The Future of HCV Therapy: NS4B as an Antiviral Target

Chronic hepatitis C virus (HCV) infection is a major worldwide cause of liver disease, including cirrhosis and hepatocellular carcinoma. It is estimated that more than 170 million individuals are infected with HCV, with three to four million new cases each year. The current standard of care, combination treatment with interferon and ribavirin, eradicates the virus in only about 50% of chronically infected patients. Notably, neither of these drugs directly target HCV. Many new antiviral therapies that specifically target hepatitis C (e.g. NS3 protease or NS5B polymerase inhibitors) are therefore in development, with a significant number having advanced into clinical trials. The nonstructural 4B (NS4B) protein, is among the least characterized of the HCV structural and nonstructural proteins and has been subjected to few pharmacological studies. NS4B is an integral membrane protein with at least four predicted transmembrane (TM) domains. A variety of functions have been postulated for NS4B, such as the ability to induce the membranous web replication platform, RNA binding and NTPase activity. This review summarizes potential targets within the nonstructural protein NS4B, with a focus on novel classes of NS4B inhibitors

Caffeine vs. carbamazepine as indicators of wastewater pollution in a karst aquifer

This paper presents the analysis of caffeine and carbamazepine transport in the subsurface as a result of wastewater release in the Sorek creek over the outcrops of the carbonate, Yarkon-Taninim, aquifer in Israel. Both caffeine and carbamazepine were used as indicators of sewage contamination in the subsurface. While carbamazepine is considered conservative, caffeine is subject to sorption and degradation. The objective of the study was to quantify differences in their transport under similar conditions in the karst aquifer. Water flow and pollutant transport in a “vadose zone–aquifer” system were simulated by a quasi-3-D dual permeability numerical model. The results of this study show that each of these two pollutants can be considered effective tracers for characterization and assessment of aquifer contamination. Carbamazepine was found to be more suitable for assessing the contamination boundaries, while caffeine can be used as a contaminant tracer only briefly after contamination occurs. In instances where there are low concentrations of carbamazepine which appear as background contamination in an aquifer, caffeine might serve as a better marker for detecting new contamination events, given its temporal nature. The estimated caffeine degradation rate and the distribution coefficient of a linear sorption isotherm were 0.091&thinsp;d−1 and 0.1&thinsp;L&thinsp;kg−1, respectively, which imply a high attenuation capacity. The results of the simulation indicate that by the end of the year most of the carbamazepine mass (approximately 95&thinsp;%) remained in the matrix of the vadose zone, while all of the caffeine was completely degraded a few months after the sewage was discharged.</p

NS5A Resistance-Associated Substitutions in Patients with Genotype 1 Hepatitis C Virus:Prevalence and Effect on Treatment Outcome

Background & Aims The efficacy of NS5A inhibitors for the treatment of patients chronically infected with hepatitis C virus (HCV) can be affected by the presence of NS5A resistance-associated substitutions (RASs). We analyzed data from 35 phase I, II, and III studies in 22 countries to determine the pretreatment prevalence of various NS5A RASs, and their effect on outcomes of treatment with ledipasvir-sofosbuvir in patients with genotype 1 HCV. Methods NS5A gene deep sequencing analysis was performed on samples from 5397 patients in Gilead clinical trials. The effect of baseline RASs on sustained virologic response (SVR) rates was assessed in the 1765 patients treated with regimens containing ledipasvir-sofosbuvir. Results Using a 15% cut-off, pretreatment NS5A and ledipasvir-specific RASs were detected in 13% and 8% of genotype 1a patients, respectively, and in 18% and 16% of patients with genotype 1b. Among genotype 1a treatment-naïve patients, SVR rates were 91% (42/46) vs. 99% (539/546) for those with and without ledipasvir-specific RASs, respectively. Among treatment-experienced genotype 1a patients, SVR rates were 76% (22/29) vs. 97% (409/420) for those with and without ledipasvir-specific RASs, respectively. Among treatment-naïve genotype 1b patients, SVR rates were 99% for both those with and without ledipasvir-specific RASs (71/72 vs. 331/334), and among treatment-experienced genotype 1b patients, SVR rates were 89% (41/46) vs. 98% (267/272) for those with and without ledipasvir-specific RASs, respectively. Conclusions Pretreatment ledipasvir-specific RASs that were present in 8–16% of patients have an impact on treatment outcome in some patient groups, particularly treatment-experienced patients with genotype 1a HCV. Lay summary The efficacy of treatments using NS5A inhibitors for patients with chronic hepatitis C virus (HCV) infection can be affected by the presence of NS5A resistance-associated substitutions (RASs). We reviewed results from 35 clinical trials where patients with genotype 1 HCV infection received treatments that included ledipasvir-sofosbuvir to determine how prevalent NS5A RASs are in patients at baseline, and found that ledipasvir-specific RASs were present in 8–16% of patients prior to treatment and had a negative impact on treatment outcome in subset of patient groups, particularly treatment-experienced patients with genotype 1a HCV

Efficacy of Sofosbuvir, Velpatasvir, and GS-9857 in Patients With Hepatitis C Virus Genotype 2, 3, 4, or 6 Infections in an Open-Label, Phase 2 Trial

Background & Aims Studies are needed to determine the optimal regimen for patients with chronic hepatitis C virus (HCV) genotype 2, 3, 4, or 6 infections whose prior course of antiviral therapy has failed, and the feasibility of shortening treatment duration. We performed a phase 2 study to determine the efficacy and safety of the combination of the nucleotide polymerase inhibitor sofosbuvir, the NS5A inhibitor velpatasvir, and the NS3/4A protease inhibitor GS-9857 in these patients. Methods We performed a multicenter, open-label trial at 32 sites in the United States and 2 sites in New Zealand from March 3, 2015 to April 27, 2015. Our study included 128 treatment-naïve and treatment-experienced patients (1 with HCV genotype 1b; 33 with HCV genotype 2; 74 with HCV genotype 3; 17 with genotype HCV 4; and 3 with HCV genotype 6), with or without compensated cirrhosis. All patients received sofosbuvir-velpatasvir (400 mg/100 mg fixed-dose combination tablet) and GS-9857 (100 mg) once daily for 6–12 weeks. The primary end point was sustained virologic response 12 weeks after treatment (SVR12). Results After 6 weeks of treatment, SVR12s were achieved by 88% of treatment-naïve patients without cirrhosis (29 of 33; 95% confidence interval, 72%–97%). After 8 weeks of treatment, SVR12s were achieved by 93% of treatment-naïve patients with cirrhosis (28 of 30; 95% CI, 78%–99%). After 12 weeks of treatment, SVR12s were achieved by all treatment-experienced patients without cirrhosis (36 of 36; 95% CI, 90%–100%) and 97% of treatment-experienced patients with cirrhosis (28 of 29; 95% CI, 82%–100%). The most common adverse events were headache, diarrhea, fatigue, and nausea. Three patients (1%) discontinued treatment due to adverse events. Conclusions In a phase 2 open-label trial, we found sofosbuvir-velpatasvir plus GS-9857 (8 weeks in treatment-naïve patients or 12 weeks in treatment-experienced patients) to be safe and effective for patients with HCV genotype 2, 3, 4, or 6 infections, with or without compensated cirrhosis

Sofosbuvir and Velpatasvir for HCV Genotype 2 and 3 Infection

BACKGROUND: In phase 2 trials, treatment with the combination of the nucleotide polymerase inhibitor sofosbuvir and the NS5A inhibitor velpatasvir resulted in high rates of sustained virologic response in patients chronically infected with hepatitis C virus (HCV) genotype 2 or 3. METHODS: We conducted two randomized, phase 3, open-label studies involving patients who had received previous treatment for HCV genotype 2 or 3 and those who had not received such treatment, including patients with compensated cirrhosis. In one trial, patients with HCV genotype 2 were randomly assigned in a 1:1 ratio to receive sofosbuvir-velpatasvir, in a once-daily, fixed-dose combination tablet (134 patients), or sofosbuvir plus weight-based ribavirin (132 patients) for 12 weeks. In a second trial, patients with HCV genotype 3 were randomly assigned in a 1:1 ratio to receive sofosbuvir-velpatasvir for 12 weeks (277 patients) or sofosbuvir-ribavirin for 24 weeks (275 patients). The primary end point for the two trials was a sustained virologic response at 12 weeks after the end of therapy. RESULTS: Among patients with HCV genotype 2, the rate of sustained virologic response in the sofosbuvir-velpatasvir group was 99% (95% confidence interval [CI], 96 to 100), which was superior to the rate of 94% (95% CI, 88 to 97) in the sofosbuvir-ribavirin group (P=0.02). Among patients with HCV genotype 3, the rate of sustained virologic response in the sofosbuvir-velpatasvir group was 95% (95% CI, 92 to 98), which was superior to the rate of 80% (95% CI, 75 to 85) in the sofosbuvir-ribavirin group (P CONCLUSIONS: Among patients with HCV genotype 2 or 3 with or without previous treatment, including those with compensated cirrhosis, 12 weeks of treatment with sofosbuvir-velpatasvir resulted in rates of sustained virologic response that were superior to those with standard treatment with sofosbuvir-ribavirin. (Funded by Gilead Sciences; ASTRAL-2 ClinicalTrials.gov number, NCT02220998; and ASTRAL-3, NCT02201953.)

STAT3 paradoxically stimulates ??catenin expression but inhibits ??catenin function

Wnt signalling and the signal transducer and activator of transcription 3 (STAT3) are oncogenic signalling pathways which are deregulated in colorectal cancer (CRC). Here we investigated the interaction of these two pathways. Firstly, we investigated biochemical interaction by inhibiting STAT3 and b-catenin (through gene knockdown and dominant-negative TCF4 expression) in nine CRC cell lines. b-catenin inhibition did not affect STAT3 levels, whereas STAT3 knock-down resulted in reduced b-catenin mRNA and protein levels. The reduction in b-catenin protein was not prevented by proteasome inhibition, and IL6-induced STAT3 activation resulted in increased b-catenin mRNA. This suggests that STAT3 positively regulates b-catenin (at a transcriptional level) and evaluation of 44 CRCs by immunostaining supported this by showing an association between nuclear STAT3 expression and nuclear b-catenin (P = 0.022). We tested the functional interaction between STAT3 and Wnt signalling by knocking down STAT3 and b-catenin individually and in combination. Knock-down of b-catenin and STAT3 individually inhibited cell proliferation (P < 0. 001 for each) through G1 arrest. However, simultaneous knockdown of STAT3 and b-catenin had a significantly weaker effect than knock-down of b-catenin alone (P < 0.01). Knock-down of STAT3 and b-catenin, individually and together, inhibited cell motility (P < 0.001) without evidence of interaction. We conclude that STAT3 regulates b-catenin but b-catenin does not regulate STAT3. The STAT3/b-catenin interaction is complex but may reduce the proliferative activity of b-catenin possibly by taking b-catenin protein beyond the optimal level. This may indicate biological differences in tumours where both STAT3 and b-catenin are activated compared to those where only one is activated

A human ribonuclease induces apoptosis associated with p21WAF1/CIP1 induction and JNK inactivation

<p>Abstract</p> <p>Background</p> <p>Ribonucleases are promising agents for use in anticancer therapy. Among the different ribonucleases described to be cytotoxic, a paradigmatic example is onconase which manifests cytotoxic and cytostatic effects, presents synergism with several kinds of anticancer drugs and is currently in phase II/III of its clinical trial as an anticancer drug against different types of cancer. The mechanism of cytotoxicity of PE5, a variant of human pancreatic ribonuclease carrying a nuclear localization signal, has been investigated and compared to that of onconase.</p> <p>Methods</p> <p>Cytotoxicity was measured by the MTT method and by the tripan blue exclusion assay. Apoptosis was assessed by flow cytometry, caspase enzymatic detection and confocal microscopy. Cell cycle phase analysis was performed by flow cytometry. The expression of different proteins was analyzed by western blot.</p> <p>Results</p> <p>We show that the cytotoxicity of PE5 is produced through apoptosis, that it does not require the proapoptotic activity of p53 and is not prevented by the multiple drug resistance phenotype. We also show that PE5 and onconase induce cell death at the same extent although the latter is also able to arrest the cell growth. We have compared the cytotoxic effects of both ribonucleases in the NCI/ADR-RES cell line by measuring their effects on the cell cycle, on the activation of different caspases and on the expression of different apoptosis- and cell cycle-related proteins. PE5 increases the number of cells in S and G₂/M cell cycle phases, which is accompanied by the increased expression of cyclin E and p21^WAF1/CIP1together with the underphosphorylation of p46 forms of JNK. Citotoxicity of onconase in this cell line does not alter the cell cycle phase distribution and it is accompanied by a decreased expression of XIAP</p> <p>Conclusions</p> <p>We conclude that PE5 kills the cells through apoptosis associated with the p21^WAF1/CIP1induction and the inactivation of JNK. This mechanism is significantly different from that found for onconase.</p

Inhibition of arachidonic acid metabolism and its implication on cell proliferation and tumour-angiogenesis

Arachidonic acid (AA) and its metabolites have recently generated a heightened interest due to growing evidence of their significant role in cancer biology. Thus, inhibitors of the AA cascade, first and foremost COX inhibitors, which have originally been of interest in the treatment of inflammatory conditions and certain types of cardiovascular disease, are now attracting attention as an arsenal against cancer. An increasing number of investigations support their role in cancer chemoprevention, although the precise molecular mechanisms that link levels of AA, and its metabolites, with cancer progression have still to be elucidated. This article provides an overview of the AA cascade and focuses on the roles of its inhibitors and their implication in cancer treatment. In particular, emphasis is placed on the inhibition of cell proliferation and neo-angiogenesis through inhibition of the enzymes COX-2, 5-LOX and CYP450. Downstream effects of inhibition of AA metabolites are analysed and the molecular mechanisms of action of a selected number of inhibitors of catalytic pathways reviewed. Lastly, the benefits of dietary omega-3 fatty acids and their mechanisms of action leading to reduced cancer risk and impeded cancer cell growth are mentioned. Finally, a proposal is put forward, suggesting a novel and integrated approach in viewing the molecular mechanisms and complex interactions responsible for the involvement of AA metabolites in carcinogenesis and the protective effects of omega-3 fatty acids in inflammation and tumour prevention