Lamivudine-resistant Hepatitis B Virus Research Articles

Virological responses to tenofovir-alafenamide-containing antiretroviral therapy in people living with HIV co-infected with lamivudine-resistant or lamivudine-susceptible hepatitis B virus

BackgroundData on the effectiveness of tenofovir alafenamide (TAF) against lamivudine-resistant (LAM-R) hepatitis B virus (HBV) among patients co-infected with human immunodeficiency virus (HIV) and HBV are limited. MethodsBetween April and December 2018, HIV-positive patients co-infected with LAM-R or lamivudine-susceptible (LAM-S) HBV who switched from tenofovir-disoproxil-fumarate-containing antiretroviral therapy (ART) to TAF-containing ART were followed for 96 weeks. Plasma HBV and HIV loads, HBV serological markers, and liver function before and after the switch were analysed. ResultsIn total, 182 patients co-infected with HIV and HBV were included in this study: 45 with LAM-R HBV and 137 with LAM-S HBV. At baseline, 28.9% and 7.4% of patients in the LAM-R and LAM-S groups, respectively, tested positive for hepatitis B virus envelope antigen (HBeAg) (P<0.001), and the respective percentages of patients who had achieved plasma HBV DNA <20 IU/mL were 95.5% and 97.1%. At weeks 48 and 96, 100% and 94.9% of patients in the LAM-R group, respectively, and 97.1% and 95.6% of patients in the LAM-S group, respectively, maintained plasma HBV DNA <20 IU/mL. Lamivudine resistance of HBV and baseline hepatitis B virus surface antigen (HBsAg) level were associated with HBsAg decrement at week 96 at a degree of 0.25 log10 IU/mL [95% confidence interval (CI) 0.059–0.246] and 0.22 log10 IU/mL (per 1-log10IU/mL increase, 95% CI 0.018–0.101), respectively. At week 96, 2.2% (4/182) of patients had HBsAg loss; no patients in the LAM-R group and 25.0% (2/8) of patients in the LAM-S group had HBeAg seroconversion. ConclusionsSwitching to TAF-containing regimens maintained high rates of HBV viral suppression in patients co-infected with either LAM-R or LAM-S HBV. The decrease in HBsAg was minimal, and HBsAg seroconversion occurred infrequently.

Aurintricarboxylic Acid Suppresses Hepatitis B Virus Replication by Inhibition of RNase H Activity

BackgroundChronic hepatitis B virus (HBV) infection is a global health threat for which there is an urgent need to develop novel therapeutics.MethodsAurintricarboxylic acid (ATA) has been demonstrated with broad-spectrum antimicrobial and antiviral activities. In this study, we implemented ATA treatment in HBV-infected and HBV-transfected hepatocytes to uncover whether ATA inhibits HBV replication and its underlying mechanism.ResultsHBV DNA levels were significantly reduced, while viral proteins or transcripts were not altered. In contrast, ATA treatment did not further deteriorate HBV DNAs, viral proteins, and transcripts in hepatocytes transfected by an HBV RNase H dead mutant. Moreover, ATA showed an inhibitory effect on DNA synthesis in hepatocytes transfected with lamivudine-resistant HBV mutants.ConclusionsATA is a potent inhibitor of HBV replication by disrupting the RNase H activity of the viral polymerase.

Inflammation Pharmacological Reaction and YMDD Mutational Patterns in Lamivudine Therapeutics Hepatitis B Virus.

Background/Aims: Emergence of tyrosine-methionine-aspartate-aspartate (YMDD) motif in reverse transcriptase is a serious problem in chronic hepatitis B(CHB) patients after Lamivudine (LAM) therapy. However, the relationship between inflammation pharmacological reaction and YMDD mutational patterns of CHB has not been well-characterized. The aim of this study was to investigate the inflammation pharmacological reaction and different YMDD mutants patterns of CHB patients. Methods: We investigated the inflammation pharmacological reaction and YMDD mutational patterns through biochemical, serological and virological detection among 83 CHB patients, including 25 YMDD mutants, 25 under detection, and 33 control patients without YMDD mutants. Results: Prevalence of YMDD mutation patterns is different. Among 25 YMDD mutants patients, YIDD was the dominant mutation (72%), followed YVDD (16%) and the hybrid YIDD + YVDD (12%). The time course during the YMDD mutations was also different. 52.4% patients developed the mutation less than 12 months after the LAM therapy. Serum hepatitis B virus (HBV) DNA level in patients with YMDD mutants were significantly higher than that in control and negative groups. Serum HbsAg and HbeAg in patients with YMDD mutants were also higher than those in control and negative groups, despite no significant difference was found forserum HbeAb. ALT and AST levels were also significantly higher in mutants group. Conclusions: Illuminating inflammation pharmacological reaction and YMDD mutational patterns of CHB during pathological process may have implications for future therapy in YMDD mutation patients. This may have impact on the choice of treatment strategies for lamivudine-resistant HBV.

Nanopore sequencing from extraction-free direct PCR of dried serum spots for portable hepatitis B virus drug-resistance typing.

Effective drug regimens for the treatment of hepatitis B virus (HBV) infections are essential to achieve the World Health Organisation commitment to eliminate viral hepatitis by 2030. Lamivudine (3TC) is widely used in countries with high levels of chronic HBV, however resistance has been shown to occur in up to 50 % of individuals receiving continuous monotherapy for 4 years. Telbivudine (LdT) is now more commonly used in place of lamivudine but is ineffective against 3TC-resistant HBV. Genotyping and identification of resistanceassociated substitutions (RAS) is not practical in many locations. A novel assay was designed to enable HBV genotyping and characterisation of resistance mutations directly from serum samples stored on filter paper, using Sanger and MinION sequencing. The assay was applied to a cohort of 30 samples stored on filter paper for several years with HBV viral loads ranging from 8.2 × 108 to 635 IU/mL. A set of 6 high-titre samples were used in a proof-of-principle study using the MinION sequencer. The assay allowed determination of HBV genotype and elucidation of RAS down to 600 IU/mL using a 550bp amplicon. Sequencing of a 1.2 kb amplicon using a MinION sequencer gave results consistent with Sanger sequencing and allowed the identification of minor populations of variants. We present two approaches for reliable HBV sequencing and RAS identification using methods suitable for resource-limited environments. This is the first demonstration of extraction-free DNA sequencing direct from DSS using MinION and these workflows are adaptable to the investigation of other DNA viruses.

361. Residual Lamivudine-Resistant Hepatitis B Virus Detected on Next-Generation Sequencing of Treatment-Experienced HIV Patients Failing Antiretrovirals

BackgroundHepatitis B is highly prevalent in the Philippines, with 17% of the population infected. With the fastest-growing HIV epidemic in the Asia-Pacific, 12% of HIV patients are HBsAg reactive. With the use of lamivudine and tenofovir-based antiretrovirals (ARVs), hepatitis B virus (HBV) treatment in co-infected HIV patients is not usually an issue. However, there is a potential to develop HBV resistance when patients are switched off tenofovir when antiretroviral resistance develops. With high rates of acquired K65R tenofovir resistance, the potential for inadvertently causing re-emergence of lamivudine-resistant HBV is present. We report two HIV patients with residual whole-genome HBV with lamivudine and telbivudine resistance mutations.MethodsAs part of a surveillance study on acquired drug resistance in the Philippines, samples with an HIV viral load >1,000 copies underwent Sanger sequencing of RT and PR for genotyping and HIV drug-resistance testing. Near-whole-genome next-generation sequencing (NGS) for HIV using Illumina HiSeq was also performed on these samples.ResultsTwo patients had coincidental whole-genome amplification of HBV on NGS (Table 1). HBV serology for both showed reactive anti-HBsAg and non-reactive HBsAg and Anti-HBc. The two HBV samples were genotype A and were resistant to lamivudine and telbivudine, with intermediate resistance to entecavir.ConclusionResidual HBV may be present in patients on ARVs. Antibody responses for HBV serology may not be very reliable in highly immunosuppressed patients. The potential of lamivudine-resistant HBV to emerge when HIV patients are shifted off tenofovir due to resistance in patients should be considered when deciding on second-line ARVs. Disclosures All authors: No reported disclosures.

A peptide-based inhibitor of gp96 suppresses HBsAg expression and HBV replication by upregulation of p53.

In hepatitis B virus (HBV) infection, the virus produces redundant hepatitis B surface antigen (HBsAg) that plays a key role in driving T-cell tolerance and viral persistence. However, currently available anti-HBV agents have no direct effect on HBsAg transcription and protein expression. In this study, we designed a heat shock protein gp96 inhibitor p37 with the cell penetrating peptide PTD (protein transduction domain of trans-activator of transcription), which mediated p37 internalization into hepatocytes. PTD-p37 effectively suppressed HBsAg expression and viral replication both in vitro and in vivo. We further provide evidence that PTD-p37 suppressed HBV enhancer/promoter activity via p53 upregulation. Moreover, PTD-p37 had antiviral activity against a lamivudine-resistant HBV strain. Considering that suppression of HBsAg expression is a major goal for treatment of HBV infection, our results provide a basis for developing a new therapeutic approaches targeting host factors against viral expression.

Assessment of quinazolinone derivatives as novel non-nucleoside hepatitis B virus inhibitors

Hepatitis B virus (HBV) infection is a worldwide public health issue. Search for novel non-nucleoside anti-HBV agents is of great importance. In the present study, a series of quinazolinones derivatives (4a-t and 5a-f) were synthesized and evaluated as novel anti-HBV agents. Among them, compounds 5e and 5f could significantly inhibit HBV DNA replication with IC50 values of 1.54 μM and 0.71 μM, respectively. Interestingly, the selective index values of 5f was higher than that of lead compound K284–1405, suggesting 5f possessed relatively safety profile than K284–1405. Notably, 5e and 5f exhibited remarkably anti-HBV activities against lamivudine and entecavir resistant HBV strain with IC50 values of 1.90 and 0.84 μM, confirming their effectiveness against resistant HBV strain. In addition, molecular docking studies indicated that compounds 5e and 5f could well fit into the dimer-dimer interface of HBV core protein dominated by hydrophobic interactions. Notably, their binding modes were different from the lead compound K284–1405, which may be attributed to the additional substituent groups in the quinazolinone scaffold. Taken together, 5e and 5f possessed novel chemical structure and potent anti-HBV activity against both drug sensitive and resistant HBV strains, thus warranting further research as potential non-nucleoside anti-HBV candidates.

Design, synthesis, and biological evaluation of novel 2′-deoxy-2′-fluoro-2′-C-methyl 8-azanebularine derivatives as potent anti-HBV agents

Hepatitis B virus (HBV) is a global health problem requiring more efficient and better tolerated anti-HBV agent. In this paper, a series of novel 2′-deoxy-2′-fluoro-2′-C-methyl-β-d-arabinofuranosyl 8-azanebularine analogues (1 and 2a) and N4-substituted 8-azaadenosine derivatives (2b-g) were designed, synthesized and screened for in vitro anti-HBV activity. Two concise and practical synthetic routes were developed toward the structural motif construction of 2′-deoxy-2′-fluoro-2′-C-methyl-β-d-arabinofuranosyl 8-azainosine from the ribonolactone 3 under mild conditions. The in vitro anti-HBV screening results showed that these 8-azanebularine analogues had a significant inhibitory effect on the expression of HBV antigens and HBV DNA at a concentration of 20 μM. Among them, halogen-substituted 8-azaadenosine derivative 2g displayed activities comparable to that of 3TC. In particular, 2g retained excellent activity against lamivudine-resistant HBV mutants.

Naturally occurring core protein mutations compensate for the reduced replication fitness of a lamivudine-resistant HBV isolate

Hepatitis B virus (HBV) replicates its DNA genome through reverse transcription of an RNA intermediate. The lack of proofreading capacity of the viral DNA polymerase results in a high mutation rate of HBV genome. Under the selective pressure created by the nucleos(t)ide analogue (NA) antiviral drugs, viruses with resistance mutations are selected. However, the replication fitness of NA-resistant mutants is markedly reduced compared to wild-type. Compensatory mutations in HBV polymerase, which restore the viral replication capacity, have been reported to arise under continuous treatment with lamivudine (LMV). We have previously identified a highly replicative LMV-resistant HBV isolate from a chronic hepatitis B patient experiencing acute disease exacerbation. Besides the common YMDD drug-resistant mutations, this isolate possesses multiple additional mutations in polymerase and core regions. The transcomplementation assay demonstrated that the enhanced viral replication is due to the mutations of core protein. Further mutagenesis study revealed that the P5T mutation of core protein plays an important role in the enhanced viral replication through increasing the levels of capsid formation and pregenomic RNA encapsidation. However, the LMV-resistant virus harboring compensatory core mutations remains sensitive to capsid assembly modulators (CpAMs). Taken together, our study suggests that the enhanced HBV nucleocapsid formation resulting from core mutations represents an important viral strategy to surmount the antiviral drug pressure and contribute to viral pathogenesis, and CpAMs hold promise for developing the combinational antiviral therapy for hepatitis B.

Novel fluoronucleoside analog NCC inhibits lamivudine-resistant hepatitis B virus in a hepatocyte model

Antiviral drug resistance is the most important factor contributing to treatment failure using nucleos(t)ide analogs such as lamivudine for chronic infection with hepatitis B virus (HBV). Development of a system supporting efficient replication of clinically resistant HBV strains is imperative, and new antiviral drugs are needed urgently to prevent selection of drug-resistant HBV mutants. A novel fluorinated cytidine analog, NCC (N-cyclopropyl-4′-azido-2′-deoxy-2′-fluoro-β-d-cytidine), was recently shown to strongly inhibit human HBV in vitro and in vivo. This study was designed to evaluate the antiviral activity of NCC against lamivudine-resistant HBV. We generated a stable cell line encoding the major pattern of lamivudine-resistant mutations rtL180M/M204V and designated it “HepG2.RL1”. Immuno-transmission electron microscopic examination and enzyme-linked immunosorbent assay were used to detect secretion of HBV-specific particles and antigens. Quantification of extracellular DNA and intracellular DNA of HepG2.RL1 cells by quantitative real-time polymerase chain reaction revealed >625-fold and >5556-fold increases in the 50% inhibitory concentration of lamivudine, respectively, compared with that for the wild-type virus. The results showed that NCC inhibited DNA replication and HBeAg production in wild-type or lamivudine-resistant HBV in a dose-dependent manner. In conclusion, screening for antiviral compounds active against lamivudine-resistant HBV can be carried out with relative ease using hepG2.RL1 cells. NCC is a potential antiviral agent against wild-type HBV and clinical lamivudine-resistant HBV and deserves evaluation for the treatment of HBV infection.

Characteristics of HBV infection in 705 HIV-infected patients under lamivudine-based antiretroviral treatment from three regions in China.

PurposeThis study aimed to investigate the HIV and hepatitis B virus (HBV) co-infection in three HIV high endemic areas with different modes of HIV transmission and explore the HBV nucleos(t)ide analogue resistance (NUCr) substitutions in this cohort receiving antiretroviral therapy (ART).Patients and methodsThe enrolled 705 HIV-infected patients were from three different regions in China and received lamivudine-based ART for at least 1 year. After screening for hepatitis B surface antigen (HBsAg), the hepatitis B e antigen (HBeAg), and antibody against hepatitis B core antigen (anti-HBc and anti-HBc IgM), HBV DNA in plasma of patients positive for HBsAg was tested. The reverse transcriptase (RT) sequences of HBV were analyzed by direct sequencing.ResultsThe overall HBsAg-positive rate was 7.1% (50/705) (Guangxi [25/170, 14.7%], Xinjiang [13/257, 5.1%], and Henan [12/278, 4.3%]). The age, transmission route, and ethnic status were found to be associated with HIV/HBV co-infection. We obtained 23 HBV RT sequences belonging to genotypes B (9/23, 39.1%), C (13/23, 56.5%), and D (1/23, 4.4%). About 65.2% (15/23) of RT sequences harbored NUCr substitutions, all of which had combination substitution patterns. Patients with HBV NUCr had significantly higher HBV DNA level and ratio of HBeAg-positive than those without NUCr. None of the patients was found to have both lamivudine-resistant HBV and HIV.ConclusionOur results suggested that HBsAg-positive rate in the studied patients was similar to that of the general population in each of the studied regions, where the age, transmission route, and ethnic status might also play roles in HIV/HBV co-infection. The HBV combination NUCr substitutions were common in co-infected patients under ART. Monitoring of HBV infection and NUCr substitutions in HIV-infected patients would help in providing better clinical decisions and management, thus lowering patients’ risks to develop end-stage liver diseases.

Comparison of replication competence of wild-type and lamivudine-resistant hepatitis B virus isolates from a chronic hepatitis B patient

In lamivudine-refractory chronic hepatitis B (CHB) patients, discontinuation of lamivudine therapy may lead to loss of lamivudine-resistant hepatitis B virus (HBV) and reappearance of wide-type HBV as dominant strains, yet the underlying mechanism remains unclear. In this study, we cloned wide-type and lamivudine-resistant HBV genomes from the sera of a CHB patient who stopped lamivudine therapy after occurrence of resistant virus and determined the biologic properties of the two isolates in hepatoma cell lines. Sequencing reverse transcriptase region of HBV revealed that the patient developed lamivudine-resistant mutations (rtV173 L, rtL180 M, and rtM204 V) 36 months after the start of lamivudine therapy, and lamivudine-resistant mutants reversed to wild-type after the treatment was stopped for 8 months. Our data showed that the wild-type and mutant isolates had similar transcriptional and translational activity. However, comparison of intracellular and released HBV DNA levels showed that replication efficiency of the mutant virus was approximately 50% of wild-type HBV, while the infectivity of released virus was not affected by the lamivudine-resistant mutations. In conclusion, the reversion of lamivudine-resistant mutants to wild-type HBV after discontinuation of lamivudine in hepatitis B patients may be attributed to better replication fitness of wild-type HBV.

Design, synthesis, and biological evaluation of new 1,2,3-triazolo-2′-deoxy-2′-fluoro- 4′-azido nucleoside derivatives as potent anti-HBV agents

Novel drugs are urgently needed to combat hepatitis B virus (HBV) infection due to drug-resistant virus. In this paper, a series of novel 4-monosubstituted 2′-deoxy-2′-β-fluoro-4′-azido-β-d-arabinofuranosyl 1,2,3-triazole nucleoside analogues (1a-g) were designed, synthesized and screened for in vitro anti-HBV activity. At 5.0 μM in the cellular model, all the synthetic compounds display activities comparable to that of the positive control, lamivudine at 20 μM. Of the compounds tested, the amide-substituted analogue (1a) shows the most promising anti-HBV activity and low cytotoxicity in the cell model. In particular, it retains excellent activity against lamivudine-resistant HBV mutants. In duck HBV (DHBV)-infected duck models, both the serum and liver DHBV DNA levels (67.4% and 53.3%, respectively) were reduced markedly by the treatment with 1a. Analysis of the structure of HBV polymer/1a-triphosphate (1a-TP) complex shows that 1a-TP is stabilized by specific van der Waals interactions with the enzyme residues arising from 4-amino-1,2,3-triazole and the 4′-azido group.

Virological Response to Tenofovir Disoproxil Fumarate in HIV-Positive Patients with Lamivudine-Resistant Hepatitis B Virus Coinfection in an Area Hyperendemic for Hepatitis B Virus Infection

BackgroundSequential addition of tenofovir disoproxil fumarate (TDF) is often needed for patients coinfected with HIV and hepatitis B virus (HBV) who develop HBV resistance to lamivudine after combination antiretroviral therapy (cART) containing only lamivudine for HBV. We aimed to assess the virological response of HBV to add-on TDF in patients coinfected with lamivudine-resistant HBV.MethodsBetween November 2010 and December 2014, 33 HIV/HBV-coinfected patients with lamivudine-resistant HBV and 56 with lamivudine-susceptible HBV were prospectively included. TDF plus lamivudine was used to substitute zidovudine or abacavir plus lamivudine contained in cART in patients with lamivudine-resistant HBV infection, while patients with lamivudine-susceptible HBV infection received TDF plus lamivudine as backbone of cART. Serial determinations of plasma HBV DNA load, HBV serologic markers, and liver and renal functions were performed after initiation of TDF-containing cART.ResultsOf 89 patients included, 38.6% tested positive for HBV envelope antigen (HBeAg) at baseline. The plasma HBV DNA level at enrollment of lamivudine-resistant and lamivudine-susceptible group were 6.1 ± 2.2 log10 and 6.0 ± 2.2 log10 copies/mL, respectively (p = 0.895). The cumulative percentage of HBV viral suppression in lamivudine-resistant and lamivudine-susceptible group was 81.8% and 91.1% at 48 weeks, respectively (p = 0.317), which increased to 86.7% and 96.2% at 96 weeks, respectively (p = 0.185). At 48 weeks, 11 patients testing HBeAg-positive at baseline failed to achieve viral suppression. In multivariate analysis, the only factor associated with failure to achieve viral suppression at 48 weeks was higher HBV DNA load at baseline (odds ratio, per 1-log10 copies/mL increase, 1.861; 95% CI, 1.204–2.878). At 48 weeks, HBeAg seroconversion was observed in 5 patients (1 in the lamivudine-resistant group and 4 in the lamivudine-susceptible group; p = 0.166). During the study period, HBsAg levels decreased over time, regardless of lamivudine resistance. Loss of HBsAg was observed in 3 (3.4%) patients in the lamivudine-susceptible group.ConclusionsAdd-on TDF-containing cART in patients coinfected with lamivudine-resistant HBV achieved a similar rate of HBV viral suppression compared to TDF-containing cART as initial regimen in patients coinfected with lamivudine-susceptible HBV. A higher baseline HBV DNA load and HBeAg positivity were associated with failure to achieve HBV viral suppression.

Tenofovir disoproxil fumarate (TDF) vs. emtricitabine (FTC)/TDF in lamivudine resistant hepatitis B: A 5-year randomised study

Long-term treatment with tenofovir disoproxil fumarate (TDF) alone, or in combination with emtricitabine (FTC) is associated with sustained viral suppression in patients with lamivudine resistant (LAM-R) chronic hepatitis B (CHB). LAM-R CHB patients were randomised 1:1 to receive TDF 300mg or FTC 200mg and TDF 300mg once daily in a prospective, double blind, study. The proportion of patients with plasma hepatitis B virus (HBV) DNA<69IU/ml (<400copies/ml) at week 96 (primary efficacy endpoint) was reported previously. Here we present week 240 follow-up data. Overall, 280 patients were randomised to receive TDF (n=141) or FTC/TDF (n=139), and 85.4% completed 240weeks of treatment. At week 240, 83.0% of patients in the TDF arm, and 82.7% of patients in the FTC/TDF treatment arm had HBV DNA<69IU/ml (p=0.96). Rates of normal alanine aminotransferase (ALT) and normalised ALT were similar between groups (p=0.41 and p=0.97 respectively). Hepatitis B e antigen loss and seroconversion at week 240 were similar between groups, (p=0.41 and p=0.67 respectively). Overall, six patients achieved hepatitis B surface antigen (HBsAg) loss and one patient (FTC/TDF arm) had HBsAg seroconversion by week 240. No TDF resistance was observed up to week 240. Treatment was generally well tolerated, and renal events were mild and infrequent (∼8.6%). The mean change in bone mineral density at week 240 was -0.98% and -2.54% at the spine and hip, respectively. TDF monotherapy was effective and well tolerated in LAM-R CHB patients for up to 240weeks. The goal of oral antiviral treatment for chronic hepatitis B (CHB) is to achieve and maintain undetectable HBV DNA levels. Treatment options with enhanced potency, and low risk of resistance development for patients infected with lamivudine resistant (LAM-R) HBV are required. Tenofovir disoproxil fumarate (TDF) monotherapy was effective and well tolerated without TDF resistance development in CHB patients with LAM-R, for up to 240weeks. Clinical trial number: NCT00737568.

Inhibition of hepatitis B virus replication by targeting ribonucleotide reductase M2 protein

Chronic hepatitis B virus (HBV) infection is a key factor for hepatocellular carcinoma worldwide. Ribonucleotide reductase (RR) regulates the deoxyribonucleoside triphosphates biosynthesis and serves as a target for anti-cancer therapy. Here, we demonstrate that RR is essential for HBV replication and the viral covalently-closed-circular DNA (cccDNA) synthesis in host liver cells. By performing computer-assisted virtual screening against the crystal structure of RR small subunit M2 (RRM2), osalmid, was identified as a potential RRM2-targeting compound. Osalmid was shown to be 10-fold more active in inhibiting RR activity than hydroxyurea, and significantly inhibited HBV DNA and cccDNA synthesis in HepG2.2.15 cells. In contrast, hydroxyurea and the RR large subunit (RRM1)-inhibitory drug gemcitabine showed little selective activity against HBV replication. In addition, osalmid also was shown to possess potent activity against a 3TC-resistant HBV strain, suggesting utility in treating drug-resistant HBV infections. Interestingly, osalmid showed synergistic effects with lamivudine (3TC) in vitro and in vivo without significant toxicity, and was shown to inhibit RR activity in vivo, thus verifying its in vivo function. Furthermore, 4-cyclopropyl-2-fluoro-N-(4-hydroxyphenyl) benzamide (YZ51), a novel derivative of osalmid, showed higher efficacy than osalmid with more potent RR inhibitory activity. These results suggest that RRM2 might be targeted for HBV inhibition, and the RRM2-targeting compound osalmid and its derivative YZ51 could be a novel class of anti-HBV candidates with potential use for hepatitis B and HBV-related HCC treatment.

Emergence of lamivudine-resistant hepatitis B virus during combination antiretroviral therapy that includes lamivudine for patients co-infected with HIV and hepatitis B virus in China: a 2-year pilot cohort study

BackgroundIn China, more than 10% of individuals infected with HIV are carriers of hepatitis B virus (HBV). Patients infected with HIV-1 are usually given combination antiretroviral therapy that includes lamivudine (ART-3TC) as a reverse-transcriptase inhibitor. Previous studies from some developed counties showed that, in ART-3TC, 3TC-resistant HBV progressively emerges at a rate of 15–20% per year in patients co-infected with HIV-1 and HBV. We investigated the occurrence of 3TC-resistant HBV during ART-3TC for double infections and tested the use of tenofovir disoproxil fumarate (TDF) as an additional reverse-transcriptase inhibitor (ART-3TC/TDF) in a 2-year cohort study in China. Methods172 plasma samples from 43 Chinese patients co-infected with HIV-1 and HBV (from 134 cases positive for HBsAg) were examined for the prevalence of 3TC-resistant HBV by direct sequencing of PCR products covering the HBV reverse transcriptase gene. The patients were randomised (7:1), using a randomised number table, to receive either ART-3TC or ART-3TC/TDF (because ART-3TC/TDF was not a standard first-line treatment in China at that time). HIV-1 RNA and HBV DNA loads were detected at baseline, 12 weeks, 48 weeks, and 96 weeks from plasma samples. All samples were tested for HIV and HBV drug resistance against 3TC. The efficacy of viral suppression and incidence of drug-resistance mutation were compared between the two groups. This study was approved by the ethics committee at Peking Union Medical College Hospital, Beijing, China, and registered at ClinicalTrials.gov (identifiers NCT00618176 and NCT00872417). Patients were enrolled in Chinese cohort studies (National Key Technologies R&D Program for the tenth and 11th Five-year Plans). FindingsMedian HIV-1 RNA loads of the ART-3TC group and the ART-3TC/TDF group significantly decreased from 26 205 copies/mL (IQR 917; p<0·0001) to fewer than 20 copies/mL (<20; p=0·0278) and from 58 972 copies/mL (3395; p<0·0001) to fewer than 20 copies/mL (<20; p=0·0207), respectively, after 96 weeks of treatment. Median HBV DNA loads also significantly decreased from 3·4 × 106 IU/mL (IQR <20; p<0·0001) to less than 20 IU/mL (<20; p<0·0001) in the ART-3TC group and from 2·7×105 IU/mL (399; p<0·;0001) to less than 20 IU/mL (<20; p=0·0152) in the ART-3TC/TDF group after 96 weeks of treatment. Although median HIV-1 viral load did not differ significantly (p>0·05) between the two groups after 96 weeks, the rate of HIV-1 RNA decline in the ART-3TC/TDF group was slightly lower than that in the ART-3TC group (RNA load range <20–905 copies/mL vs <20–4932 copies/mL at 12 weeks, <20–157 copies/mL vs <20–398 copies/mL at 48 weeks). ART-3TC/TDF had a higher efficacy than ART-3TC in controlling amounts of HBV. At 96 weeks, the median HBV DNA load was less than 20 IU/mL (IQR <20–1·2 × 108) in the ART-3TC group, suggesting that some patients had rebound or uncontrolled HBV DNA loads, but no such case was reported in the ART-3TC/TDF group, the median HBV DNA load of which was <20 IU/mL (<20; p=0<0119). Ten (23%) of 43 patients, all from the ART-3TC group, had HBV breakthrough after 96 weeks of treatment, and eight of these patients were resistant to ART-3TC (60% tr204 mutation and 80% rt180 mutation; appendix). InterpretationAlthough both regimens were able to control replication of HIV and HBV during the 2-year treatment, our findings showed that, in China, ART-3TC is likely to lead to the emergence of 3TC-resistant HBV, lending support to the benefit of the inclusion of TDF for patients co-infected with HIV-1 and HBV.

Epidemiology of HBV infection in a cohort of Ugandan HIV-infected patients and rate and pattern of lamivudine-resistant HBV infection in patients receiving antiretroviral therapy.

Many HIV-infected patients in sub-Saharan Africa are not routinely screened for hepatitis B virus (HBV) infection and are on antiretroviral therapy (ART) regimens containing only lamivudine as anti-HBV active drug. In 2009-2011, we screened for hepatitis B surface antigen (HBsAg) in 2820 HIV-infected adults patients at the Mbarara Hospital Uganda and investigated risk factors for HBV infection. Using samples of dried plasma or blood spots, we tested for HBV viral load and HBV drug resistance mutations in all HBsAg-positive patients on ART for ≥ 12 months. In this study, 109 patients tested HBsAg positive (3.9%; 109/2820). HBsAg-positive patients were more likely to have had >4 lifetime sexual partners (p<0.01). Of the 55 HBsAg-positive patients on ART for ≥ 12 months, 53 were only on lamivudine as anti-HBV active drug and two were on tenofovir and lamivudine. HBV-DNA was detected in 30 patients (54.5%; 30/55), all on lamivudine-monotherapy. Of the 23 patients in whom HBV-DNA sequencing was successful, 17 had lamivudine-resistant HBV strains harbouring rtM204V/I mutations accompanied by secondary/compensatory mutations. Our study suggests that sexual transmission may represent a major mode of spread of HBV in southwest Uganda and confirms the importance of screening for HBV and of using ART regimens containing tenofovir in HIV/HBV co-infected patients.

The case of chronic hepatitis B treatment with tenofovir: an update for nephrologists.

Tenofovir is a nucleotide acting both as an inhibitor of human immunodeficiency (HIV) reverse transcriptase and as a competitor for hepatitis B virus (HBV) RNA-directed DNA polymerase. Approved worldwide in 2001, tenofovir is used as a component of highly active antiretroviral therapy (HAART) in patients with HIV infection. Since 2008, it has also been indicated for treatment of chronic HBV infection or HIV/HBV co-infection. The aim of the treatment consists in suppressing viral replication, thus reducing hepatic complications and improving patient survival. Furthermore, tenofovir could represent an effective therapeutic option in lamivudine-resistant HBV patients. Tenofovir is eliminated unchanged through urine via glomerular filtration (80%) and proximal tubular secretion (20%). Thus, alterations in renal clearance may interfere with tenofovir pharmacokinetics and systemic drug concentrations, modifying the therapeutic response. Hence, a renal overload of tenofovir in patients with a pre-existing kidney impairment could result in a worsening of renal function. Following a brief introduction on HBV infection and its therapeutic options, we review the latest evidence, to our knowledge, on renal toxicity of tenofovir in HBV patients and on drug management.

Quantification of 2′-deoxy-2′-β-fluoro-4′-azidocytidine in rat and dog plasma using liquid chromatography-quadrupole time-of-flight and liquid chromatography-triple quadrupole mass spectrometry: Application to bioavailability and pharmacokinetic studies

2′-Deoxy-2′-β-fluoro-4′-azidocytidine (FNC) is a novel pyrimidine analog that inhibits not only the replication of the hepatitis B virus (HBV), hepatitis C virus (HCV) and HIV but also the replication of lamivudine-resistant HBV, 4′-azidocytidine or 2′-β-methylcytidine-resistant HCV, and nucleoside reverse-transcriptase inhibitor-resistant HIV variants. The present study was undertaken to evaluate the absolute oral bioavailability of FNC in rats and the pharmacokinetic properties of FNC after intragastric administration of single and multiple doses in rats and dogs. A sensitive high-performance liquid chromatography tandem quadrupole time-of-flight mass spectrometry (HPLC/Q-TOF MS) method and a reliable high-performance liquid chromatography tandem triple quadrupole mass spectrometry (HPLC/QqQ MS/MS) method were established for the determination of FNC in the rat and dog plasmas, respectively. The sample preparation involved a protein-precipitation method with methanol after the addition of lamivudine as an internal standard. FNC was analyzed by LC using a YMC-Pack Pro C18 column (150mm×4.6mm, 3μm) with methanol (containing 0.3% formic acid): 10mM ammonium acetate (containing 0.3% formic acid, pH 2.8) (35:65, v/v) as the mobile phase. Both mass spectrometers were equipped with an electrospray ionization interface in the positive-ion mode. The linear range was from 2.00 to 2000.00ngmL−1 in rat plasma and 0.50 to 400.00ngmL−1 in dog plasma. The intraday and interday precision were less than 10.55%, and the accuracy was in the range of −5.86 to 5.13%. The mean recoveries were greater than 82.70% and 82.97% for FNC and IS, respectively. The HPLC/Q-TOF MS and HPLC/QqQ MS/MS methods were both successfully applied in the pharmacokinetic studies of FNC in rats and dogs.