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[Display omitted] • Adefovir accumulated in the renal interstitium causes renal interstitial fibrosis when Oat1/3 were inhibited by probenecid for long-term. • Adefovir accumulation in the renal interstitium triggers mast cell degranulation and promotes renal interstitial fibrosis. • Mast cell membrane stabilizer sodium cromoglycate and angiotensin receptor antagonist valsartan ameliorate adefovir-associated fibrosis. Organic anion transporters 1 (OAT1) and OAT3 are responsible for transporting adefovir (ADV) into renal tubular epithelial cells. Our previous research found that ADV accumulated in the renal interstitium and caused renal interstitial fibrosis when Oat1/3 were inhibited by OATs inhibitor probenecid for long-term. Mast cells (MCs) in the interstitial space are considered to be key drivers of renal fibrosis. The current work investigated the effect of ADV on MCs in vitro and during the development of interstitial fibrosis in rats. Results indicate that ADV triggers chymase release from cultured RBL-2H3 mast cells in a time-and concentration-dependent manner. Angiotensin II (Ang II) in renal interstitium is generated mainly by chymase, renin and other products released from MCs, and has a direct effect on fibrosis through the angiotensin receptor. The concentrations of Ang II and fibrosis was significantly increased after administration of ADV alone or with probenecid for 4 weeks. The MCs membrane stabilizer sodium cromoglycate (SCG) and the angiotensin receptor antagonist Valsartan (VAL) could ameliorate ADV-induced nephrotoxicity. Additionally, SCG or VAL could reduce the accumulation of ADV in the renal interstitium by upregulating the expression of Oat1/3 and multidrug resistance-associated protein 4. Therefore, ADV accumulation in the renal interstitium could promote the degranulation of interstitial MCs and drive the development of renal fibrosis. SCG or VAL could ameliorate ADV-associated fibrosis by decreasing degranulation of MCs and accelerating renal clearance of ADV. [ABSTRACT FROM AUTHOR]

The deficiency of nucleos(t)ide analogues (NAs) as anti-hepatitis B virus (HBV) drugs in clinical use is attributable to their insufficient enrichment in liver and non-target organ toxicity. We aimed to develop potent anti-HBV adefovir derivatives with hepatotrophic properties and reduced nephrotoxicity. A series of adefovir mono l -amino acids, mono cholic acid-drug conjugates were designed and synthesized, and their antiviral activity and uptake in rat primary hepatocytes and Na+-dependent taurocholate co-transporting polypeptide (NTCP)-HEK293 cells were evaluated. We isolated compound 6c as the optimal molecular candidate, with the highest antiviral activity (EC 50 0.42 μmol/L, SI 1063.07) and highest cellular uptake in primary hepatocytes and NTCP-HEK293 cells. In-depth mechanistic studies demonstrated that 6c exhibited a lower toxicity in HK-2 cells when compared to adefovir dipivoxil (ADV). This is because 6c cannot be transported by the human renal organic anion transporter 1 (hOAT1). Furthermore, pharmacokinetic characterization and tissue distribution of 6c indicates it has favorable druggability and pharmacokinetic properties. Further docking studies suggested compounds with ursodeoxycholic acid and l -amino acid groups are better at binding to NTCP due to their hydrophilic properties, indicating that 6c is a potential candidate as an anti-HBV therapy and therefore merits further investigation. [ABSTRACT FROM AUTHOR]

Abstract Background Entecavir (ETV) is a nucleoside analogue (NA) that is effective for treatment of chronic hepatitis B (CHB) due to its low resistance rates and potent antiviral effects. We aimed to evaluate the clinical, biochemical and virological response to ETV in patients without a prior use of nucleos(t)ide (NA-naïve) vs. those who failed prior NA use (NA-experienced) in the treatment of CHB. Methods Patients treated between April 2012 and December 2017 were retrospectively studied. A comparison was made between patients treated with ETV in NA-naïve Vs. NA-experienced. Complete virological response (CVR) was defined as achieving undetectable HBV-DNA level, up to 15 IU/ml, partial virological response (PVR) as 15–200 IU/ml and >200 IU/ml for no virological response (NVR) after one year of therapy. Results Overall, 148 patients were included (69 NA-naïve and 79 NA-experienced). In NA-naïve group, 51%, 17% and 32% achieved CVR, PVR and NVR vs. 17%, 9% and 75% in NA-experienced group, respectively (p < 0.001). HBsAg seroconversion was achieved in 5.8% in NA-naïve group vs. 6.3% in NA-experienced group (p = 1.00). HBeAg seroconversion was 17% in NA-naïve group and 25% in NA-experienced group (p = 0.24). There was no significant difference in alanine transaminase normalization or in mortality rate between both groups; p = 0.87 and p = 1.00 respectively. Conclusion ETV therapy in CHB results in a better virological response in NA-naïve patients compared to NA-experienced. There were no differences between both groups in regards to the rate of HBsAg or HBeAg seroconversions, biochemical improvements or mortality. [ABSTRACT FROM AUTHOR]

[Display omitted] Adefovir based acyclic nucleoside phosphonates were previously shown to modulate bacterial and, to a certain extent, human adenylate cyclases (mACs). In this work, a series of 24 novel 7-substituted 7-deazaadefovir analogues were synthesized in the form of prodrugs. Twelve analogues were single-digit micromolar inhibitors of Bordetella pertussis adenylate cyclase toxin with no cytotoxicity to J774A.1 macrophages. In HEK293 cell-based assays, compound 14 was identified as a potent (IC 50 = 4.45 μM), non-toxic, and selective mAC2 inhibitor (vs. mAC1 and mAC5). Such a compound represents a valuable addition to a limited number of small-molecule probes to study the biological functions of individual endogenous mAC isoforms. [ABSTRACT FROM AUTHOR]

Background: Efficacy of sequential therapy with nucleos(t)ide analogues and interferons versus monotherapy in patients with hepatitis B e antigen (HBeAg)-positive chronic hepatitis B (CHB) remains unexplored. We aimed to assess efficacy and safety of sequential therapy with adefovir (ADV) or entecavir (ETV) followed by peginterferon (PEG-IFN) alfa-2a in Taiwanese patients with HBeAg-positive.Methods: This randomized, placebo-controlled, double-blind trial was conducted at nine sites in Taiwan from April 2010 to October 2013. Patients (N = 280) were randomized 1:1:1 to receive placebo, ETV or ADV alone for four weeks, combined with PEG-IFN alfa-2a for two weeks, then PEG-IFN alfa-2a alone for 46 weeks. The primary efficacy end point was HBeAg seroconversion at 48 weeks post-treatment.Results: No significant differences were observed among groups for HBeAg seroconversion (PEG-IFN alfa-2a+placebo, 36.3%; PEG-IFN alfa-2a+ETV, 29.5%; and PEG-IFN alfa-2a+ADV, 27.4%), HBeAg loss (37.4%, 32.2%, and 28.6%, respectively) or change in hepatitis B surface antigen (HBsAg) levels from baseline (-0.56 IU/mL, -0.60 IU/mL, and -0.41 IU/mL, respectively). However, hepatitis B virus DNA levels were higher with PEG-IFN alfa-2a+placebo than PEG-IFN alfa+ETV at week 64 (p = 0.0412), 76 (p = 0.0311), and 88 (p = 0.0113), and alanine aminotransferase (ALT) normalization rate was higher with PEG-IFN alfa-2a+placebo than PEG-IFN alfa-2a+ADV (p = 0.0283) or PEG-IFN alfa-2a+ETV (p = 0.0369) at week 88. Sub-analysis of results revealed an association between on-treatment HBsAg and ALT levels and efficacy 48 weeks post-treatment. Safety was comparable among treatment groups.Conclusion: Pre-therapy with ADV or ETV followed by PEG-IFN alfa-2a is not superior to PEG-IFN alfa-2a monotherapy in Taiwanese patients with HBeAg-positive CHB.Clinical Trial Id: NCT: 00922207. [ABSTRACT FROM AUTHOR]

For patient convenience, sustained release Adefovir Poly-d,l-lactic- co -glycolic acid (PLGA) microspheres were formulated to relieve the daily use of the drug which is a problem for patients treated from chronic hepatitis-B. PLGA microspheres were prepared and characterized by entrapment efficiency, particle size distribution and scanning electron microscopy (SEM). In-vitro release and in-vivo studies were carried out. Factors such as drug: polymer ratio, polymer viscosity and polymer lactide content were found to be important variables for the preparation of PLGA Adefovir microspheres. Fourier transform infrared (FTIR) analysis and differential scanning calorimetry (DSC) were performed to determine any drug-polymer interactions. One way analysis of variance (ANOVA) was employed to analyze the pharmacokinetic parameters after intramuscular injection of the pure drug and the selected PLGA microspheres into rats. FTIR and DSC revealed a significant interaction between the drug and the polymer. Reports of SEM before and after 1 and 24 h release showed that the microspheres had nonporous smooth surface even after 24 h release. The entrapment efficiency ranged between 55.83 and 86.95% and in-vitro release studies were continued for 16, 31 and 90 days. The pharmacokinetic parameters and statistical analysis showed a significant increase in the T max, AUC 0–t and MRT, and a significant decrease in the C max of the tested formulation ( p  < 0.05). Results demonstrated that PLGA Adefovir microspheres could be used for long-term treatment of chronic hepatitis-B instead of the daily dose used by the patient. [ABSTRACT FROM AUTHOR]

The study aimed to characterize rtA181T/sW172stop (*) and rtA181T/sW172non-stop mutations of hepatitis B virus (HBV). Total of 22,009 patients who visited Beijing 302 Hospital from 2007 to 2016 were enrolled. These patients all received nucleos(t)ide analogues (NAs) treatment and their serum samples were collected for sequence analysis of HBV reverse-transcriptase (RT) and S regions. The rtA181T mutation was detected in 5.37% (1182/22,009) of the patients' samples. The rtA181T-causative sW172*, sW172non-stop (sW172 L/S), and mixed sW172*/non-stop mutations occupied 82.91%, 7.70%, and 9.39%, respectively. The patients with rtA181T/sW172non-stop mutants had a higher HBV DNA level compared to those with rtA181T/sW172* mutants. 44.33% (524/1182) rtA181T-positive samples were detected with signature drug-resistant mutations, including 325 with adefovir-resistant mutation rtA181V/N236T, 57 with lamivudine-resistant mutation rtM204V/I, 99 with entecavir-resistant mutation rtM204V/I plus rt184/202/250 substitution(s), and 43 with multidrug-resistant mutation rtA181V/N236T + rtM204V/I ± rt184/202/250 substitution(s). The rtA181T/sW172non-stop mutation had a higher ratio of coexistence with adefovir-resistant mutation compared to rtA181T/sW172* mutation (42.86% vs. 24.59%, P < 0.05). rtA181T/sW172S + rtN236T and rtA181T/sW172L + rtN236T mutants exhibited higher HBV DNA production and adefovir resistance fold than that of rtA181T/sW172* + rtN236T mutant (98.02% and 85.5% vs. 42.1% in HBV DNA production, and 7.38-fold and 5.49-fold vs. 3.69-fold in half maximal effective concentration of wild-type strain); rtA181T/sW172L + rtS202G + rtM204V strain exhibited higher HBV DNA production and entecavir resistance fold than that of rtA181T/sW172* + rtS202G + rtM204V strain (50.98% vs. 34.49%, 524.00-fold vs. 69.33-fold). In conclusion, rtA181T/sW172non-stop mutation may increase resistance fold of adefovir- and entecavir-resistant mutants compared to rtA181T/sW172* mutation and might influence clinical presentation of NAs-treated patients. [ABSTRACT FROM AUTHOR]

Background and aim Precore (PC) and basal core promoter (BCP) mutations are associated with responses to interferon-based treatment in HBeAg-positive chronic hepatitis B (CHB) patients. Here, we identify viral minority variants in these regions and assess association with response to peginterferon-alfa (Peg-IFN) and adefovir combination therapy. Patients and methods Ultra-deep pyrosequencing analysis of the BCP and PC region was performed for 89 CHB patients (42 HBeAg-positive; 47 HBeAg-negative), at baseline and during treatment. Specifically, associations of individual positions with the HBeAg-negative phenotype were studied, as well as the association of the most prevalent mutations with combined response in HBeAg-positive and –negative patients at week 72 (HBeAg negativity, HBV-DNA <2000 IU/mL and ALT normalization at 24 weeks of treatment-free follow-up). Results The mutations most strongly correlated with the HBeAg-negative phenotype were at positions 1762/1764 and 1896/1899 in the BCP and PC region, respectively. No major changes in nucleotide composition of these positions were observed during treatment. In HBeAg-negative patients, a combined presence of 1764A and 1896A was correlated with lower ALT levels (p = 0.004), whereas the presence of 1899A was correlated with higher age (p = 0.030), lower HBV-DNA level (p = 0.036), and previous IFN therapy (p = 0.032). The presence of 1764A/1896A or the absence of 1899A at baseline, was associated with lower response rates, after adjustment for HBV genotype (p = 0.031 and p = 0.017) and HBsAg level (p = 0.035 and p = 0.022). Conclusion We identified novel correlations between common BCP and PC variants with response to Peg-IFN and adefovir in HBeAg-negative patients. Ultimately, this may guide the selection of those patients most likely to benefit from Peg-IFN-based treatment. [ABSTRACT FROM AUTHOR]

Background and aims Adefovir, an acyclic nucleotide reverse transcriptase inhibitor used to treat hepatitis B viral infection, is primarily eliminated renally through cooperation of glomerular filtration with active tubular transport. Nonalcoholic steatohepatitis is a variable in drug disposition, yet the impact on renal transport processes has yet to be fully understood. The goal of this study was to determine the effect of nonalcoholic steatohepatitis on the pharmacokinetics of adefovir in rats given a control or methionine and choline deficient diet to induce nonalcoholic steatohepatitis. Methods Animals received a bolus dose of 7 mg/kg (35 μCi/kg) [ 3 H] adefovir with consequent measurement of plasma and urine concentrations. Inulin clearance was used to determine glomerular filtration rate. Results Methionine and choline deficient diet-induced nonalcoholic steatohepatitis prolonged the elimination half-life of adefovir. This observation occurred in conjunction with reduced distribution volume and hepatic levels of adefovir. Notably, despite these changes, renal clearance and overall clearance were not changed, despite markedly reduced glomerular filtration rate in nonalcoholic steatohepatitis. Alteration of glomerular filtration rate was fully compensated for by a significant increase in tubular secretion of adefovir. Analysis of renal transporters confirmed transcriptional up-regulation of Mrp4, the major transporter for adefovir tubular secretion. Conclusions This study demonstrates changes to glomerular filtration and tubular secretion that alter pharmacokinetics of adefovir in nonalcoholic steatohepatitis. Nonalcoholic steatohepatitis-induced changes in renal drug elimination processes could have major implications in variable drug response and the potential for toxicity. [ABSTRACT FROM AUTHOR]

Introduction Interferon-y–inducible protein-10 (IP-10), also called CXCL10, is produced by different types of cells such as monocytes, neutrophils and hepatocytes. IP-10 functions as an inflammatory cytokine, which after binding to its receptor CXCR3, expressed on T-lymphocytes, leads to immune activation. We aimed to establish if IP-10 expression in liver tissue and in plasma of chronic hepatitis B (CHB) patients correlated with each other and further to investigate if IP-10 levels before and during therapy with peginterferon and adefovir could predict treatment outcome in CHB patients. Patients and methods A total of 86 CHB patients (41 HBeAg-positive and 45 HBeAg-negative) received combination therapy of peginterferon and adefovir for 48 weeks. Combined Response (CR) (HBeAg-negativity, HBV-DNA ≤ 2000 IU/mL, ALT normalization) and non-response (NR) were assessed at Week 72. Plasma IP-10 levels were measured at baseline and during treatment at Day 3 (D3) and Week 1 (W1). Pre-treatment liver biopsies from 40 of 86 patients were obtained and stored in liquid nitrogen for the analysis of intrahepatic IP-10 mRNA expression. Results CR was achieved in 14/41 HBeAg-positive and 17/45 HBeAg-negative patients. Mean baseline plasma IP-10 levels were significantly higher in HBeAg-positive patients with CR than NR (3.20 vs 3.00 log pg/mL p = 0.03); but not in HBeAg-negative patients. Baseline IP-10 levels correlated with ALT-levels in HBeAg-positive and -negative patients (both p < 0.001), and with a decline of HBsAg-levels of ≥0.5 log IU/mL at Week 12 in HBeAg-positive patients (p = 0.001). Plasma IP-10 levels were associated with intrahepatic IP-10 mRNA expression, however, more strongly in HBeAg-positive (R = 0.79, p < 0.001) than in HBeAg-negative patients (R = 0.53, p = 0.011). IP-10 levels only correlated with HAI-scores in HBeAg-positive patients (R = 0.40 p = 0.025). Mean plasma IP-10 levels of both HBeAg-positive and -negative patients increased significantly at D3 compared to baseline (+0.30 log pg/mL p = 0.003), to then decline subsequently at W1 to a level still significantly higher than baseline (+0.14 log pg/mL p < 0.001). The increase of IP-10 was significantly higher in HBeAg-positive patients with NR than in those with CR (+0.35 versus +0.11 log pg/mL p = 0.003). Conclusions Baseline plasma IP-10 levels and IP-10 mRNA expression in the liver are correlated with each other, suggesting that plasma IP-10 reflects intrahepatic immune activation. Higher IP-10 levels at baseline seem to be associated with CR in HBeAg-positive patients treated with peginterferon and adefovir, but not in HBeAg-negative patients. [ABSTRACT FROM AUTHOR]

We report here three novel cocrystals, which are composed of adefovir as the API (Active Pharmaceutical Ingredient) with p -aminobenzoic acid ( 1, 2C 8 H 12 N 5 O 4 P·C 7 H 6 NO 2· 3H 2 O), 3,5-dihydroxybenzoic acid ( 2, C 8 H 12 N 5 O 4 P·C 7 H 6 O 4· H 2 O) and 2,6-pyridinedicarboxlic acid ( 3, C 8 H 12 N 5 O 4 P·C 7 H 5 NO 4 ) as CCFs (cocrystal formers) respectively by crystal engineering strategy. Their structures were characterized by single crystal X-ray diffraction, powder X-ray diffraction (PXRD) analysis, thermogravimetric analyses (TGA), elemental analysis (EA) and infrared spectral analysis (IR). The analysis of single crystal X-ray diffraction demonstrate that cocrystal 1 and 2 form a strong hydrogen-bonded assembly through the phosphoric acids of API with water in the lattice and carboxylic acids of CCF respectively. Cocrystal 3 is formed in which the phosphoric acid groups of API are also held by the carboxylic acid groups of CCF. The PXRD results indicate their high purity of as-synthesized samples. The TGA, EA, IR and dissolution study of API and the cocrystals were also measured and discussed. [ABSTRACT FROM AUTHOR]

An analytical method based on solid phase extraction was developed and validated for analysis of adefovir in human plasma. Adefovir-d 4 was used as an internal standard and Synergi MAX RP80A (150 mm×4.6 mm, 4 µm) column provided the desired chromatographic separation of compounds followed by detection with mass spectrometry. The method used simple isocratic chromatographic condition and mass spectrometric detection in the positive ionization mode. The calibration curves were linear over the range of 0.50–42.47 ng/mL with the lower limit of quantitation validated at 0.50 ng/mL. Matrix effect was assessed by post-column infusion experiment to monitor phospholipids and post-extraction addition experiment was performed. The degree of matrix effect for adefovir was determined as 7.5% and ion-enhancement in five different lots of human plasma was 7.1% and had no impact on study samples analysis with 4.5 min run time. The intra- and inter-day precision values were within 7.7% and 7.8%, respectively, for adefovir at the lower limit of quantification level. Validated bioanalytical method was successfully applied to clinical sample analysis. [ABSTRACT FROM AUTHOR]

Adefovir is a commonly used anti-hepatitis B virus nucleoside analog. Four N-acyl derivatives of adefovir phosphonate with different lipid chains were prepared, including N-decanoyl-1-(3-chlorophenyl)-1,3-propanyl phosphonyl adefovir (DCPA), N-lauroyl-1-(3-chlorophenyl)-1,3-propanyl phosphonyl adefovir (LCPA), N-myristoyl-1-(3-chlorophenyl)-1,3-propanyl phosphonyl adefovir (MCPA) and N-stearoyl-1-(3-chlorophenyl)-1,3-propanyl phosphonyl adefovir (SCPA). They formed the Langmuir monolayers at the air/water interface with a little difference of the surface pressure–molecular area isotherms. Self-assembled nanoassemblies of the derivatives were prepared by injecting their methanol solutions into water. The sizes were 148.2, 169.6 and 159 nm for the nanoassemblies of DCPA, LCPA and MCPA, respectively, and the zeta potentials were −22.2, −16.8 and −13.8 mV, respectively. Flocky precipitates appeared in the suspension of the SCPA nanoassemblies due to aggregation of the nanoassemblies and the strong hydrophobic interaction of SCPA. Dissipative particle dynamics was used to simulate molecular self-assembly of the derivatives in water based on the coarse-grained models. The simulation result was consistent with the experimental results. The nanoassemblies of the derivatives would be promising nanomedicines. [ABSTRACT FROM AUTHOR]

Background & Aims Achievement of HBsAg loss remains the hallmark of chronic hepatitis B treatment. In order to identify host factors contributing to treatment-induced HBsAg loss, we performed a genome-wide screen of single nucleotide polymorphisms (SNPs) and studied its immunological consequence. Methods Chronic hepatitis B patients (40 HBeAg-positive and 44 HBeAg-negative) treated with peginterferon alfa-2a and adefovir were genotyped for 999,091 SNPs, which were associated with HBsAg loss at week 96 (n = 9). Plasma carnitine levels were measured by tandem-mass spectrometry, and the effect of carnitine on the proliferative capacity of hepatitis B virus (HBV)-specific and non-specific CD8 T cells was studied in vitro . Results One polymorphism, rs12356193 located in the SLC16A9 gene, was genome-wide significantly associated with HBsAg loss at week 96 ( p = 1.84 × 10 −8 ). The previously reported association of rs12356193 with lower carnitine levels was confirmed in our cohort, and baseline carnitine levels were lower in patients with HBsAg loss compared to patients with HBsAg persistence ( p = 0.02). Furthermore, we demonstrated that carnitine suppressed HBV-specific CD8 T cell proliferation. Conclusions In chronic hepatitis B patients treated with peginterferon and adefovir, we identified strong associations of SLC16A9 gene variation and carnitine levels with HBsAg loss. Our results further suggest that a lower baseline plasma carnitine level increases the proliferative capacity of CD8 T cells, making patients more susceptible to the immunological effect of this treatment. These novel findings may provide new insight into factors involved in treatment-induced HBsAg loss, and play a role in the prediction of treatment outcome. [ABSTRACT FROM AUTHOR]

Abstract: Probe substrates for, and inhibitors of, specific transporters are desired to evaluate quantitatively the in vivo functions of transporters in humans. Based on published data, adefovir and benzylpenicillin were selected as organic anion transporter (OAT) 1- and OAT3-selective probe substrates, respectively. In human kidney slices, probenecid potently inhibited the uptake of both adefovir and benzylpenicillin with inhibition constant (K i) values of 18.6±5.1 and 12.6±4.2μM, respectively, whereas p-aminohippurate (PAH) preferentially inhibited adefovir uptake. A clinical drug-interaction study involving healthy subjects was performed to investigate the dose-dependent inhibition potencies of probenecid and PAH on the renal clearance of the probe substrates. Adefovir or benzylpenicillin was coadministered with different oral doses of probenecid (500, 750, or 1500mg) or intravenous PAH infusion rates (70, 120, or 210mg/min/person) to the same subject using a crossover design. The renal clearance of adefovir was reduced by 45% and 46% in the subjects treated with the maximum dose of probenecid and PAH, respectively, which was in accordance with the results of in vitro inhibition study. On the other hand, renal clearance of benzylpenicillin was reduced by 78% in the subjects treated with the maximum dose of probenecid (1500mg), which could be explained by its in vitro K i values. However, PAH unexpectedly increased the renal clearance of benzylpenicillin by 47%. These results suggest that adefovir and benzylpenicillin can be used as probe drugs for OAT1 and OAT3, respectively, and that PAH can be used to investigate the role of OAT1 in the urinary excretion of drugs in humans, whereas it may modulate other transport processes in the kidney. [Copyright &y& Elsevier]

Background & Aims: Suboptimal virologic response to nucleos(t)ide analogs may represent a significant risk factor for resistance development in patients with chronic hepatitis B virus infection; treatment options have not been well studied. We evaluated long-term efficacy and safety of tenofovir alone and in combination with emtricitabine in a prospective, placebo-controlled trial in patients who remained viremic on adefovir therapy. Methods: Hepatitis B e antigen-positive and -negative patients with hepatitis B virus DNA ⩾1000 copies/ml despite up to 96weeks of adefovir were randomized to double-blind tenofovir or emtricitabine/tenofovir for 168weeks. Patients with hepatitis B virus DNA ⩾400 copies/ml (⩾69IU/ml) at or after week 24 could switch to open-label emtricitabine/tenofovir. Results: Overall, 90/105 (86%) patients (46/53 tenofovir and 44/52 emtricitabine/tenofovir) completed the 168-week study period, including 74/105 (70%) patients (35/53 tenofovir and 39/52 emtricitabine/tenofovir) who completed the study on their initial randomized treatment. Long-term viral suppression (hepatitis B virus DNA <400 copies/ml) was maintained at week 168 in 84% and 82% of patients receiving either emtricitabine/tenofovir combination or tenofovir monotherapy, respectively (non-completer equal to failure analysis). Baseline viral load as well as the presence of lamivudine and/or adefovir resistance-associated mutations at baseline had no impact on long-term treatment response. No resistance to tenofovir was observed through 168weeks. Both treatments had a favorable safety profile. Conclusions: Tenofovir monotherapy is as effective as emtricitabine/tenofovir combination therapy in maintaining long-term viral suppression in patients with a suboptimal response to adefovir, and is well tolerated in this population. [Copyright &y& Elsevier]

Abstract: Background and study aims: The development of antiviral-resistant mutations with long-term treatment remains a major concern in the treatment of chronic hepatitis B virus (HBV) infection. The study aimed to compare the therapeutic efficacy of entecavir 1mg versus combined lamivudine/adefovir dipivoxil (Lam/Adv) in chronic HBV patients resistant to lamivudine monotherapy. Patients and methods: This study included two groups of lamivudine-resistant patients who received lamivudine 100mg for 1–3years. Group 1 was composed of 25 cases (52% HBeAg+ve) who received combined Lam/Adv, and group 2 was composed of 13 patients (30.8% HBeAg+ve) who received entecavir 1mg. Pre-enrolment assessment included biochemical, serological and quantitative HBV-DNA testing as well as HBeAg and hepatitis B envelope antibody (HBeAb) assessment. Evaluation was done at 3, 6, 12, 24 and 36months of treatment by the same parameters. Hepatitis B surface antigen and antibody (HbsAg and HBsAb) were assessed after each year of treatment. Results: At the end of 36months of treatment, 16 cases (69%) in group 1 completed the study period, versus 13 (100%) in group 2. Two cases in group 1 underwent HBeAg seroconversion, accompanied by HBV-DNA undetectability, at 6 and 12months, respectively; no cases were seroconverted in group 2. Both treatments achieved improvement in alanine aminotransferase (ALT), bilirubin and alpha-foetoprotein equally at the end of the study. HBV-DNA undetectability was better achieved in group 2 when compared to group 1. HBeAg seroconversion was only achieved in two cases in group 1, whereas no cases lost HBeAg in group 2. None of our cases achieved HbsAg seroconversion or loss at the end of the study period. Conclusion: The entecavir 1-mg monotherapy group achieved better HBV-DNA undetectability starting at 3months of treatment when compared to the Lam/Adv group; however, both lines of treatment showed almost similar results over the rest of the study period. HBeAg seroconversion was only achieved in two cases in the combined Lam/Adv group, whereas no cases lost HBeAg in the other group. [Copyright &y& Elsevier]

Abstract: The dissociation constants of Adefovir ({[2-(6-amino-9H-purine-9-l)ethoxi]methyl}phosphoric acid) used in the treatment of hepatitis B and Cidofovir ({[2-(6-amino-9H-purine-9-l)ethoxi]methyl}phosphoric acid), used in the treatment of cytomegalovirus retinitis in AIDS patients, together with the stability constants of its Cu2+, Ni2+, Zn2+, Co2+, Ca2+, and Mg2+ complexes were studied by potentiometric titration. Adefovir and Cidofovir were abbreviated to PMEA and HPMPC. All measurements were obtained under two sets of conditions: at 298K and ionic strength (I) of 0.1moldm−3 (NaCl) and at 310K and I of 0.16moldm−3 (NaCl), corresponding to the conditions of human blood. Equilibrium constants in aqueous solution were calculated using the program SUPERQUAD. Also, solvent effects on the equilibrium constants of the ligands were examined. Moreover, all coordination studies were carried out with the ligand/metal molar ratios of 1:1. This ligand/metal stoichiometric ratio was spectrometrically determined using Job's method. [Copyright &y& Elsevier]

Abstract: Objectives: Health care decision makers are increasingly concerned about the value of chronic hepatitis B (CHB) treatments in China. This analysis aims at estimating the relative value of entecavir as a first-line option at treatment initiation and for different treatment durations from a holistic perspective. Methods: CHB was simulated by using a Markov disease transition model with disease states based on available natural history data. The model assumed 5-year treatment duration with entecavir, lamivudine, telbivudine, and adefovir based on published clinical data. The speed of disease progression varies by viral load and hepatitis B “e” antigen status. Direct medical costs included medication and management of liver complications. The primary output was the estimated cost savings of entecavir per patient per day versus the comparator. Results: For treatment duration of 5 years and a follow-up period of 30 years, entecavir treatment was translated into specific patient benefit of an estimated cost saving of $2.69 per day compared with no treatment. In addition, long-term usage of entecavir resulted in daily $2.33 and $1.73 cost saving compared with short-term usage (1-year and 2-year, respectively). Among available treatment options in China, entecavir treatment exhibited about $0.90 to $1.81 daily cost saving versus the comparators. The detailed daily cost saving of entecavir is summarized as follows—entecavir versus lamivudine: $1.81, entecavir versus telbivudine: $0.90, entecavir versus adefovir: $2.02, and entecavir versus generic adefovir: $1.37. Conclusions: Long-term usage of entecavir exhibits the characteristics of a favorable CHB treatment, which translates into economic value as opposed to either no treatment or alternative strategies. [Copyright &y& Elsevier]

Abstract: In this review article, a number of milestones in the antiviral research field on nucleosides and nucleotides are reviewed in which the author played a significant part, especially in the initial stages of their development. Highlighted are the amino acyl esters of acyclovir, particularly valacyclovir (VACV), brivudin (BVDU) and the valine ester of Cf1743 (FV-100), the 2′,3′-dideoxynucleosides (nucleoside reverse transcriptase inhibitors, NRTIs), the acyclic nucleoside phosphonates (S)-HPMPA, (S)-HPMPC (cidofovir) and alkoxyalkyl esters thereof (HDP-, ODE-CDV), adefovir and adefovir dipivoxil, tenofovir and tenofovir disoproxil fumarate (TDF), combinations containing TDF and emtricitabine, i.e., Truvada®, Atripla®, Complera®/Eviplera® and the Quad pill, and the phosphonoamidate derivatives GS-7340, GS-9131, GS-9191 and GS-9219. [Copyright &y& Elsevier]

Abstract: Electrochemical reduction behavior of Adefovir was studied using Hanging Mercury Drop Electrode (HMDE) in Britton–Robinson (BR) buffer solution. Voltammetric study showed one well-defined reduction peak in the potential range −1.2 to −1.4V (vs. Ag/AgCl) due to reduction of C=N bond of the imidazole ring. Solid-phase extraction and protein-precipitation techniques were employed for extraction of Adefovir from human plasma. The proposed method allows quantification of Adefovir in human plasma over the concentration range 0.50–5.00μg/mL with the detection limit 0.17μg/mL, whereas in pharmaceutical formulation 0.25–2.25μg/mL with the detection limit 0.08μg/mL. [Copyright &y& Elsevier]

Abstract: A series of phosphonate ester prodrugs of adefovir incorporating l -amino (thio)acid and non-steroidal anti-inflammatory drug (NSAID) moieties were synthesized and their anti-HBV activity and renal cell toxicity were evaluated in HepG2 2.2.15 and HK-2 cells respectively. Bioactivity evaluation results revealed that this kind of adefovir prodrug have lower renal cell toxicity than adefovir dipivoxil. Compounds 8a and 8b, incorporating the NSAID ketoprofen and the l -amino acid (Val or Ile) structural fragments, exhibited more potent anti-HBV activity than adefovir dipivoxil with IC50 = 0.51 and 0.73 μM, SI = 1697.64 and 881.92 respectively. In vitro stability studies showed that the synthesized prodrugs have higher chemical and plasma stability than the positive control adefovir dipivoxil. [Copyright &y& Elsevier]

Abstract: Amino acid substitutions at positions rtN238T/D of the hepatitis B virus (HBV) polymerase have been reported as potential mutations associated with adefovir (ADV) resistance. In this study, we characterized the prevalence of the rtN238H mutation and determined the susceptibility to LAM and ADV using phenotypic analyzes in vitro. One thousand eight hundred and sixty-five HBsAg-positive patients with chronic HBV (CHB) infection were included in this study. HBV genotypes and reverse transcriptase (RT) mutations were determined by direct sequencing. Replication-competent HBV constructs containing the naturally occurring rtN238H mutation were generated and replication capacity and susceptibility to LAM and ADV in transiently transfected hepatoma cell lines were determined. Among 1865 enrolled HBV infected patients, 8.8% (165/1865) showed mutations in the rtN238 locus (143 males/22 females, 91 treatment-naive, 42 ADV-treated, 16 LAM-treated and 16 ADV+LAM-treated), namely 86% rtN238H (142/165), 5.5% rtN238S (9/165), 5.5% rtN238T (9/165) and 3% rtN238D (5/165). Among the rtN238H mutant strains, there were no significant differences between ADV- or/and LAM- treated patients and treated-naive patients (42% vs. 58%). Compared with wild-type HBV, this mutant displayed an equivalent susceptibility to LAM or ADV in phenotypic assays. Importantly, we found that the incidence rate of rtN238H was higher in HBV genotype B infected patients than HBV genotype C subsets (80.3% vs. 19.7%), even without exogenous selection pressures. As rtN238H did neither impair the viral replication efficiency nor susceptibility to LAM or ADV in vitro, rtN238H likely represents background polymorphisms rather than resistance mutations with clinical implications. The incidence of rtN238H may be associated with HBV genotype. [Copyright &y& Elsevier]

Abstract: Adefovir dipivoxil (ADV) is an anti-viral drug having low bioavailability due to low permeability and pH dependent solubility. In this study, self-nanoemulsifying drug delivery systems (SNEDDS) of ADV were developed with the objective of increasing its bioavailability by enhancing its intestinal permeability and minimizing the effect of pH. Preliminary screening was carried out to select oil, surfactant and co-surfactant. Ternary phase diagrams were constructed to identify the area of nanoemulsification. The nanoemulsion system selected from the phase diagram was transformed into solid-SNEDDS (S-SNEDDS) by lyophilization using D-mannitol as cryoprotectant. The formulations were characterized for transmittance, globule size, polydispersity index, zeta potential, cloud point, robustness to dilution, effect of pH and temperature, microscopic properties, in vitro and ex vivo drug release parameters. The liquid SNEDDS (L-SNEDDS) showed mean globule size of 110±10nm while mean globule size of 150±16nm was obtained with S-SNEDDS. The formulations were found to be robust to dilution and showed cloud point at 80–85°C. TEM and SEM studies of nanoemulsion reconstituted from S-SNEDDS demonstrated the spherical shape and size of the globules. Results of DSC and XRD studies confirmed that the drug was incorporated in the S-SNEDDS. No significant difference was observed in the globule size within physiological variations of pH and temperature. The in vitro and ex vivo drug release from ADV SNEDDS was found to be significantly higher in comparison to that from plain drug suspension, irrespective of pH. Thus, SNEDDS were found to be instrumental in reducing the effect of pH variability of ADV and improving the release performance of ADV, indicating their potential to improve the oral bioavailability and thus the therapeutic efficacy of ADV. [Copyright &y& Elsevier]

Background & Aims: Therapy of chronic hepatitis B with HBV-polymerase inhibitors, in particular tenofovir or adefovir, may affect renal function. To assess renal function more accurately in the normal range, we used the recently validated, Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) formula to calculate the estimated glomerular filtration rate (eGFR). Methods: Patient subgroups included: patients with HBV-monoinfection treated with lamivudine (n =36), adefovir (n =32), entecavir (n =32), or tenofovir (n =37). HBsAg-positive untreated patients (n =60) served as control. For comparison HIV-monoinfected patients treated with tenofovir (n =120) or zidovudine (n =52) based antiretroviral therapy and antiretroviral naive patients (n =109) were assessed. CKD-EPI equation was used to calculate eGFR. In a more sensitive approach, we modeled the individual change in eGFR over time with linear mixed effects models (LME). Results: Yearly predicted median changes in individual eGFR according to the LME model were: HBV untreated −2.05ml/min, HBV lamivudine −0.92ml/min, HBV adefovir −1.02ml/min, HBV entecavir −1.00ml/min, and HBV tenofovir −0.92ml/min (p <0.01 for HBV untreated vs. HBV treated). In HIV-monoinfected patients: HIV untreated −0.62ml/min, HIV treated with tenofovir −2.64ml/min, HIV treated with zidovudine −1.0ml/min (p =0.017 for tenofovir vs. no treatment, p <0.001 for tenofovir vs. zidovudine). Conclusions: Therapy of HBV infection irrespective of medication seems to result in a milder decrease of renal function. In contrast tenofovir as part of HIV combination therapy seems to impair renal function in this Caucasian population. [ABSTRACT FROM AUTHOR]

Abstract: Liver transplantation remains the ultimate cure for patients with hepatitis B virus-related end-stage liver disease. Clinical Practice Guidelines currently recommend that patients with decompensated HBV cirrhosis should be treated without delay with nucleos(t)ide analogues regardless of the patient''s serum ALT, HBV DNA level, and HBeAg status. The main goal of pre-transplantation antiviral therapy is to achieve a rapid and prolonged suppression of viral replication and thus decrease the risk of hepatitis B virus reinfection of the graft. In addition, sustained negativization of serum HBV DNA may also result in clinical stabilization which can decrease the mortality rate while on the waiting list and delay or avoid liver transplantation in some cases. Clinical improvement may take from 3 to 6 months to become evident. Lamivudine and adefovir are no longer considered first-line therapy in these patients. Potent nucleos(t)ide analogues with good resistance profiles such as entecavir or tenofovir should be used instead. Preliminary data suggest that these agents are effective and have a good safety profile in patients with decompensated cirrhosis. More safety data, however, are needed, particularly in patients with severe impairment of liver function. More data are needed to determine which of the newer antiviral agents offer the best risk–benefit ratio in this challenging patient population. Although a tenofovir-based regimen may be preferred in decompensated patients with lamivudine-resistant hepatitis B virus, there are some concerns about the long-term safety of tenofovir including nephrotoxicity and metabolic bone disease. [Copyright &y& Elsevier]

Abstract: New Adefovir (PMEA) prodrugs with a pro-moiety consisting of decyl or decyloxyethyl chain bearing hydroxyl function(s), hexaethyleneglycol or a (5-methyl-2-oxo-1,3-dioxolen-4-yl)methyl unit were prepared starting from the tetrabutylammonium salt of the phosphonate drug and an appropriate alkyl bromide or tosylate. Analogously, two esters of Cidofovir [(S)-HPMPC] bearing a hexaethyleneglycol moiety were prepared. The activity of the prodrugs was evaluated in vitro against different virus families. A loss in the antiviral activities of the hydroxylated decyl or decyloxyethyl esters and hexaethyleneglycol esters of PMEA against human immunodeficiency virus (HIV) and herpesviruses [including herpes simplex virus (HSV), varicella-zoster virus (VZV), and human cytomegalovirus (CMV)] occurred in comparison with the parent compound. On the other hand, the (5-methyl-2-oxo-1,3-dioxolen-4-yl)methyl ester of PMEA showed significant activities against HIV and herpesviruses. (S)-HPMPC prodrugs exhibited anti-cytomegalovirus activities in the same range as the parent drug, whereas the anti-HSV and anti-VZV activities were one- to seven-fold lower than that of Cidofovir. [Copyright &y& Elsevier]

Abstract: The Italian recommendations for the therapy of hepatitis B virus (HBV)-related disease were issued in 2008. Subsequently in 2008 the nucleotide analogue (NA) Tenofovir was approved for antiviral treatment. The introduction of this important new drug has called for the current guidelines update, which includes some additional revisions: (a) the indication for therapy is extended to mild liver fibrosis and the indication for treatment is graded as “possible”, “optional” or “mandatory” according to the fibrosis stage; (b) two different treatment strategies are described: first line definite duration treatment with interferon, long-term treatment of indefinite duration with NA; (c) the indication to follow either strategy is also based on the stage of liver fibrosis; (d) virological monitoring is modified to include the definitions of failure and of sustained virological response to interferon therapy; (e) the recommendation to use HBV DNA assays with high sensitivity and wide linear ranges is underlined (f) guidelines on post-treatment follow-up after finite treatment with NA, potential side effects of therapy and non-virological monitoring are defined; (g) definitions and treatment of patients without optimal response to NA are reported; (f) treatment and monitoring of compensated or decompensated cirrhosis and hepatocellular carcinoma are updated. [Copyright &y& Elsevier]

Background & Aims: Little is known about the optimal management of patients with chronic hepatitis B (CHB) who developed multiple-drug resistance. Methods: We assessed 91 patients with compensated CHB who developed adefovir-resistant mutations during adefovir monotherapy for lamivudine-resistant CHB. Of these, 41 were treated with a combination of lamivudine plus adefovir (LAM+ADV group) and 50 were treated with entecavir monotherapy (ETV group). Results: There were no significant differences between the two groups in baseline characteristics, including serum HBV DNA levels (p>0.05). The rate of virologic non-response (HBV DNA reduction <1 log10 IU/ml at 6months) was significantly greater in the LAM+ADV than in the ETV group (51.2% vs. 16.0%, p<0.01). At 12months, HBV DNA declined less in the LAM+ADV than in the ETV group (−1.49±1.78 vs. −3.47±2.13 log10 IU/ml, p<0.01). Only 12.2% and 22.0% of patients in the LAM+ADV and ETV groups, respectively, achieved complete virologic response (HBV DNA <60IU/ml) at 12months. Multivariable analysis showed that LAM+ADV group (OR=0.08, CI=0.02–0.28) and the presence of the rtA181V/T mutation (OR=0.21, CI=0.05–0.91) were independently associated with a decreased rate of virologic response (HBV DNA <2000IU/ml) at 12months. Conclusions: In patients with CHB resistant to lamivudine and adefovir, combination therapy with these two drugs was not effective and was inferior to entecavir monotherapy in suppressing HBV DNA. However, the response to entecavir monotherapy was also not optimal. These results emphasize the importance of preventing the development of multidrug-resistant HBV and of exploration for adequate combination therapy in treatment of multidrug-resistant CHB. [ABSTRACT FROM AUTHOR]

Abstract: A selective, rapid and sensitive hydrophilic interaction liquid chromatography–tandem mass spectrometry (HILIC–MS/MS) method was developed for the first time to determine adefovir in human plasma and applied to a pharmacokinetic study. Plasma samples were prepared by protein precipitation with methanol followed by a further cleaning using dichloromethane. The chromatographic separation was carried out on an ACQUITY UPLC™ BEH HILIC column with the mobile phase of methanol–water–formic acid (85:15:0.2, v/v/v). The detection was performed on a triple-quadrupole tandem mass spectrometer with multiple reaction monitoring (MRM) mode via electrospray ionization (ESI) source. The method was rapid with a run time of 3min per sample. The linear calibration curves were obtained in the concentration range of 1.02–102ng/mL (r 2 ≥0.99) with the lower limit of quantification (LLOQ) of 1.02ng/mL. The intra- and inter-day precision (relative standard deviation, R.S.D.) values were below 12% and the accuracy (relative error, R.E.) was from 0.6% to 3.2% at all quality control (QC) levels. The method was applicable to clinical pharmacokinetic study of adefovir in healthy volunteers after oral administration of adefovir dipivoxil tablet. [Copyright &y& Elsevier]

Background & Aims: Entecavir is a potent inhibitor of viral replication in nucleos(t)ide analogue (NA)-naïve chronic hepatitis B patients, but data on the efficacy in NA-experienced subjects are limited. Methods: In a multi-center cohort study we investigated 161 chronic hepatitis B patients (34% NA-experienced) treated with entecavir monotherapy. Results: During a median follow-up of 11 (3–23)months, 82 (79%) of 104 NA-naïve patients achieved virologic response (VR), defined as HBV DNA <80IU/ml, and none of the patients (0%) developed genotypic entecavir-resistance. VR was demonstrated in 31 (54%) of 57 NA-experienced patients during a median follow-up of 12 (3–31)months. Patients with lamivudine-resistant mutations at the start of entecavir monotherapy had a reduced probability of achieving VR compared to lamivudine-naïve patients (HR 0.14; 95% CI 0.04–0.58; p =0.007). Antiviral efficacy was not decreased by prior treatment with lamivudine when lamivudine-resistance had never developed (HR 0.81; 95% CI 0.43–1.52; p =0.52). Prior adefovir therapy without development of adefovir-resistance (HR 0.84; 95% CI 0.43–1.64; p =0.61) and presence of adefovir-resistance (HR 0.86; 95% CI 0.27–2.71; p =0.80) did not influence antiviral response to entecavir. Switching to a tenofovir-containing treatment regimen resulted in viral load decline in patients with entecavir-resistance associated mutations. Conclusions: Entecavir proved to be efficacious in NA-naïve patients. The antiviral efficacy of entecavir was not influenced by prior treatment with adefovir or presence of adefovir-resistance. Entecavir should not be used in patients with previous lamivudine-resistance, yet it may still be an option in lamivudine-experienced patients in case lamivudine-resistance never developed. [Copyright &y& Elsevier]

Abstract: Chronic hepatitis B virus (HBV) infection is overall recognised in 10% of HIV+ persons worldwide, with large differences according to geographical region. Chronic hepatitis C virus (HCV) infection affects 25% of HIV+ individuals, with greater rates (∼75%) in intravenous drug users and persons infected through contaminated blood or blood products. HIV-hepatitis co-infected individuals show an accelerated course of liver disease, with faster progression to cirrhosis. The number of anti-HBV drugs has increased in the last few years, and some agents (e.g. lamivudine, emtricitabine, tenofovir) also exert significant activity against HIV. Emergence of drug resistance challenges the long-term benefit of anti-HBV monotherapy, mainly with lamivudine. The results using new more potent anti-HBV drugs (e.g. tenofovir) are very promising, with prospects for stopping or even revert HBV-related liver damage in most cases. With respect to chronic hepatitis C, the combination of pegylated interferon plus ribavirin given for 1 year permits to achieve sustained HCV clearance in no more than 40% of HIV–HCV co-infected patients. Thus, new direct anti-HCV drugs are eagerly awaited for this population. Although being a minority, HIV+ patients with delta hepatitis and those with multiple hepatitis show the worst prognosis. Appropriate diagnosis and monitoring of chronic viral hepatitis, including the use of non-invasive tools for assessing liver fibrosis and measurement of viral load, may allow to confront adequately chronic viral hepatitis in HIV+ patients, preventing the development of end-stage liver disease, for which the only option available is liver transplantation. This article forms part of a special issue of Antiviral Research marking the 25th anniversary of antiretroviral drug discovery and development, Vol 85, issue 1, 2010. [Copyright &y& Elsevier]

Background/Aims: The rtQ215S mutation in the hepatitis B virus (HBV) polymerase has been described as a secondary mutation associated with resistance to lamivudine (LAM) and adefovir (ADV). We aimed at assessing the prevalence of substitutions at rtQ215 of the HBV polymerase and determining the molecular and functional consequences using phenotypic analyses in vitro. Methods: The polymerase region was directly sequenced in HBV isolates from a cohort of 249 HBV genotype D-infected patients from Iran (174 males/ 75 females, 194 treatment-naı¨ve/ 55 LAM-treated). Replication-competent HBV constructs containing the naturally occurring rtQ215H, rtQ215P and rtQ215S mutations were generated, and compared to wild-type, LAM- (rtM204I, rtL180M/rtM204V) and ADV-resistant (rtN236T) clones. Results: In an Iranian cohort of 249 HBV infected patients, 14.5% (36/249) showed mutations in the rtQ215 locus, namely 6.8% rtQ215S, 3.6% rtQ215P and 4.1% rtQ215H. The frequency of rtQ215 substitutions was higher in LAM-treated than treatment-naı¨ve patients (25% vs. 11%), but not associated with clinical complications. In phenotypic assays, rtQ215S, rtQ215P and rtQ215H constructs showed equivalent levels of viral replication as wild-type HBV, whereas LAM- and ADV-resistant mutants had significantly impaired replicative capacities. Furthermore, rtQ215S, rtQ215P and rtQ215H harbouring constructs remained susceptible towards treatment with LAM or ADV in vitro. Conclusions: Our study reveals that rtQ215 substitutions in the HBV polymerase frequently occur in chronic hepatitis B, even without exogenous selection pressures. As these substitutions do neither impair the viral replication efficiency nor susceptibility to LAM or ADV in vitro, rtQ215 substitutions likely represent background polymorphisms rather than resistance mutations with clinical implications. [Copyright &y& Elsevier]

Abstract: Background: Data concerning the outcome of lamivudine-resistant (LAM-R) chronic hepatitis B (CHB) patients with compensated cirrhosis under adefovir (ADV) treatment are limited. The aim of our study was to evaluate the medium term outcome of these, high-risk for fatal events, patients. Methods: 31 LAM-R patients with compensated cirrhosis who had been treated with ADV monotherapy (n =8) or ADV plus LAM (n =23) for a mean of 27.6 months, were evaluated. Virological response (VR) was defined as HBV-DNA levels <104 copies/ml within the first year of treatment. Results: Twenty-three patients (74.19%) achieved VR. Six patients (19.35%) developed ADV-related mutations (annual incidence 11%). Liver-related death, liver decompensation and hepatocellular carcinoma (HCC) were observed in 12.9%, 16.12% and 16.12% of patients, respectively. HCC (annual incidence 9.1%) was the main cause of liver decompensation (4/5, 80%) and of liver-related deaths (3/4, 75%). HCC development was not related to patients'' age (p =0.440), HBeAg status (p =0.245), HBV genotype (p =0.598), baseline ALT levels (p =0.981), baseline viral load (p = 0.464), VR (p =0.504) as well as emergence of ADV resistance (p =0.871). Conclusions: ADV suppresses viral replication in more than 70% of LAM-R cirrhotic patients during the first year of treatment. Despite that, HCC is frequently observed in these high-risk patients, irrespective of virological response or emergence of ADV resistance. [Copyright &y& Elsevier]

Background/Aims: We investigated the efficacy of entecavir in lamivudine-experienced and -naïve patients with persistently high HBV DNA during adefovir treatment. Methods: Fourteen chronic hepatitis B patients (57% lamivudine-experienced) with a viral load above 5log10 copies/mL after 12months of adefovir therapy and thereafter were treated with entecavir 1mg daily. Results: During a median follow-up of 15months (range: 8–23months) one of six lamivudine–naïve and none of the eight lamivudine-experienced patients achieved undetectable HBV DNA (<373copies/mL). HBeAg loss occurred in none of the subjects. Two lamivudine-experienced patients demonstrated the rtM204I mutation; no other entecavir-resistant substitutions were detected (rtI169, rtT184, rtS202, and rtM250). Two of three patients with genotypic adefovir resistance at baseline demonstrated a rapid virologic response to entecavir, but undetectable HBV DNA was not achieved. To attain a better antiviral response the dosage of entecavir was increased to 2mg daily in two patients, resulting in further viral load decline for both of them. Conclusions: Entecavir monotherapy dosed at 1mg resulted in a slow reduction of viral load in both lamivudine-experienced and -naïve patients with persistently high HBV DNA during adefovir therapy. Increasing the dosage of entecavir led to further HBV DNA decline. [Copyright &y& Elsevier]

Abstract: Telbivudine, a nucleoside analog inhibitor of the viral polymerase of hepatitis B virus (HBV), has been approved for the treatment of chronic HBV infection, along with the nucleoside inhibitors lamivudine and entecavir, and the nucleotide inhibitors adefovir and tenofovir. The resistance profiles of these agents were investigated via drug treatment of HepG2 cells stably transfected with wild-type or mutant HBV genomes bearing known resistance mutations. Telbivudine was not active against HBV strains bearing lamivudine mutations L180M/M204V/I but remained active against the M204V single mutant in vitro, potentially explaining the difference in resistance profiles between telbivudine and lamivudine. Against HBV genomes with known telbivudine-resistance mutations, M204I and L80I/M204I, telbivudine, lamivudine and entecavir lost 353- to >1000-fold activity whereas adefovir and tenofovir exhibited no more than 3–5-fold change. Conversely, against HBV cell lines expressing adefovir resistance mutations N236T and A181V, or the A194T mutant associated with resistance to tenofovir, telbivudine remained active as shown by respective fold-changes of 0.5 (N236T) and 1.0 (A181V and A194T). These in vitro results indicate that nucleoside and nucleotide drugs have different cross-resistance profiles. The addition of telbivudine to ongoing adefovir therapy could provide effective antiviral therapy to patients who develop adefovir resistance. [Copyright &y& Elsevier]

Background/Aims: Early detection of antiviral drug-resistant mutations enables prompt initiation of rescue therapy. The aim of this study was to determine the accuracy and sensitivity of a new line probe assay in the detection of antiviral drug-resistant HBV mutations. Methods: One-hundred samples from 54 patients with virologic breakthrough during entecavir, lamivudine or adefovir treatment and 21 samples from 21 nucleoside-naïve patients were tested by direct sequencing and an updated line probe assay (Innogenetics, HBV DR v.3) which incorporates probes that can detect mutations at 11 positions of the reverse transcriptase region of the HBV polymerase gene. Results: Complete concordance between line probe and sequencing results was observed for 90/121 samples (74.3%) and 1291/1331 amino acid positions (96.9%). Testing of follow-up samples and clonal analysis of discordant samples confirmed the presence of mutations where line probe assay but not direct sequencing detected mutations. HBV DR v.3 assay consistently detected mutations present in ⩾5% of the virus population when HBV DNA concentration was ⩾4log10 copies/mL. Conclusions: The updated version of the line probe assay (HBV DR v.3) has high concordance with direct sequencing in detecting antiviral drug-resistant mutations but its sensitivity in detecting mutations at some positions needs to be improved. [Copyright &y& Elsevier]

Abstract: Hepatitis B surface antigen (HBsAg) loss under antiviral therapy is rare in chronic hepatitis B patients and the dynamics of serum HBsAg in these patients are not available. The changes in serum HBsAg following treatment with adefovir (n =31) or peg-interferon-α-2a (n =23) were studied in hepatitis B e-antigen (HBeAg) positive chronic hepatitis B patients. Abbott Architect HBsAg assay was used to quantify serum HBsAg. HBsAg levels were significantly decreased during the first 12 weeks of treatment with median change of −397.0IU/ml and −555.4IU/ml, respectively for adefovir and peg-interferon-α-2a (p =0.005 and 0.001, respectively). Beyond 12 weeks, no further significant HBsAg reductions were found even in patients with sustained viral replication inhibition in either group. Three distinct patterns of HBsAg changes were observed in most patients in both treatment groups: biphasic pattern (rapid HBsAg reduction from baseline to week 12); assurgent pattern (higher HBsAg level at week 12 than at baseline); and wavy pattern (HBsAg reduction from baseline to week 12, followed by relapse at week 24 or week 28). These results might offer insights into the possible mechanism(s) underlying the unusual occurrences of HBsAg loss under antiviral therapy. [Copyright &y& Elsevier]

Studies in the past decades have shown that active hepatitis B virus (HBV) replication is the key driver of liver injury and disease progression, and thus sustained viral suppression is of paramount importance in the management of chronic HBV infection. The nucleos(t)ide analogues lamivudine, adefovir, entecavir, telbivudine and tenofovir are potent inhibitors of HBV polymerase/reverse transcriptase activity and are highly effective in the suppression of HBV replication, but rarely eliminate the virus. Long-term therapy is usually required to achieve sustained hepatitis B e antigen seroconversion, HBV DNA suppression, ALT normalization and fibrosis reversal. Maintained long-term therapy has been demonstrated to significantly lower the rate of hepatic decompensation and development of cirrhosis or hepatocellular carcinoma. However, drug resistance is a serious risk on prolonged nucleos(t)ide analogue therapy, and this poses a critical challenge. Prevention and proper management of drug resistance are crucial to ensure long-term success. [Copyright &y& Elsevier]

The practising clinician is currently faced with a number of effective treatment options for chronic hepatitis B, including two formulations of interferon (standard IFN and pegylated IFN) and five nucleos(t)ide analogues (lamivudine, adefovir, entecavir, telbivudine and tenofovir). Treatment strategies can be divided into those aiming for sustained response after discontinuation of therapy and those that need to be maintained by prolonged antiviral therapy. Sustained response is particularly achieved with interferon-based therapy, while treatment-maintained response can be achieved with long-term nucleos(t)ide analogue therapy in the majority of patients. Of currently available drugs for the treatment of chronic hepatitis B, PEG-IFN seems to result in the highest rate of off-treatment sustained response after a 1-year course of therapy. Sustained transition to the immune-control phase (inactive HBsAg carrier state) can be achieved in 30–35% of HBeAg-positive patients and 20–25% of HBeAg-negative patients. Loss of HBsAg has been observed in 11% of both HBeAg-positive and HBeAg-negative patients after 3–4years. Since hepatitis B virus (HBV) genotype is an important predictor of response to PEG-IFN, determination of HBV genotype is essential in patients in whom sustained off-treatment response is pursued. Aiming for sustained response is of particular interest because many HBV-infected patients are in need of antiviral therapy at a young age and may otherwise require indefinite antiviral therapy. [Copyright &y& Elsevier]

Abstract: Adefovir (9-(2-phosphonomethoxyethyl)adenine) is an acyclic nucleoside phosphonate currently used for the treatment of hepatitis B. The aim of this study was to evaluate the effect of permeation enhancer DDAK (6-dimethylaminohexanoic acid dodecyl ester) on the transdermal and topical delivery of adefovir. In porcine skin, DDAK enhanced adefovir flux 42 times with maximum at pH 5.8 suggesting ion pair formation. DDAK increased thermodynamic activity and stratum corneum/vehicle distribution coefficient of adefovir, as well as it directly decreased the skin barrier resistance. Maximal flux was observed already at 2% adefovir+1% DDAK. The results were confirmed in freshly excised human skin where DDAK enhanced adefovir flux 179 times to 8.9μg/cm2/h. This rate of percutaneous absorption would allow for reaching effective plasma concentrations. After the topical application, adefovir concentrated in the stratum corneum with low penetration into the deeper skin layers from either aqueous or isopropyl myristate vehicle without the enhancer. With 1% DDAK, adefovir concentrations in the viable epidermis and dermis were 33–61 times higher. These results offer an attractive alternative to established routes of administration of adefovir and other acyclic nucleoside phosphonates. [Copyright &y& Elsevier]

Abstract: The objective of this work was to investigate feasibility of transdermal and dermal delivery of adefovir (9-(2-phosphonomethoxyethyl)adenine), a broad-spectrum antiviral from the class of acyclic nucleoside phosphonates. Transport of 2% adefovir through and into porcine skin and effects of various solvents, pH, and permeation enhancers were studied in vitro using Franz diffusion cell. From aqueous donor samples, adefovir flux through the skin was 0.2–5.4μg/cm2/h with greatest permeation rate at pH 7.8. The corresponding adefovir skin concentrations reached values of 120—350μg/g of tissue. Increased solvent lipophilicity resulted in higher skin concentration but had only minor effect on adefovir flux. A significant influence of counter ions on both transdermal and dermal transport of adefovir zwitterion was observed at pH 3.4. Permeation enhancer dodecanol was ineffective, 1-dodecylazepan-2-one (Azone) and dodecyl 2-(dimethylamino)propionate (DDAIP) showed moderate activity. The highest adefovir flux (11.3±3.6μg/cm2/h) and skin concentration (1549±416μg/g) were achieved with 1% Transkarbam 12 (5-(dodecyloxycarbonyl)pentylammonium 5-(dodecyloxycarbonyl)pentylcarbamate) at pH 4. This study suggests that, despite its hydrophilic and ionizable nature, adefovir can be successfully delivered through the skin. [Copyright &y& Elsevier]

Background/Aims: We sought to identify mutations associated with treatment failure to adefovir (ADV) and to determine virologic response to tenofovir (TDF) alone and in combination with emtricitabine (FTC) in these patients. Methods: Serum samples prior to and after the change in treatment to TDF/TDF+FTC from 13 patients were analyzed by direct sequencing and clonal analysis. Results: ADV-resistant mutations, rtA181V and rtN236T, were detected on direct sequencing in 3 of 8 patients who had virologic breakthrough. Among patients with suboptimal virologic response, rtA181T, rtI233V, and rtN236T were present on clonal analysis in 3 patients. Ten patients received TDF, 8 achieved virologic response. One had ADV-resistance at baseline and persistence of ADV-resistant mutations during TDF treatment, addition of FTC resulted in a further decrease in HBV DNA. Another patient had no ADV-resistance at baseline, and selection of ADV-resistant mutations during TDF treatment. All 3 patients who received TDF+FTC had undetectable HBV DNA within 3–12months including 2 who had ADV-resistance at baseline. Conclusions: TDF monotherapy is effective for patients with virologic breakthrough or suboptimal response to ADV, but combination therapy with a nucleoside analogue should be considered in patients with ADV-resistance. No novel mutations were detected. [Copyright &y& Elsevier]

Abstracts: Objectives:The aim of this study was to assess the therapeutic effectiveness of adefovir dipivoxil (ADV), administered in combination with lamivudine (LAM) or as monotherapy, and the rate of resistance to ADV, in hepatitis B e antigen (HBeAg)-negative adult patients with chronic hepatitis B virus (HBV) infection and clinical or virologic resistance to LAM. Furthermore, we evaluated in these selected patients the clinical co-variates associated with a sustained virologic response. Methods:Data from adult outpatients aged >18 years with chronic HBV infection and clinical or virologic resistance to LAM were used in this retrospective, multicenter, nonrandomized, open-label study. Patients were selected if they received ADV 10 mg PO QD + LAM 100 mg QD PO or ADV 10 mg PO QD as monotherapy for 24 to 32 months between June 2003 and July 2006. End points were the proportions of patients who achieved virologic response (undetectable HBV-DNA [<3.3 log10 copies/mL]) and biochemical response (normalization [<40 IU/L] of alanine aminotransferase [ALT]), and the proportions in whom resistance to ADV (rebound serum HBV-DNA >1 log10 copies/mL compared with on-treatment nadir, as confirmed on molecular analysis) was found. HBV-DNA and ALT levels were checked every month during the first 3 months of treatment and every 3 months thereafter until 28 months. Data from each center were stored in a centralized database and analyzed by a blinded independent investigator. Results:Data from 70 patients were included (48 men, 22 women; median age, 51 years; ADV + LAM, 36 patients; ADV monotherapy, 34). The median duration of the pharmacologic treatment in the 2 groups of patients was 28 months (range, 24–32 months). By month 3, virologic response was achieved in 30 patients (83%) in the ADV + LAM group and in 26 patients (76%) in the ADV monotherapy group. At 12 months, virologic response was achieved in 5 additional patients in the ADV + LAM group and 2 additional patients in the ADV monotherapy group. Biochemical response was found to be time dependent: in the 2 groups, the rates of biochemical response were, respectively, 56% and 54% at month 3, 80% and 71% at month 6, and 96% and 79% at month 12, persisting up to the end of the study period. The rates of clinical resistance to ADV were 3% with ADV + LAM and 18% with ADV monotherapy (with a 6% rate of resistance due to rtA181 mutation in the monotherapy group). Logistic regression analysis found that pre-treatment levels of HBV-DNA <5 log10 copies/mL, ALT levels >150 IU/L, an inflammation score >7, and a fibrosis score <2 were the strongest covariates independently associated with a sustained virologic response in both groups of patients. No adverse events were reported in any of the patients. Conclusion:ADV, administered in combination with LAM or as monotherapy, appeared to be effective in this small, selected group of HBeAg-negative patients with clinical or virologic resistance to LAM, especially in those with low pretreatment HBV-DNA levels, high ALT levels, and low fibrosis scores. [Copyright &y& Elsevier]

Background/Aims: The rtA181V and rtN236T mutations have been associated with resistance to adefovir dipivoxil (ADV). Recent reports have proposed other ADV resistance (ADV-R) mutations. The aims of this study were to confirm the role of rtA181V and rtN236T in clinical resistance to ADV and to screen for other potential ADV-R mutations. Methods: Patients from ADV studies (n =998) were screened for viral breakthrough and/or insufficient HBV DNA suppression after at least 48 weeks of ADV therapy [virologic failure, VF]. McNemar’s exact test was used to test for differences in the proportion of patients with switches from consensus amino acid (AA) at baseline to non-consensus AA at VF and vice versa. Results: Data obtained from 172 paired HBV polymerase sequences demonstrated that only positions rt181 and rt236 had significantly more changes among patients with VF after adjusting for multiple comparisons (p <0.0005). When tested separately, the mutations rtA181V and rtN236T were statistically significant (p <0.0005); no other AA position was associated with VF. Patients who had HBV DNA breakthrough were more likely to develop ADV-R mutations than patients with insufficient HBV DNA suppression (36% vs. 5%). Conclusions: rtA181V and rtN236T were the only HBV polymerase mutations significantly associated with virologic failure to adefovir dipivoxil. [Copyright &y& Elsevier]

Background/Aims: To determine the factors associated with virological response (VR), HBeAg loss or the emergence of adefovir (ADV)-related mutations in ADV-treated chronic hepatitis B (CHB) patients with lamivudine (LAM) resistance. Methods: Fifty-four LAM-resistant CHB patients (46% HBeAg-positive) were treated with ADV monotherapy (n =28) or ADV plus LAM (n =26) for a mean of 30.4 months. Results: Thirty-eight patients (70.4%) achieved VR defined as HBV-DNA levels <104 copies/ml within the first 12 months of treatment. Six (24%) of 25 HBeAg-positive patients exhibited HBeAg loss and 20% seroconverted to anti-HBe. Eight patients (14.8%) developed ADV-related mutations. In the multivariate analysis, female gender (HR=0.20, 95% CI: 0.05–0.76, p =0.018), HBeAg-negative (HR=0.37, 95% CI: 0.14–0.96, p =0.040) and low baseline HBV-DNA levels (HR=0.65, 95% CI: 0.45–0.95, p =0.027) were independent predictors of VR, whereas low HBV-DNA levels (HR=0.36, 95% CI: 0.11–1.20, p =0.095) and HBV-genotype D (HR=0.06, 95% CI: 0.004–0.84, p =0.037) independently predicted HBeAg loss. Conclusions: ADV therapy suppresses viral replication in more than 70% of LAM-R patients. Factors associated with virologic response are female gender, HBeAg-negative status and low baseline serum HBV-DNA levels. Genotype D HBV infection and low baseline HBV-DNA levels independently predict HBeAg loss. [Copyright &y& Elsevier]

Abstract: Esterification of cidofovir (CDV), an antiviral nucleoside phosphonate, with alkyl or alkoxyalkyl groups increases antiviral activity by enhancing cell uptake and conversion to CDV diphosphate. Hexadecyloxypropyl-CDV (HDP-CDV) has been shown to be 40–100 times more active than CDV in vitro in cells infected with herpes group viruses, variola, cowpox, vaccinia or ectromelia viruses. Since the first phosphorylation of CDV may be rate limiting, we synthesized the hexadecyloxypropyl-phosphate (HDP-P-) and octadecyloxyethyl-phosphate (ODE-P-) conjugates of CDV and phosphonomethoxy-ethyl-adenine (PMEA, adefovir). We tested the CDV analogs in cells infected with human cytomegalovirus, herpes simplex virus, cowpox virus and vaccinia virus; the analogs of PMEA were tested in cells infected with the human immunodeficiency virus, type 1. In general, the alkoxyalkyl-phosphate conjugates of CDV were substantially more active than CDV. HDP-P-CDV and ODE-P-CDV were 4.6–40 times more active against HCMV and 7–30 times more active against cowpox and vaccinia in vitro. Although the compounds of this type were more cytotoxic than the unmodified bases, their selectivity for virally infected cells was generally greater than the parent nucleotides except that HDP-P-PMEA showed little or no selectivity in HIV-1 infected MT-2 cells. Although the new compounds with an interposed phosphate were generally less active than the corresponding alkoxyalkyl esters of CDV and PMEA, the present approach provides a possible alternative method for enhancing the antiviral activity of drugs of this class. [Copyright &y& Elsevier]

Abstract: A simple HPLC/UV method for the determination of the transdermal permeation and dermal penetration of a broad-spectrum antiviral drug adefovir (PMEA) was developed. The separation was achieved on a C18 column with the mobile phase composed of 10mM KH2PO4 and 2mM Bu4NHSO4 at pH 6.0 and 7% acetonitrile. The method was validated with respect to selectivity, linearity (0.1–50μg/ml), precision, accuracy, and stability. Transdermal permeation of 2% PMEA was studied in vitro using the Franz diffusion cell and porcine skin. The flux values were 1.8, 3.0, and 0.6μg/cm2/h from aqueous donor samples at pH 3.4 and 7.4, and isopropyl myristate, respectively. The respective skin concentrations at 48h were 294, 263, and 971μg/g from these vehicles. These results will serve as a lead for further studies on transdermal and topical delivery of antivirals from the group of acyclic nucleoside phosphonates. [Copyright &y& Elsevier]

Background/Aims: Some patients receiving adefovir at the approved dose of 10mg daily for chronic hepatitis B have a “suboptimal” virological response characterized by a slow and moderate decrease in viral replication. Methods: We assessed the efficacy and safety of adefovir 20mg daily in patients with hepatitis B e antigen-positive chronic hepatitis B resistant to lamivudine and a suboptimal virological response to adefovir 10mg daily add-on. Results: No amino acid substitutions known to confer adefovir resistance were found in these patients. In the five treated patients, the switch from 10mg to 20mg of adefovir daily significantly improved antiviral efficacy (−1.78±0.28 log international units/mL versus −3.73±0.51 log international units/mL, respectively, p =0.0039), and alanine aminotransferase levels normalized in all but one of the patients. No signs of renal dysfunction occurred. Conclusions: These results suggest: (i) that suboptimal responses to adefovir 10mg daily are due to underdosing; and (ii) that increasing the adefovir dose to 20mg daily is beneficial and safe in patients with lamivudine-resistant HBV and a suboptimal response to adefovir 10mg daily, especially when alanine aminotransferase levels are elevated and/or the liver disease is severe or rapidly progressive. Careful monitoring of renal function is necessary. [Copyright &y& Elsevier]

Abstract: The nucleotide analog adefovir is an important therapy for hepatitis B viral infection. The study of nucleoside/tide pharmacology has been hampered by difficulties encountered when trying to develop LC/MS/MS methods for these polar analytes. In an attempt to identify a more convenient, selective and sensitive alternative to the analysis of the metabolism of radiolabled parent nucleotide traditionally used for in vitro cell culture studies, an LC/MS/MS method was developed for the quantitative detection of adefovir and its phosphorylated metabolites in cellular samples. Ion-pairing reversed phase LC using tetrabutylammonium (TBA) and ammonium phosphate had the best compromise between chromatographic separation and positive mode MS/MS detection. Using microbore reverse phase columns and a low flow acetonitrile gradient it was possible to quantitate adefovir, its metabolites and 2′-deoxyadenosine triphosphate. A cross-validation showed comparable levels of adefovir and its metabolites were determined using either LC/MS/MS or radioactivity detection. However, initial methods were conducted at high pH and utilized an acetonitrile step gradient causing unacceptable column life and unpredictable equilibration. Further method optimization lowered the concentration of TBA and phosphate, decreased pH and applied a linear gradient of acetonitrile. This work resulted in a method that was found to have sensitivity, accuracy and precision sufficient to be a useful tool in the study of the intracellular pharmacology of adefovir in vitro and may be more broadly applicable. [Copyright &y& Elsevier]