Antiviral drug susceptibility of human herpesvirus 8.

We studied the susceptibility of human herpesvirus 8 (HHV-8) to a number of antiherpesvirus agents. The acyclic nucleoside phosphonate (ANP) analogs cidofovir and HPMPA [(S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)adenine] effected potent inhibition of HHV-8 DNA synthesis, with 50% effective concentrations (EC50) of 6.3 and 0.6 microM, respectively. Adefovir, an ANP with both antiretrovirus and antiherpesvirus activity, blocked HHV-8 DNA replication at a fourfold-lower concentration than did foscarnet (EC50 of 39 and 177 microM, respectively). The most potent inhibitory effect was obtained with the N-7-substituted nucleoside analog S2242 (EC50, 0.11 microM). The nucleoside analogs acyclovir, penciclovir, H2G ((R)-9-[4-hydroxy-2-(hydroxymethyl) butyl]guanine), and brivudine had weak to moderate effects (EC50 of > or =75, 43, 42, and 24 microM, respectively, and EC90 of > or =75 microM), whereas ganciclovir elicited pronounced anti-HHV-8 activity (EC50, 8.9 microM).     

Antiviral effects of lamivudine, emtricitabine, adefovir dipivoxil, and tenofovir disoproxil fumarate administered orally alone and in combination to woodchucks with chronic woodchuck hepatitis virus infection

Adefovir dipivoxil (ADV) and tenofovir disoproxil fumarate (TDF) are nucleotide analogs that inhibit the replication of wild-type hepatitis B virus (HBV) and lamivudine (3TC)-resistant virus in HBV-infected patients, including those who are coinfected with human immunodeficiency virus. The combination of ADV or TDF with other nucleoside analogs is a proposed strategy for managing antiviral drug resistance during the treatment of chronic HBV infection. The antiviral effect of oral ADV or TDF, alone or in combination with 3TC or emtricitabine (FTC), against chronic woodchuck hepatitis virus (WHV) infection was evaluated in a placebo-controlled study in the woodchuck, an established and predictive model for antiviral therapy. Once-daily treatment for 48 weeks with ADV plus 3TC or TDF plus FTC significantly reduced serum WHV viremia levels from the pretreatment level by 6.2 log₁₀ and 6.1 log₁₀ genome equivalents/ml serum, respectively, followed by TDF plus 3TC (5.6 log₁₀ genome equivalents/ml), ADV alone (4.8 log₁₀ genome equivalents/ml), ADV plus FTC (one survivor) (4.4 log₁₀ genome equivalents/ml), TDF alone (2.9 log₁₀ genome equivalents/ml), 3TC alone (2.7 log₁₀ genome equivalents/ml), and FTC alone (2.0 log₁₀ genome equivalents/ml). Individual woodchucks across all treatment groups also demonstrated pronounced declines in serum WHV surface antigen, characteristically accompanied by declines in hepatic WHV replication and the hepatic expression of WHV antigens. Most woodchucks had prompt recrudescence of WHV replication after drug withdrawal, but individual woodchucks across treatment groups had sustained effects. No signs of toxicity were observed for any of the drugs or drug combinations administered. In conclusion, the oral administration of 3TC, FTC, ADV, and TDF alone and in combination was safe and effective in the woodchuck model of HBV infection.     

Effect of oxetanocin G, a novel nucleoside analog, on DNA synthesis by hepatitis B virus virions.

The novel nucleoside oxetanocin G, 9-(2-deoxy-2-hydroxymethyl-beta-D-erythro-oxetanosyl)guanine (OXT-G), that is a derivative of oxetanocin A, was studied in relation to its action on the synthesis of hepatitis B virus (HBV) DNA and cellular DNA in an HBV-producing cell line, HB611 (T. Tsurimoto, A. Fujiyama, and K. Matsubara, Proc. Natl. Acad. Sci. USA 84:444-448, 1987). The median effective concentration of OXT-G against HBV replication was 1.5 microM, and the median cytotoxic concentration was more than 1,000 microM. At the same concentration, OXT-G did not inhibit cellular DNA synthesis or viral RNA synthesis. Chemically synthesized OXT-GTP inhibited the HBV endogenous DNA polymerase reaction and was incorporated into HBV DNA strands at a low efficiency compared with the incorporation of dGTP. A synthetic primer-template study revealed that OXT-GTP was incorporated into DNA strands at a low efficiency and that further extension of the DNA strand by using the 2' position of the incorporated OXT-G could take place.     

Identification of BMS-200475 as a potent and selective inhibitor of hepatitis B virus.

BMS-200475 is a novel carbocyclic 2'-deoxyguanosine analog found to possess potent and selective anti-hepatitis B virus (anti-HBV) activity. BMS-200475 is distinguished from guanosine by replacement of the natural furanose oxygen on the sugar moiety with an exo carbon-carbon double bond. In the HepG2 stably transfected cell line 2.2.15, BMS-200475 had a 50% effective concentration (EC50) of 3.75 nM against HBV, as determined by analysis of secreted HBV DNA. Structurally related compounds with adenine, iodouracil, or thymine base substitutions were significantly less potent or were inactive. Direct comparison of the antiviral activities of BMS-200475 with those of a variety of other nucleoside analogs, including lamivudine (EC50 = 116.26 nM), demonstrated the clearly superior in vitro potency of BMS-200475 in 2.2.15 cells. Intracellular HBV replicative intermediates were uniformly reduced when cells were treated with BMS-200475, but rebounded after treatment was terminated. The concentration of BMS-200475 causing 50% cytotoxicity in 2.2.15 cell cultures was 30 microM, approximately 8,000-fold greater than the concentration required to inhibit HBV replication in the same cell line. Treatment with BMS-200475 resulted in no apparent inhibitory effects on mitochondrial DNA content.     

In vitro susceptibility of lamivudine-resistant hepatitis B virus to adefovir and tenofovir

Emergence of lamivudine-resistant hepatitis B virus (HBV) is a major concern in human immunodeficiency virus (HIV) and HBV coinfected patients. Following selection of resistant mutants, hepatitis flare or rapid progression to cirrhosis may occur. Treatment of patients with new nucleotide analogues such as adefovir dipivoxil (ADV) or tenofovir disoproxil fumarate (TDF) has shown good efficacy in controlling wild-type or lamivudine-resistant HBV replication. The purpose of this study was to assess the in vitro efficacy of new nucleotide analogues on HBV strains isolated from lamivudine-treated patients. After purification of HBV DNA from patient sera, the whole HBV genome was PCR-amplified and cloned. Drug sensitivity was measured after transfection of the isolated full genomes into HepG2 cells and measurement of HBeAg, HBsAg and viral replication in the culture media under increasing drug concentrations. A wild-type strain isolated from an untreated patient served as control. In a clinical study of ADV (Gilead 460i study), seven of the 35 patients carried HBV strains with the triple lamivudine resistance-associated amino-acid changes rtV173L/L180M/M204V at baseline. Although all patients responded to ADV in this clinical study, the serum HBV reduction was lower in the seven patients with the triple mutation (median -3.3 log copies/ml) compared to the patients who had only the rtL180M/M204V mutations (median -4.1 log copies/ml) at week 48 (P=0.04, Mann-Whitney test). In our in vitro system, lamivudine IC50 on lamivudine-resistant HBV carrying amino-acid substitutions rtL180M and rtM204V within the polymerase encoding region increased by more than 16,000-fold (from 6 nM to over 100 microM) when compared to wild-type HBV. For ADV and TDF, comparison of wild-type and lamivudine-resistant HBV IC50 (rtL180M-M204V) showed, respectively, 2.85-fold (from 0.07 to 0.2 microM) and 3.3-fold (from 0.06 to 0.2 microM) increases, indicating a mild decrease of both drug activities, in vitro. At the ADV concentration of 0.1 microM, presence of the V173L mutation reduced the inhibition of HBsAg production from 50 to 30% (P<0.01) and the viral replication from 45 to 32% (P<0.01, Mann-Whitney). Conversely, tenofovir had similar potency on both HBV mutation profiles with 60% inhibition of HBsAg production and 45% inhibition of viral replication at 0.1 microM. Our study supports the high efficacy of ADV and TDF seen in patients after lamivudine breakthrough. The excellent activity of TDF on lamivudine-resistant virus independently of the resistance mutation profile offers an interesting treatment alternative to HIV-HBV coinfected patients.     

Antiviral activities and cellular toxicities of modified 2',3'-dideoxy-2',3'-didehydrocytidine analogues

The antiviral efficacies and cytotoxicities of 2',3'- and 4'-substituted 2',3'-didehydro-2',3'-dideoxycytidine analogs were evaluated. All compounds were tested (i) against a wild-type human immunodeficiency virus type 1 (HIV-1) isolate (strain xxBRU) and lamivudine-resistant HIV-1 isolates, (ii) for their abilities to inhibit hepatitis B virus (HBV) production in the inducible HepAD38 cell line, and (iii) for their abilities to inhibit bovine viral diarrhea virus (BVDV) production in acutely infected Madin-Darby bovine kidney cells. Some compounds demonstrated potent antiviral activities against the wild-type HIV-1 strain (range of 90% effective concentrations [EC(90)s], 0.14 to 5.2 micro M), but marked increases in EC(90)s were noted when the compounds were tested against the lamivudine-resistant HIV-1 strain (range of EC(90)s, 53 to >100 micro M). The beta-L-enantiomers of both classes of compounds were more potent than the corresponding beta-D-enantiomers. None of the compounds showed antiviral activity in the assay that determined their abilities to inhibit BVDV, while two compounds inhibited HBV production in HepAD38 cells (EC(90), 0.25 micro M). The compounds were essentially noncytotoxic in human peripheral blood mononuclear cells and HepG2 cells. No effect on mitochondrial DNA levels was observed after a 7-day incubation with the nucleoside analogs at 10 micro M. These studies demonstrate that (i) modification of the sugar ring of cytosine nucleoside analogs with a 4'-thia instead of an oxygen results in compounds with the ability to potently inhibit wild-type HIV-1 but with reduced potency against lamivudine-resistant virus and (ii) the antiviral activity of beta-D-2',3'-didehydro-2',3'-dideoxy-5-fluorocytidine against wild-type HIV-1 (EC(90), 0.08 micro M) and lamivudine-resistant HIV-1 (EC(90) = 0.15 micro M) is markedly reduced by introduction of a 3'-fluorine in the sugar (EC(90)s of compound 2a, 37.5 and 494 micro M, respectively).     

Acyclic guanosine analogs as inhibitors of human cytomegalovirus.

The acyclic guanosine analogs R- and S-enantiomers of 9-(3,4-dihydroxybutyl)guanine [(R)- and (S)-DHBG], 9-(4-hydroxybutyl)guanine (HBG), and 9-(2-hydroxyethoxymethyl)guanine (ACV) were examined for their effects on human cytomegalovirus (CMV) replication and on CMV DNA synthesis in cell culture as well as for their ability as triphosphates to interact with CMV DNA polymerase. Production of early CMV antigens was not affected. All analogs inhibited CMV DNA synthesis and late viral antigen synthesis. Primary CMV isolates were less susceptible to all tested analogs than was the laboratory strain CMV Ad.169. The triphosphate of ACV was the most potent inhibitor of CMV DNA polymerase, with an observed Ki of 0.0076 microM. The corresponding Ki values of the triphosphates of (R)-DHBG, (S)-DHBG, and HBG were 3.5, 13.0 and 0.23 microM, respectively. All triphosphates of the analogs given above inhibited CMV DNA polymerase in a competitive manner with respect to dGTP. The triphosphates of the analogs also inhibited reactions when the synthetic template poly(dC)oligo(dG)12-18 was used, whereas no inhibition was observed with poly(dA)oligo(dT)12-18. None of the triphosphate analogs supported DNA synthesis in the absence of dGTP, showing that no analog was an alternative substrate to dGTP.     

Inhibition of replication of hepatitis B virus by cytallene in vitro.

The acyclic cytosine nucleoside analog cytallene [1-(4'-hydroxy-1',2'-butadienyl)cytosine], which has both (+)- and (-)-enantiomers, was evaluated for its anti-hepatitis B virus (HBV) activity in 2.2.15 cells and was found to have potent activity against HBV DNA synthesis. The R-(-)-enantiomer was found to be the more active of the cytallene enantiomers, with a 50% inhibition concentration against HBV synthesis (HBIC50) of 0.08 microM. Its antiviral activity could be reversed by deoxycytidine (dC) and less efficiently by cytidine. Upon removal of the R-(-)-enantiomer from culture medium, the synthesis of HBV DNA could reinitiate, which suggested that the antiviral action is reversible. The R-(-)-enantiomer was also found to be more cytotoxic than the S-(+)-enantiomer. The degree of cytotoxicity varied among the cell lines, with a 50% inhibition of cell growth at greater than 10 microM. The R-(-)-enantiomer had no effect on HBV RNA synthesis and mitochondrial DNA synthesis at a concentration of 10 times or more than the HBIC50. The two enantiomers cannot be deaminated by dC deaminase, and they can be phosphorylated by cytoplasmic dC kinase. The R-(-)-enantiomer of cytallene is the first acyclic cytosine analog with potent inhibitory activity against HBV similar to those of other L-(-)-ddC analogs.     

Suboptimal response to adefovir dipivoxil therapy for chronic hepatitis B in nucleoside-naive patients is not due to pre-existing drug-resistant mutants

BACKGROUND: Adefovir dipivoxil (ADV) has demonstrated activity against wild-type and lamivudine-resistant hepatitis B virus (HBV). After 1 year of therapy, a median 3.5-4.0 log10 decrease in viral load is observed. Our aim was to characterize the different profiles of response to ADV in relation to the in vitro susceptibility of viral strains to ADV. METHODS: In an international Phase III randomized, placebo-controlled study of ADV in patients positive for hepatitis B virus e antigen (HBeAg), different profiles of virological response to ADV 10 mg/day were identified at week 48. The top 25% patients (quartile 1, Q1) showed > 4.91 log10 reduction in serum HBV DNA at week 48, in Q2 patients demonstrated a 3.52 to 4.90 log10 reduction of viral load, whereas in Q3 a 2.22 to 3.51 log10 reduction in viral load was observed. The bottom 25% of patients (Q4) showed < 2.22 log10 reduction in HBV DNA levels. The influence of baseline characteristics and drug compliance on response was investigated. The replication capacity and drug susceptibility of HBV genomes of selected clinical isolates that were considered representative of the treatment response quartiles were analysed using a phenotypic assay. RESULTS: The lowest quartile of response (Q4) appears to have worse compliance. Higher alanine aminotransferase levels at baseline are associated with improved response. Phenotypic analysis of viral strains in vitro in Huh7 and HepG2 cells showed that HBV genomes remained susceptible to ADV, regardless of treatment response observed in patients. CONCLUSION: Suboptimal response to ADV might result from a host pharmacological effect or from patient compliance issues rather than from a reduced susceptibility of HBV to ADV.     

Novel nucleoside analogue MCC-478 (LY582563) is effective against wild-type or lamivudine-resistant hepatitis B virus

The emergence of resistant hepatitis B virus (HBV) with the L528M mutation and/or the M552V and M552I mutations in the polymerase gene following long-term lamivudine treatment is becoming an important clinical problem. The aim of this study was to investigate the susceptibility of wild-type and lamivudine-resistant HBV to MCC-478 (LY582563), a novel nucleoside analogue derivative of phosphonomethoxyethyl purine. The susceptibility of wild-type HBV and lamivudine-resistant mutants (M552I, M552V, and L528M/M552V) to MCC-478 was examined by transient transfection of full-length HBV DNA into human hepatoma cells. HBV DNA replication was monitored by Southern blot hybridization, and the effective concentration required to reduce replication by 50% (EC(50)) was determined. The replicative intermediates of wild-type and lamivudine-resistant mutants were progressively diminished by treatment with increasing doses of MCC-478. The MCC-478 EC(50)s were 0.027 microM for wild-type HBV (about 20 times more efficient than lamivudine), 2.6 microM for M552I, 3.3 microM for M552V, and 2.0 microM for L528M/M552V. Wild-type HBV and lamivudine-resistant mutants are susceptible to MCC-478. MCC-478 appears to be a candidate for the treatment of HBV infection and exhibits potent activity against lamivudine-resistant HBV.     

Inhibition of severe acute respiratory syndrome-associated coronavirus (SARSCoV) by calpain inhibitors and beta-D-N4-hydroxycytidine

We evaluated two types of compounds for efficacy in inhibiting SARSCoV replication in vitro: calpain inhibitors (a class of cellular cysteine proteinases) and a number of nucleoside analogues. Cytopathic effect reduction assays visually determined with spectrophotometric verification by neutral red (NR) uptake assay were used to evaluate cytotoxicity and antiviral potency of the compounds. Significantly inhibitory compounds were then evaluated in virus yield reduction assays. Two calpain inhibitors, Val-Leu-CHO (calpain inhibitor VI) and Z-Val-Phe-Ala-CHO (calpain inhibitor III), were the most potent inhibitors of SARSCoV. By virus yield reduction assay, calpain inhibitor VI had a 90% effective concentration (EC90) of 3 microM and calpain inhibitor III had an EC90 of 15 microM. Beta-D-N4-hydroxycytidine was the most selective nucleoside analogue inhibitor with an EC90 of 6 microM by virus yield reduction assay. These compounds or analogues warrant further evaluation as potential therapies for treating SARS or could be used as lead compounds for discovery of more potent SARSCoV inhibitors.     

Phosphorylation of beta-D-ribosylbenzimidazoles is not required for activity against human cytomegalovirus.

We have previously reported that 2,5,6-trichloro-1-(beta-D-ribofuranosyl)benzimidazole (TCRB) and its 2-bromo analog (2-bromo-5,6-dichloro-1-(beta-D-ribofuranosy)benzimidazole [BDCRB]) are potent and selective inhibitors of human cytomegalovirus (HCMV) replication that block viral DNA maturation via HCMV gene products UL89 and UL56. To determine if phosphorylation is required for antiviral activity, the in vitro metabolism of BDCRB was examined and the antiviral activities of nonphosphorylatable 5'-deoxy analogs were determined. Reverse-phase high-performance liquid chromatography (HPLC) analysis of extracts from uninfected and HCMV-infected cells incubated with [(3)H]BDCRB revealed two major metabolites. Both were less polar than naturally occurring nucleoside monophosphates, but one peak coeluted with a BDCRB-5'-monophosphate (BDCRB-5'-MP) standard. Further analysis revealed, however, that neither metabolite partitioned with BDCRB-5'-MP on anion-exchange HPLC. Their retention patterns were not affected by incubation with alkaline phosphatase, thereby establishing that the compounds were not nucleoside 5'-monophosphates. Both compounds were detected in uninfected and HCMV-infected cells and in mouse live extracts, but neither has been identified. Like TCRB and BDCRB, the nonphosphorylatable 5'-deoxy analogs were potent and selective inhibitors of HCMV replication. The 5'-deoxy analogs maintained inhibition of HCMV replication upon removal of BDCRB, whereas an inhibitor of DNA synthesis did not. Similar to TCRB, its 5'-deoxy analog (5'-dTCRB) did not affect viral DNA synthesis, but 5'-dTCRB did inhibit viral DNA maturation to genome-length units. Additionally, virus isolates resistant to TCRB were also resistant to 5'-dTCRB and the 5'-deoxy analog of BDCRB. Taken together, these results confirm that TCRB, BDCRB, and their 5'-deoxy analogs have common mechanisms of action and establish that these benzimidazole ribonucleosides, unlike other antiviral nucleosides, do not require phosphorylation at the 5' position for antiviral activity.     

Penciclovir is a selective inhibitor of hepatitis B virus replication in cultured human hepatoblastoma cells.

Penciclovir [9-(4-hydroxy-3-hydroxymethylbut-1-yI)guanine], an effective antiherpesvirus agent, was found to be a potent and selective antiviral agent against intracellular hepatitis B virus (HBV) replication (drug concentration at which a 10-fold decrease in HBV DNA from the average level in an untreated culture was observed [EC90], 1.6 microM) and extracellular virion release (EC90, 0.7 microM) by cultured human hepatoblastoma (2.2.15) cells. Acyclovir and three other related 9-alkoxypurines with activity against either herpesviruses or human immunodeficiency virus were uniformly inactive against HBV. The activity of penciclovir is discussed in relation to recent findings related to its mode of action against HBV.     

Enzymology and pathogenicity in mice of a herpes simplex virus type 1 mutant resistant to 2''-fluoro-2''-deoxy-1-beta-D-arabinofuranosyl-5-iodocytosine.

The deoxypyrimidine nucleoside analog 2'-fluoro-2'-deoxy-1-beta-D-arabinofuranosyl-5-iodocytosine (FIAC) is a potent and selective inhibitor of herpes simplex virus type 1 in vitro (C. Lopez, K. A. Watanabe, and J. J. Fox, Antimicrob. Agents Chemother. 17:803-806, 1980). Isopycnographic analysis demonstrated that 1 microM FIAC inhibited herpes simplex virus DNA replication by more than 95% but inhibited cellular DNA replication by only 32%. Mutant herpes simplex virus type 1 selected resistant to FIAC displayed linked resistance to other nucleoside analogs, including arabinosylthymine and acyclovir. Lysates derived from Vero cells infected with FIAC-resistant virus showed markedly lower levels of thymidine kinase activity and were unable to phosphorylate selectively arabinosylthymine or FIAC, in contrast to lysates from cells infected with wild-type herpes simplex virus type 1. Finally, drug-resistant virus displayed a 6,000-fold decrease in pathogenicity when inoculated intraperitoneally into genetically susceptible A/J mice. These results indicate that resistance to deoxypyrimidine nucleoside analogs is due, at least in part, to alterations in viral thymidine kinase and is accompanied by decreased pathogenicity in vivo.     

New drugs for chronic hepatitis B: a review

Three nucleotide/nucleoside analogs are used for chronic hepatitis B (HBV): lamivudine, adefovir dipivoxil, and entecavir. Lamivudine and adefovir are advantageous for oral administration and safety but induce a sustained response after withdrawal of therapy in only a minority of patients. Thus, the treatment should be given in trials in a majority of patients for a long period of time. In addition, the long-term efficacy of lamivudine is limited by the frequent emergence of drug-resistant HBV mutants. Adefovir is associated with a low frequency of resistance, but its antiviral effect is not optimal. Entecavir, a cyclopentyl guanosine analog, is a potent inhibitor of HBV-DNA polymerase and it inhibits both priming and elongation steps of viral DNA replication. In phase II and III clinical trials, entecavir was found to be superior to lamivudine for all primary end points evaluated in both nucleoside-naive and lamivudine-resistant patients, and it was effective in both HBeAg-positive and HBeAg-negative nucleoside-naive patients. Only one trial has shown cases of viral resistance to this drug. The approved dosage in treatment-naive patients is 0.5 mg per day orally, whereas in patients who have failed lamivudine therapy or who are known to harbor lamivudine-resistant mutants, the approved dosage is 1.0 mg per day. Recent preliminary results show that clevudine, telbivudine, and emtricitabine may be potent analogs available for the treatment of HBV. Further studies are being conducted to assess the long-term efficacy and safety of these drugs.