorylated v-Ha ras polypeptide p21, showed a reduced requirement for growth-factor supplements, and became aneuploid as an early cellular response to v-Ha ras expression. As the transfectants acquire an indefinite life-span and anchorage independence they became transplantable tumor cells and showed many phenotypic changes suggesting a pleiotropic mechanism for the role of Ha ras in human carcinogenesis.

Apparent alteration in properties of arl mutants of Escherichia coli.

The published properties of lambda phages grown in Escherichia coli arl mutants, and plasmids maintained in them, included increased homologous recombination, decreased DNA-cytosine methylation, and increased sensitivity of DNA to nuclease S1. Some of these properties now appear altered; others remain approximately as published.

Transgenic mice as a chemotherapeutic model for hepatitis B virus infection.

Many transgenic mice carrying either portions of the hepatitis B virus (HBV) genome, or the complete genome, have been developed as models because HBV does not infect any other organisms besides humans and chimpanzees to cause a productive infection and disease. Some of these models have been useful in evaluating chemotherapeutic agents such as interferon-alpha, interleukins-2 and -12, other cytokines and nucleoside analogues for efficacy against HBV. A recently developed transgenic mouse (Guidotti et al., Journal of Virology 69:6158-6169.) which supports the replication of high levels of infectious HBV, provides the opportunity to evaluate the effect of antiviral drugs on various portions of the HBV life cycle in the whole animal. Evaluation of lamivudine, zidovudine and interferon-alpha B/D (IFN-alpha) in this HBV transgenic mouse model are described. Lamivudine and IFN-alpha were highly efficacious in reducing serum HBV DNA. As might be predicted, zidovudine was not efficacious. IFN-alpha was more effective in reducing virus titres in male mice as compared to female mice. This gender difference was not due to lower ability of female mice to express the virus. One anticipates that as this high level HBV transgenic-expressing mouse becomes more fully developed as a chemotherapeutic model, questions about the efficacy of different agents, routes of administration, synergy of antiviral combinations and novel drug therapies will be answered.

Antiviral activity of a conjugate of adenine-9-beta-D-arabinofuranoside 5'-monophosphate and a 9 kDa fragment of arabinogalactan.

An arabinogalactan conjugate containing a 9 kDa fragment of arabinogalactan and adenine-9-beta-D-arabinofuranoside 5'-monophosphate (araAMP), denoted AG(9 kDa)-araAMP, has been synthesized and characterized. In 2.2.15 (human hepatoblastoma) cells, the attachment of araAMP to AG(9 kDa), a ligand of the asialoglycoprotein receptor, decreased the effective concentration for inhibiting extracellular hepatitis B virus (HBV) production by 90% (EC90) from 17 to 0.9 microM adenine arabinoside (araA) equivalents, and increased the cytotoxic concentration (CC50) from 188 to > 17 300 microM araA equivalents. Hence, the selectivity index (CC50/EC90) of araA was improved from 11 (188/17) to > 19200 (17 300/0.9) by conjugation with the 9 kDa fragment of arabinogalactan. AG(9 kDa)-araAMP did not affect the production of viral RNA or viral proteins. In the woodchuck hepatitis model, AG(9 kDa)-araAMP inhibited woodchuck hepatitis virus (WHV) DNA replication at a dose of 0.3 mg of araA equivalents per kg; in this case, AG(9 kDa)-araAMP was 20-30 times more potent than was unconjugated araA. AG(9 kDa)-araAMP was effective by intramuscular or subcutaneous administration. The reduction in HBV DNA levels obtained in 2.2.15 cells and of WHV DNA levels in woodchucks was sustained after treatment with AG(9 kDa)-araAMP ceased. In both cases, viral DNA gradually returned to pre-treatment levels.

Enhanced antiviral benefit of combination therapy with lamivudine and alpha interferon against WHV replication in chronic carrier woodchucks.

Cell culture studies in our laboratory previously demonstrated synergistic antiviral activity for the combinations of lamivudine and a novel recombinant hybrid human alpha B/D interferon (rHu alpha B/D IFN) against hepatitis B virus (HBV) replication. Based on these results, a study was designed to determine if an enhanced antiviral effect with this drug combination could be demonstrated in vivo using the woodchuck hepatitis virus (WHV)/woodchuck experimental model of chronic HBV infection. Both antiviral agents have been shown to be effective against WHV replication in WHV chronic carriers during previous studies by our laboratories. Two combination treatment regimens were compared to matched monotherapies in a placebo-controlled trial. The first used simultaneous administration of rHu alpha B/D IFN and lamivudine for 24 weeks. The other combination treatment regimen used a staggered dosing schedule of 12 weeks of administration of lamivudine alone, followed by 12 weeks of simultaneous dosing with both drugs, followed by 12 weeks of therapy with rHu alpha B/D IFN alone. Both treatment regimens with combinations of lamivudine and rHu alpha B/D IFN were more effective at reducing WHV replication in chronically infected wood-chucks than the corresponding monotherapies. Both combination treatments produced antiviral effects that were at least equal to that expected for additive activity based on estimations generated by Bliss Independence calculations. The staggered treatment regimen reduced viraemia and intrahepatic WHV replication significantly more than that expected for additive interactions, indicating synergistic antiviral effects. These studies demonstrate that combination therapy of chronic WHV infection has enhanced antiviral benefit over corresponding monotherapies and indicate that combination treatment of chronic HBV infection can be superior to therapies using a single antiviral agent.

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.

Does the anti-hepatitis B virus activity of (+)-5'-noraristeromycin exist in its 4'-epimer and 4'-deoxygenated derivatives?

To begin an exploration of the structural parameters responsible for the activity of (+)-5'-noraristeromycin toward hepatitis B virus (HBV), three derivatives varied at the C-4' position have been prepared and evaluated. The syntheses began with a Mitsunobu coupling reaction of an appropriate cyclopentanol with 6-chloropurine. The products of these reactions were synthetically altered by standard ammonolysis and deprotection procedures to give the desired products. Evaluation of the new derivatives indicated that removal of the C-4' hydroxyl of (+)-5'-noraristeromycin increased its potency toward HBV by approximately 10-fold.

Enhanced oral absorption and antiviral activity of 1-O-octadecyl-sn-glycero-3-phospho-acyclovir and related compounds in hepatitis B virus infection, in vitro.

Acyclovir (ACV) triphosphate and azidothymidine (AZT) triphosphate inhibit the DNA polymerase of human hepatitis B virus (HBV) by 50% at submicromolar concentrations, but no effects of ACV or AZT treatment have been noted on the clinical manifestations of hepatitis B. We synthesized 1-O-octadecyl-sn-glycero-3-phospho-acyclovir (ODG-P-ACV), 1-O-hexadecylpropanediol-3-phospho-acyclovir (HDP-P-ACV), and 1-O-octadecyl-sn-glycero-3-phospho-azidothymidine (ODG-P-AZT), and evaluated their antiviral activity in human hepatoma cells that constitutively produce HBV (2.2.15 cells). ACV and AZT up to 100 microM caused only slight inhibition of HBV replication in 2.2.15 cells. However, HDP-P-ACV and ODG-P-ACV inhibited viral replication by 50% at 0.5 and 6.8 microM, respectively. ODG-P-AZT also showed increased antiviral activity, with a 50% reduction in HBV replication at 2.1 microM. Based on the EC50, HDP-P-ACV, ODG-P-ACV, and ODG-P-AZT were > 200, > 14.7, and > 48 times more active than their free nucleosides in reducing HBV replication in 2.2.15 cells. To evaluate the biochemical basis for the increased antiviral activity, we studied the uptake and metabolism of 1-O-octadecyl-sn-glycero-3-phospho-[3H]acyclovir (ODG-P-[3H]ACV) in HepG2 cells. Cellular uptake of ODG-P-[3H]ACV was found to be substantially greater than that of [3H]ACV, and cellular levels of ACV-mono-, -di-, and -triphosphate were much higher with ODG-P-ACV. ODG-P-[3H]ACV was well absorbed orally. Based on urinary recovery of tritium after oral or parenteral administration of the radiolabeled compounds, oral absorption of ODG-P-ACV in mice was 100% versus 37% for ACV. ODG-P-ACV plasma area under the curve was more than 7-fold greater than that of ACV. Lipid prodrugs of this type may be useful orally in treating viral diseases.

In vitro evaluation of combination therapies against hepatitis B virus replication.

The HBV-producing human hepatoblastoma cell line, 2.2.15, has been shown to be an accurate model of chronic cellular viral infection and a predictive model of antiviral response for in vivo hepadnaviral infection. Our laboratory has utilized the 2.2.15 cell line in a standardized assay to examine treatment schemes which use combinations of clinically relevant nucleoside analogues, novel methods to deliver potentially useful nucleoside combinations, and treatments which simultaneously target different parts of the HBV replication pathway. For example, the combination of 3TC (lamivudine) with either alpha interferon or penciclovir significantly enhances the antiviral effectiveness of these agents against HBV replication in 2.2.15 cell culture.

Antisense oligonucleotides are effective inhibitors of hepatitis B virus replication in vitro.

Antisense oligonucleotides are currently being used in numerous laboratories as potential anticancer and antiviral agents. The unique replication cycle of hepatitis B virus (HBV) contains several different steps which are potentially amenable to modulation by these molecules. We have examined the ability of 56 different single-stranded, oligodeoxyribonucleotides (14-23 nucleotides in length), which target several HBV-specific functions, to inhibit HBV replication in the human hepatoblastoma cell line, 2.2.15. None of the oligonucleotides examined were toxic at concentrations up to 500 microM. Oligonucleotides directed against the HBV surface antigen (HBsAg) gene (S gene), the preS1 open reading frame, and the HBV core antigen (HBcAg) gene (C gene) were effective at depressing virus production, while molecules targeting the HBV e antigen (HBeAg) open reading frame and the HBV polymerase (POL) gene were ineffective. Oligonucleotides directed against the HBV encapsidation signal/structure (epsilon) comprised some of the most effective antiviral molecules against HBV. None of 5 oligonucleotides complementary (i.e., 'sense' orientation) to the antisense oligonucleotides targeting HBsAg, HBcAg, HBeAg, preS1 and POL had any measurable effect on HBV production. The relative effectiveness of oligonucleotides targeting the S and C genes on HBV replication was highly correlated with an effect on HBsAg or HBcAg levels, respectively. None of the antisense oligonucleotides examined affected either the levels or the sizes of HBV-specific RNA transcripts. Since antisense oligonucleotides can exert biologic effects on HBV in 2.2.15 cell cultures in a sequence-specific manner which are consistent with predicted modes of action, such molecules may have practical applications in the therapy of chronic HBV infection.

Use of a standardized cell culture assay to assess activities of nucleoside analogs against hepatitis B virus replication.

A cell culture system for the evaluation of compounds which inhibit HBV replication (Korba and Milman, Antiviral Res. 15:217, 1991) has been developed into a standardized assay. Toxicity of test compounds was assessed by the uptake of neutral red dye under culture and treatment conditions which were identical to those used for the antiviral assays. A total of 667 separate cultures of 2.2.15 cells were evaluated for this study. In 86 untreated cell cultures, representing 15 experiments over a 24-month period, the levels of extracellular HBV virion DNA and intracellular HBV DNA forms were found to vary by less than 2.5-fold overall. Virion DNA in serum and intracellular viral DNA replication intermediates [RI] are the two most reliable and commonly followed markers of hepadnavirus replication in patients and experimental animals. In these assays, levels of extracellular HBV virion DNA and intracellular HBV RI were well correlated in 2.2.15 cells. Less correlation was observed between the levels of HBV virion DNA and the 3.2-kb episomal HBV genomes present in the cells. A threshold level of 22-37 intracellular replicating HBV genomes appeared to be required before virions were detected in the culture medium. The activities of several 2'-substituted and 3'-substituted deoxynucleoside analogs against HBV replication were compared using this standardized assay. Dideoxycytosine [ddC] and dideoxyguanosine [ddG] were the most selective 2',3'-dideoxynucleosides against HBV in 2.2.15 cells. Substitution of fluorine at the 2' position abolished the antiviral activity of ddC, but enhanced the selective antiviral activities of dideoxythymidine and dideoxyuracil. Several 2'-fluorinated pyrimidine arabinosyl furanosides, reported to be potent (but toxic) inhibitors of hepadnaviruses in vivo, demonstrated relatively low selective antiviral activities in 2.2.15 cells. The current data base allows for validation of any given set of test evaluations through statistical analysis of both the positive and the negative treatment controls present in each experiment; thus, relevant comparisons of the selectivity of anti-HBV activities for different compounds examined in future experiments can be made.

Enhanced antiviral benefit of combination therapy with lamivudine and famciclovir against WHV replication in chronic WHV carrier woodchucks.

Cell culture studies in our laboratory and others have previously demonstrated synergistic antiviral activity for combinations of 3TC (lamivudine) and penciclovir against Hepatitis B Virus (HBV) replication and the Duck Hepatitis B Virus (DHBV). Based on these results, a study was designed to determine if an enhanced antiviral effect with combinations of 3TC and famciclovir (FCV, oral prodrug of penciclovir) could be demonstrated in vivo using the Woodchuck Hepatitis Virus (WHV)/woodchuck experimental model of chronic HBV infection. Both antiviral agents have been shown to be effective against WHV replication in WHV chronic carriers in previous studies by our laboratories. The antiviral effects of four different combinations of lamivudine and FCV were found to be greater than those observed for the corresponding monotherapies. All four combination treatments produced antiviral effects that were at least equal to that expected for additive activity based on estimations generated by Bliss Independence calculations. Two of the combination treatments produced antiviral effects that were significantly greater than that expected for additive effects, indicative of synergistic antiviral interactions. These studies demonstrate that combination therapy of chronic WHV infection has enhanced antiviral benefit over corresponding monotherapies and indicate that combination treatment of chronic HBV infection can be superior to therapies using a single antiviral agent.

Antiviral activity of phosphatidyl-dideoxycytidine in hepatitis B-infected cells and enhanced hepatic uptake in mice.

Dideoxycytidine (ddC) inhibits the replication of hepatitis B virus (HBV) but its clinical use is limited by peripheral neuropathy. We synthesized dioleoylphosphatidyl-ddC (DOP-ddC), a phospholipid prodrug of ddC which forms lipid bilayers and is readily incorporated into liposomes. The 90% effective dose (ED90) of DOP-ddC was 18 microM vs. 7 microM for ddC. However, in HBV-infected human hepatoma cells (2.2.15 cells), DOP-ddC was less toxic in vitro. When liposomal DOP-[5,6-3H]ddC was administered intraperitoneally to mice, drug levels in liver were 40 times greater than [5,6-3H]ddC when expressed as area under curve. Liposomal DOP-ddC also provided higher levels of drug in lymph nodes and spleen, important accessory sites of HBV replication. Plasma levels of drug remained above the ED90 six times longer with DOP-ddC than with ddC. DOP-ddC levels in sciatic nerve, the major site of toxicity, were not significantly different from levels observed with free ddC. The phospholipid prodrug approach is a general one which may readily be applied to other antiviral nucleosides for HBV.

The S-acyl-2-thioethyl pronucleotide approach applied to acyclovir: part I. Synthesis and in vitro anti-hepatitis B virus activity of bis(S-acyl-2-thioethyl)phosphotriester derivatives of acyclovir.

The synthesis and in vitro anti-hepatitis B virus (HBV) activity of two mononucleoside phosphotriester derivatives of acyclovir incorporating S-acyl-2-thioethyl (SATE) groups are reported. In contrast to the parent nucleoside, the described phosphotriesters emerged as potent and selective inhibitors of HBV replication in HepG2.2.15 cells. This result can be attributed to the unique cellular metabolism of the SATE pronucleotides giving rise to the delivery to acyclovir 5'-monophosphate inside the infected cells. Moreover, the in vitro anti-HBV activities of one of these bis(SATE)phosphotriesters and of (-)-beta-L-2',3'-dideoxy-3'-thiacytidine (lamivudine, 3TC) were compared alone and in combination. Analysis of the combination data indicates that 3TC and the studied SATE pronucleotide of acyclovir exhibited strong synergistic interactions. The present study provides an example where the use of a pronucleotide approach extends the antiviral spectrum of a nucleoside analogue. Given the potency of SATE pronucleotides of acyclovir against HBV in HepG2.2.15 cells, further studies including animal experiments seem warranted to evaluate the potential of these compounds as anti-HBV agents.

Robustaflavone, a potential non-nucleoside anti-hepatitis B agent.

Robustaflavone, a naturally occurring biflavanoid isolated from Rhus succedanea, was found to be a potent inhibitor of hepatitis B virus (HBV) replication in 2.2.15 cells, with an effective concentration (EC50) of 0.25 microM, and a selectivity index (SI, IC50/EC90) of 153. Robustaflavone hexaacetate inhibited HBV replication with an EC50 of 0.73 microM, but exhibited no cytotoxicity at concentrations up to 1000 microM. Combinations of robustaflavone with penciclovir and lamivudine displayed synergistic anti-HBV activity, having the most pronounced effects when the combination ratios were similar to the ratio of EC50 potencies. Thus, a 1:1 combination of robustaflavone and penciclovir exhibited an EC50 of 0.11 microM and an SI of 684, while a 10:1 combination of robustaflavone and lamivudine exhibited an EC50 of 0.054 microM and an SI of 894. Statistical analyses of the combination data using the Combostat program confirmed that robustaflavone exhibited synergism with both penciclovir and lamivudine.

Utilization of transgenic mice replicating high levels of hepatitis B virus for antiviral evaluation of lamivudine.

A recently developed transgenic mouse strain which expresses high levels of hepatitis B virus (HBV) was studied as a model for evaluation of potential chemotherapeutic agents. Lamivudine ([-]2'-deoxy-3'-thiacytidine), known to reduce hepatitis B viremia in human patients, and zidovudine (3'-azido-3'-deoxythymidine), previously shown to be ineffective for HBV infections in man, were used in parallel in this transgenic animal model. Orally administered lamivudine at dosages of 100, 50, and 25 mg/kg per day given once a day for 21 days significantly decreased serum and liver HBV DNA titers in a dose-responsive manner. Zidovudine (approximately 22 mg/kg per day) administered in the drinking water for 21 days was not effective in reducing these HBV parameters as compared to transgenic placebo-treated controls. The serum HBV DNA titers rebounded to high levels 1 week after cessation of lamivudine treatment. Male and female mice responded in a similar manner to these therapies. The results using this transgenic mouse model were similar to what would be predicted from treatment of HBV-infected human patients with lamivudine and zidovudine, and indicate these mice may be useful as a small animal chemotherapeutic model for study of potential HBV inhibitors.